@article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/107508, title ="Physiological, genomic, and sulfur isotopic characterization of methanol metabolism by Desulfovibrio carbinolicus", author = "Sim, Min Sub and Skennerton, Connor T.", journal = "PLoS ONE", volume = "16", number = "1", pages = "Art. No. e0245069", month = "January", year = "2021", doi = "10.1371/journal.pone.0245069", issn = "1932-6203", url = "https://resolver.caltech.edu/CaltechAUTHORS:20210115-120616827", note = "© 2021 Sim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. \n\nReceived: September 6, 2020; Accepted: December 21, 2020; Published: January 14, 2021. \n\nFunding: This work was supported by the Mid-Career Researcher Program (2019R1A2C1087039) through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) to MSS, and Gordon and Betty Moore Foundation Grant GBMF 3306 and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research program DE-SC0020373 to VJO. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. \n\nThe authors have declared that no competing interests exist. \n\nData Availability Statement: All relevant data are included within the paper. \n\nAuthor Contributions: Conceptualization: Min Sub Sim, Victoria J. Orphan. Data curation: Min Sub Sim, Connor T. Skennerton. Formal analysis: Min Sub Sim, Connor T. Skennerton. Investigation: Min Sub Sim, Connor T. Skennerton, Victoria J. Orphan. Resources: Victoria J. Orphan. Writing – original draft: Min Sub Sim, Connor T. Skennerton. Writing – review & editing: Min Sub Sim, Connor T. Skennerton, Victoria J. Orphan.", revision_no = "6", abstract = "Methanol is often considered as a non-competitive substrate for methanogenic archaea, but an increasing number of sulfate-reducing microorganisms (SRMs) have been reported to be capable of respiring with methanol as an electron donor. A better understanding of the fate of methanol in natural or artificial anaerobic systems thus requires knowledge of the methanol dissimilation by SRMs. In this study, we describe the growth kinetics and sulfur isotope effects of Desulfovibrio carbinolicus, a methanol-oxidizing sulfate-reducing deltaproteobacterium, together with its genome sequence and annotation. D. carbinolicus can grow with a series of alcohols from methanol to butanol. Compared to longer-chain alcohols, however, specific growth and respiration rates decrease by several fold with methanol as an electron donor. Larger sulfur isotope fractionation accompanies slowed growth kinetics, indicating low chemical potential at terminal reductive steps of respiration. In a medium containing both ethanol and methanol, D. carbinolicus does not consume methanol even after the cessation of growth on ethanol. Among the two known methanol dissimilatory systems, the genome of D. carbinolicus contains the genes coding for alcohol dehydrogenase but lacks enzymes analogous to methanol methyltransferase. We analyzed the genomes of 52 additional species of sulfate-reducing bacteria that have been tested for methanol oxidation. There is no apparent relationship between phylogeny and methanol metabolizing capacity, but most gram-negative methanol oxidizers grow poorly, and none carry homologs for methyltransferase (mtaB). Although the amount of available data is limited, it is notable that more than half of the known gram-positive methanol oxidizers have both enzymatic systems, showing enhanced growth relative to the SRMs containing only alcohol dehydrogenase genes. Thus, physiological, genomic, and sulfur isotopic results suggest that D. carbinolicus and close relatives have the ability to metabolize methanol but likely play a limited role in methanol degradation in most natural environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/107283, title ="Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer", author = "Mullin, Sean W. and Wanger, Greg", journal = "Frontiers in Microbiology", volume = "11", pages = "Art. No. 536535", month = "November", year = "2020", doi = "10.3389/fmicb.2020.536535", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20201224-085808375", note = "© 2020 Mullin, Wanger, Kruger, Sackett, Hamilton-Brehm, Bhartia, Amend, Moser and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 20 February 2020. Accepted: 24 September 2020. Published: 19 November 2020. \n\nSamples were obtained under scientific research permit DEVA-2013-SCI-0069, issued to DM from the U.S. National Park Service (NPS), and we thank Terry Fisk, Richard Friese, Josh Hoines, Genne Nelson, and Kevin Wilson of the NPS and Alisa Lembke of the Inyo County Planning Commission for site access. We extend our thanks also to John Healey, Brad Lyles, and Chuck Russell of the Desert Research Institute, John Bredehoeft and Michael King of The Hydronamics Group, LLC for logistical assistance; and Rachel Gates, Stephanie Connon, Haley Sapers, Daan Speth, Erin Bertrand, Ted Present, and Elizabeth Trembath-Reichert for occasional field and lab assistance. Thanks also to Bill Willborn and others from the DOE UGTA program for allowing use of their downhole logging system. \n\nThis work was supported by the NASA Astrobiology Institute “Life Underground” project (NNA13AA92A) and the Center for Dark Energy Biosphere Investigations (C-DEBI). SM received additional support from an NIH Training Grant. JS was partially supported by a NASA Space Grant Consortium Fellowship. \n\nAuthor Contributions. SM coordinated field experiment set-up and sampling and performed DNA sequencing, sequence analysis, microscopy, and gas chromatography. GW coordinated field sampling, including the design of the in situ suspension line and provided microscopy data. BK, JS, and SH-B performed field sampling. Additionally, BK and JS coordinated retrieval and processing of geochemistry analyses. SH-B performed phase contrast cell counts. DM supervised BK, JS, and SH-B, identified and secured the field site, co-wrote the funding proposal, and organized downhole logging. RB, JA, and VO co-wrote the proposal and supervised field activities. VO helped conceive the experimental design and supervised SM. All authors contributed to the article and approved the submitted version. \n\nData Availability Statement. The datasets generated for this study can be found in the NCBI SRA # PRJNA605066. \n\nConflict of Interest. Two authors declared commercial interests at the time of publication but not during the period of data collection: GW is employed by Oberland Agriscience, Inc., and RB is employed by Photon Systems, Inc. \n\nThe remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.", revision_no = "13", abstract = "The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter, Methyloversatilis, and Pseudomonas. Subsequent experimental deployments included a methanogen-dominated stage (Methanobacteriales and Methanomicrobiales) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/102503, title ="Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy", author = "Metcalfe, Kyle S. and Murali, Ranjani", journal = "ISME Journal", month = "October", year = "2020", doi = "", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200413-101943907", note = "© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nReceived: 10 April 2020; Revised: 28 July 2020; Accepted: 21 August 2020. \n\nThe authors acknowledge the ROC-HITS science party, R/V Atlantis crew, and HOV Alvin pilots from cruise AT37-13 for their assistance with sample collection and processing. We would like to thank H. Yu for assistance with sediment incubations, and S. Lim for performing IC measurements. We are grateful to Y. Guan for his assistance with the nanoSIMS analysis, R. Hatzenpichler for early BONCAT-FACS experiments, M. Aoki (National Institute of Technology, Wakayama College, Japan) for design of the FISH probe ANME-2a-828, and M. Schwarzkopf and Molecular Technologies for designing a set of HCR-FISH probes for nifH mRNA. We thank G. Chadwick and three anonymous reviewers for their comments on this work. Funding for this work was provided by the US Department of Energy’s Office of Science (DE-SC0020373), the SIMONS Foundation Life Sciences Collaboration on Principals of Microbial Ecosystems, the National Science Foundation BIO-OCE grant (#1634002), and a Gordon and Betty Moore Foundation Marine Microbiology Investigator grant (#3780); (all to VJO). A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). KSM was supported in part by a National Science Foundation Graduate Research Fellowship and a Schlanger Ocean Drilling Fellowship. VJO is a CIFAR Fellow in the Earth 4D: Subsurface Science and Exploration Program. \n\nThese authors contributed equally: Kyle S. Metcalfe, Ranjani Murali. \n\nThe authors declare that they have no conflict of interest.", revision_no = "94", abstract = "Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH₄ and ¹⁵N₂ revealed differences in cellular ¹⁵N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in ¹⁵N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/103049, title ="Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions", author = "Fincker, Maeva and Huber, Julie A.", journal = "Environmental Microbiology", volume = "22", number = "8", pages = "3188-3204", month = "August", year = "2020", doi = "10.1111/1462-2920.15061", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200506-150820782", note = "© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.\n\nIssue Online: 17 August 2020; Version of Record online: 04 June 2020; Accepted manuscript online: 05 May 2020; Manuscript accepted: 03 May 2020; Manuscript revised: 29 April 2020; Manuscript received: 23 February 2020. \n\nThe authors thank Benjamin J. Tully for his help accessing the C‐DEBI server on which part of the analysis was run. The authors thank Connor Skennerton for his work on the Santa Monica Mounds samples as well as the members of the Spormann Group for their helpful comments. Data from the Mid‐Cayman Rise were based upon work supported by the Schmidt Ocean Institute during cruise FX008‐2013 aboard R/V Falkor. The authors thank Julie Reveillaud and Rika Anderson for providing sequence data. Data for the White Oak River were based upon work supported by the US Department of Energy and a European Research Council DARCLIFE grant; the authors thank Cassandre Lazar and Brett Baker for their work on the samples. Data for the Guaymas Basin were based upon work supported by NSF Biological Oceanography grants; the authors thank Nina Dombrowski, Kiley Seitz for their work on the samples. Data from the Juan de Fuca Ridge flank were based on work supported by the National Science Foundation grants MCB‐0604014 and OCE‐1260723 during cruise AT18‐07 aboard the R/V Atlantis; the authors thank Sean Jungbluth for initial sequence analysis. This work was funded by the US National Science Foundation through the Center for Deep Dark Energy Biosphere Investigations. This is C‐DEBI contribution number 531.", revision_no = "32", abstract = "Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin‐dependent transport‐coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO₂ reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H₂ consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/102905, title ="Resolving micron-scale heterogeneity in porewater δ³⁴S_(H₂S) by combining films for in-situ sulfide capture and secondary ion mass spectrometry", author = "Houghton, J. L. and Jones, C.", journal = "Marine Chemistry", volume = "223", pages = "Art. No. 103810", month = "June", year = "2020", doi = "10.1016/j.marchem.2020.103810", issn = "0304-4203", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200429-124352121", note = "© 2020 Elsevier B.V. \n\nReceived 19 December 2019, Revised 20 April 2020, Accepted 21 April 2020, Available online 28 April 2020.", revision_no = "10", abstract = "Sulfur cycling is ubiquitous in marine sedimentary environments and is influenced by microbial and abiotic processes that alter both the abundance and isotopic composition of sulfur species that can ultimately be captured as sedimentary minerals. Microbial metabolisms that generate sulfur isotopic (δ³⁴S) signatures in hydrogen sulfide have a spatial distribution that varies on the micron scale, yet porewater hydrogen sulfide is most often measured in bulk samples representing much larger volumes. This mismatch of scales can lead to erroneous or non-unique interpretations of biogeochemical processes and environmental conditions. Recently, an in-situ film-based technique was described that captures dissolved sulfide (H₂S) in porewaters and which can be subsectioned to reconstruct the δ³⁴S_(H₂S) profiles on the sub-cm scale within sediments. Here, we investigate the use of a Cameca 7f-GEO secondary ion mass spectrometer (SIMS) to analyze the δ³⁴S_(H₂S) captured from porewaters on these films on even smaller spatial scales and particularly in films with low sulfide abundance that could not otherwise be processed with bulk extraction techniques. We present a best-practice method for film analysis that minimizes analytical artifacts from varying sulfide abundance and interactions with silver halide nanocrystals imbedded in the organic-based film amalgam. This method was tested on several films from field deployments, including examples with heterogeneities on small (~100\u202fμm) scales, steep isotopic gradients, and very low sulfide abundance across the sediment-water interface. The results demonstrate that analysis using SIMS can accurately measure δ³⁴S of in-situ sulfide captured by film with high precision (1σ\u202f~\u202f0.3‰) in both spot and image modes and that the film itself can accurately record δ³⁴S variability down to 25\u202fμm spatial resolution, below which physical limitations of the film can create artifacts.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101179, title ="Lateral Gene Transfer Drives Metabolic Flexibility in the Anaerobic Methane-Oxidizing Archaeal Family Methanoperedenaceae\n", author = "Leu, Andy O. and McIlroy, Simon J.", journal = "mBio", volume = "11", number = "3", pages = "Art. No. e01325-20", month = "May", year = "2020", doi = "10.1128/mBio.01325-20", issn = "2150-7511", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200207-102104518", note = "© 2020 Leu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. \n\nReceived 18 May 2020; Accepted 27 May 2020; Published 30 June 2020. \n\nThis work was supported by the Australian Research Council (ARC) (grant FT170100070) and the U.S. Department of Energy’s Office of Biological Environmental Research (grant DE-SC0016469). A.O.L. was supported by an ARC Australian Postgraduate Award, and S.J.M. was partly supported by an ARC Future Fellowship (FT190100211). \n\nWe thank the AWMC team, particularly Shihu Hu and Zhiguo Yuan, for their ongoing collaboration working on various “Ca. Methanoperedens” enrichments. \n\nWe have nothing to disclose. \n\nData availability.The genomes assembled in this study have been deposited in the NCBI database under the accession numbers SAMN10961276 to SAMN10961283.", revision_no = "33", abstract = "Anaerobic oxidation of methane (AOM) is an important biological process responsible for controlling the flux of methane into the atmosphere. Members of the archaeal family Methanoperedenaceae (formerly ANME-2d) have been demonstrated to couple AOM to the reduction of nitrate, iron, and manganese. Here, comparative genomic analysis of 16 Methanoperedenaceace metagenome-assembled genomes (MAGs), recovered from diverse environments, revealed novel respiratory strategies acquired through lateral gene transfer (LGT) events from diverse archaea and bacteria. Comprehensive phylogenetic analyses suggests that LGT has allowed members of the Methanoperedenaceae to acquire genes for the oxidation of hydrogen and formate, and the reduction of arsenate, selenate and elemental sulfur. Numerous membrane-bound multi-heme c type cytochrome complexes also appear to have been laterally acquired, which may be involved in the direct transfer of electrons to metal oxides, humics and syntrophic partners.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/100444, title ="Methanotrophic bacterial symbionts fuel dense populations of deep-sea feather duster worms (Sabellida, Annelida) and extend the spatial influence of methane seepage", author = "Goffredi, Shana K. and Tilic, Ekin", journal = "Science Advances", volume = "6", number = "14", pages = "Art. No. eaay8562", month = "April", year = "2020", doi = "10.1126/sciadv.aay8562", issn = "2375-2548", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200102-085056769", note = "© 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). \n\nReceived for publication July 24, 2019. Accepted for publication January 9, 2020. \n\nWe thank the captains and crew of the R/V Atlantis, HOV Alvin pilots and technicians, AUV Sentry team, as well as scientific participants of AT37-13 and AT42-03, especially O. Pereira, L. McCormick, C. Seid, and A. Durkin, for their assistance at sea. We also thank the captain and crew of the R/V Falkor, ROV SuBastian pilots and technicians, scientific participants of FK190106, as well as J. Gonzalez, A. Crémière, J. Magyar, and S. Connon for their assistance and contributions to the isotope, ion chromatography, nanoSIMS, and microbial community analysis, respectively. C. Roman (University of Rhode Island, as co–principal investigator) and J. Cortes Nunez (University of Costa Rica, international collaborator) played significant roles as co–principal investigators during both expeditions and contributed current meter data. In addition, Occidental College undergraduates M. Cazin, K. Ruis, and C. Brzechffa assisted with microbial community and microscopy analysis, sponsored by the Oxy Undergraduate Research Center. Funding: Support for S.K.G. was provided by a Faculty Enrichment Grant through Occidental College. Support for E.T. was provided, in part, by a postdoctoral fellowship from the German Research Foundation (DFG TI 973/1-1). Support for V.J.O. was provided, in part, by the Gordon and Betty Moore Foundation (grant no. 3780). The research was primarily supported by U.S. NSF grants OCE 1635219 (to E.E.C), OCE 1634172 (to L.A.L. and G.W.R.), and OCE 1634002 (to V.J.O.). Sample collection and export permits were acquired through the Costa Rican Ministry of the Environment and Energy (SINAC-CUSBSE-PI-R-032-2018 and Academic License SINAC-SE-064-2018). \n\nAuthor contributions: S.K.G. conducted DNA analysis, including 16S rRNA barcoding and fluorescent microscope analyses, analyzed experimental data, wrote the manuscript with input from coauthors, and participated in both expeditions. E.T. performed electron microscopy analyses and participated in AT42-03. A.K. performed Sentry data analysis and wrote the manuscript. K.S.D. designed and performed the incubation experiments, participated in both expeditions, and managed the SRA submission. S.W.M. designed and performed the incubation experiments and participated in both expeditions. F.W. performed fluorescent microscopy analyses. R.W.L. performed the isotope analyses. G.W.R. was a principal investigator on the NSF-funded project, fixed the specimens for study (including seamount worms), coordinated and interpreted the electron microscopy analyses, identified and is naming the worm species, and participated in all expeditions. L.A.L. was a principal investigator on the NSF-funded project, coordinated isotope analyses, wrote the manuscript, and participated in both expeditions. E.E.C. was a principal investigator on the NSF-funded project and chief scientist on both expeditions. V.J.O. was a principal investigator on the NSF-funded project, designed the incubation experiments, and participated in both expeditions. All authors contributed to data interpretation and editing of the paper. \n\nAll authors declare that they have no competing interests. \n\nData and materials availability: 16S rRNA sequences for bacterial isolates are available from GenBank under accession numbers MN416048 through MN416065. The raw Illumina 16S rRNA barcode sequences and metadata collected in this study are available from the Dryad Digital Repository (https://doi.org/10.5061/dryad.wdbrv15jq) and the NCBI Small Read Archive (BioProject no. PRJNA599018). Animal images and specimens were vouchered (Laminatubus catalog no. A9589 and Bispira catalog no.A9598) for long-term archiving into the Benthic Invertebrate Collection at Scripps Institution of Oceanography (https://sioapps.ucsd.edu/collections/bi/).", revision_no = "36", abstract = "Deep-sea cold seeps are dynamic sources of methane release and unique habitats supporting ocean biodiversity and productivity. Here, we describe newly discovered animal-bacterial symbioses fueled by methane, between two species of annelid (a serpulid Laminatubus and sabellid Bispira) and distinct aerobic methane-oxidizing bacteria belonging to the Methylococcales, localized to the host respiratory crown. Worm tissue δ¹³C of −44 to −58‰ are consistent with methane-fueled nutrition for both species, and shipboard stable isotope labeling experiments revealed active assimilation of ¹³C-labeled methane into animal biomass, which occurs via the engulfment of methanotrophic bacteria across the crown epidermal surface. These worms represent a new addition to the few animals known to intimately associate with methane-oxidizing bacteria and may further explain their enigmatic mass occurrence at 150–million year–old fossil seeps. High-resolution seafloor surveys document significant coverage by these symbioses, beyond typical obligate seep fauna. These findings uncover novel consumers of methane in the deep sea and, by expanding the known spatial extent of methane seeps, may have important implications for deep-sea conservation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101051, title ="Anaerobic methane oxidation coupled to manganese reduction by members of the Methanoperedenaceae", author = "Leu, Andy O. and Cai, Chen", journal = "ISME Journal", volume = "14", number = "4", pages = "1030-1041", month = "April", year = "2020", doi = "10.1038/s41396-020-0590-x", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200203-085914271", note = "© 2020 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nReceived 06 June 2019; Revised 10 December 2019; Accepted 16 January 2020; Published 27 January 2020. \n\nWe thank Serene Low, Isabelle Krippner, Brian Kemish, and Nicola Angel for library preparation, Illumina sequencing, and computing support and Aharon Oren for providing the species names and etymology. This work was supported by the U.S. Department of Energy’s Office of Biological Environmental Research (DE-SC0016469) and the Australian Research Council (ARC; Discovery project DP170104038). ZY is a recipient of an Australian Laureate Fellowship (FL170100086). AOL was supported by an Australian Postgraduate Award. CC was supported by The University of Queensland International Scholarship and a China Scholarship Council Scholarship. S.H. was supported by an Advanced Queensland Research Fellowship. GWT was supported by an Australian Research Council Future Fellowship (FT170100070). \n\nData availability: Sequencing data are deposited at the NCBI Sequence Read Archive under accession numbers SAMN10868419-SAMN10868422, SAMN10868423, and SAMN11109471-SAMN11109472. All draft genome nucleotide sequences have been deposited under the NCBI Biosample accession numbers SAMN10872749-SAMN10872769. \n\nAuthor Contributions: These authors contributed equally: Andy O. Leu, Chen Cai. \n\nThe authors declare that they have no conflict of interest.", revision_no = "20", abstract = "Anaerobic oxidation of methane (AOM) is a major biological process that reduces global methane emission to the atmosphere. Anaerobic methanotrophic archaea (ANME) mediate this process through the coupling of methane oxidation to different electron acceptors, or in concert with a syntrophic bacterial partner. Recently, ANME belonging to the archaeal family Methanoperedenaceae (formerly known as ANME-2d) were shown to be capable of AOM coupled to nitrate and iron reduction. Here, a freshwater sediment bioreactor fed with methane and Mn(IV) oxides (birnessite) resulted in a microbial community dominated by two novel members of the Methanoperedenaceae, with biochemical profiling of the system demonstrating Mn(IV)-dependent AOM. Genomic and transcriptomic analyses revealed the expression of key genes involved in methane oxidation and several shared multiheme c-type cytochromes (MHCs) that were differentially expressed, indicating the likely use of different extracellular electron transfer pathways. We propose the names “Candidatus Methanoperedens manganicus” and “Candidatus Methanoperedens manganireducens” for the two newly described Methanoperedenaceae species. This study demonstrates the ability of members of the Methanoperedenaceae to couple AOM to the reduction of Mn(IV) oxides, which suggests their potential role in linking methane and manganese cycling in the environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/97550, title ="Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane‐oxidizing archaea", author = "Bird, Laurence R. and Dawson, Katherine S.", journal = "Geobiology", volume = "17", number = "6", pages = "611-627", month = "November", year = "2019", doi = "10.1111/gbi.12354", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190731-105709287", note = "© 2019 John Wiley & Sons Ltd. \n\nIssue Online: 18 October 2019; Version of Record online: 31 July 2019; Manuscript accepted: 23 June 2019; Manuscript revised: 17 June 2019; Manuscript received: 20 August 2018. \n\nWe thank Denny Walizer and Clayton Magill for help in the Penn State Organic Geochemistry Lab and Christopher Junium and Pratigya Polissar for help with the nano‐EA‐IRMS system. Anne Dekas, Stephanie Connon, and Jennifer Glass are thanked for sample collection. We also thank Sara Lincoln for constructive comments. Fenfang Wu in the Caltech Stable Isotope facility and Nathan Daleska in the Caltech Environmental Analysis Center are thanked for providing technical assistance. Funding support for this study came from Royal Dutch Shell Geosciences Energy Research Facilitation Awards, ConocoPhillips graduate student fellowship, the Penn State Astrobiology Research Center, and Pennsylvania Space Grant, the American Chemical Society petroleum research fund (54478‐ND2). Research conducted at Caltech was funded by the Gordon and Betty Moore Foundation (Grant GBMF 3306).", revision_no = "22", abstract = "Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy‐archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH_3‐tetrahydromethanopterin (H_4MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in ^(13)C (mean δ^(13)C = −27‰ for Hydrate Ridge and −80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ^(13)C = −97‰ for Hydrate Ridge and −122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl‐CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of − 73‰ to − 77‰ for F430 and − 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ^(13)C value for Hydrate Ridge was 13C‐enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/99050, title ="Peptidoglycan Production by an Insect-Bacterial Mosaic", author = "Bublitz, DeAnna C. and Chadwick, Grayson L.", journal = "Cell", volume = "179", number = "3", pages = "703-712", month = "October", year = "2019", doi = "10.1016/j.cell.2019.08.054", issn = "0092-8674", url = "https://resolver.caltech.edu/CaltechAUTHORS:20191003-113812895", note = "© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). \n\nReceived 18 June 2019, Revised 6 August 2019, Accepted 28 August 2019, Available online 3 October 2019. \n\nData and Code Availability: 16S rRNA amplicon sequences have been deposited in NCBI under BioProject ID: PRJNA546070. All membrane measurements presented in Figure 4 are included in this manuscript as Table S3. \n\nWe thank Bil Clemons for helpful discussions about PG; Denghui David Xing of the University of Montana Genomics Core for sequencing expertise; Carol Garland, Matthew Hunt, and the Caltech Kavli Nanoscience Institute for aid in maintaining the TF-30 electron microscope; and the Gordon and Betty Moore and Beckman Foundations for gifts to Caltech to support electron microscopy. PG Mass spectrometry analyses were performed by the biOMICS Facility of the Faculty of Science Mass Spectrometry Centre at the University of Sheffield. We thank Adelina E. Acosta-Martin and Ankur Patel for their help with peptidoglycan analyses. This work was supported by the Gordon and Betty Moore Foundation (GBMF5602), the National Aeronautics and Space Administration Astrobiology Institute (NNA15BB04A), the National Science Foundation (IOS-1553529), and the Biotechnology and Biological Sciences Research Council (BB/N000951/1 and 2058718). \n\nAuthor Contributions: D.C.B.: conceptualization, investigation, analysis, methodology, validation, visualization, and writing; G.L.C. and J.S.M.: investigation, methodology, analysis, validation, visualization, and writing; K.M.S.: conceptualization, analysis, methodology, investigation, resources, and writing; S.M.: analysis, methodology, resources, software, and writing; D.M.B.: investigation, methodology, and visualization; M.S.L.: investigation, methodology, resources, validation, and visualization; A.I.G.: data curation, analysis, investigation, software, and visualization; P.J.B.: methodology, resources, and administration; V.J.O.: conceptualization, funding acquisition, resources, and administration; J.P.M.: conceptualization, funding acquisition, administration, resources, visualization, and writing. \n\nThe authors declare no competing interests.", revision_no = "37", abstract = "Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/98815, title ="NanoSIMS imaging reveals metabolic stratification within current-producing biofilms", author = "Chadwick, Grayson L. and Jiménez-Otero, Fernanda", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "116", number = "41", pages = "20716-20724", month = "October", year = "2019", doi = "10.1073/pnas.1912498116", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190923-160449911", note = "© 2019 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). \n\nEdited by Susan L. Brantley, Pennsylvania State University, University Park, PA, and approved August 29, 2019 (received for review July 24, 2019). \n\nThis publication was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0016469) and the NASA Astrobiology Institute, Award NNA13AA92A (to V.J.O.) and the Simons Foundation (Program: Life Sciences-Simons Collaboration on Principles of Microbial Ecosystems; Award 542393). D.R.B. and J.A.G. were supported by National Science Foundation Dimensions of Biodiversity program DEB 1542513. G.L.C. was supported by NIH/NRSA Training Grant T32 GM007616. F.J.O. was supported by the Mexican National Council for Science and Technology and the Office of Naval Research Award N000141612194. We thank Dr. Gail Celio for training and loan of the UMN Imaging facilities, Dr. Ryan Hunter for assistance developing a biofilm staining and embedding protocol, and Dr. Yunbin Guan for assistance with the nanoSIMS analysis. \n\nAuthor contributions: G.L.C., F.J.O., J.A.G., D.R.B., and V.J.O. designed research; G.L.C. and F.J.O. performed research; G.L.C. and F.J.O. analyzed data; and G.L.C., F.J.O., J.A.G., D.R.B., and V.J.O. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1912498116/-/DCSupplemental.", revision_no = "24", abstract = "Metal-reducing bacteria direct electrons to their outer surfaces, where insoluble metal oxides or electrodes act as terminal electron acceptors, generating electrical current from anaerobic respiration. Geobacter sulfurreducens is a commonly enriched electricity-producing organism, forming thick conductive biofilms that magnify total activity by supporting respiration of cells not in direct contact with electrodes. Hypotheses explaining why these biofilms fail to produce higher current densities suggest inhibition by formation of pH, nutrient, or redox potential gradients; but these explanations are often contradictory, and a lack of direct measurements of cellular growth within biofilms prevents discrimination between these models. To address this fundamental question, we measured the anabolic activity of G. sulfurreducens biofilms using stable isotope probing coupled to nanoscale secondary ion mass spectrometry (nanoSIMS). Our results demonstrate that the most active cells are at the anode surface, and that this activity decreases with distance, reaching a minimum 10 µm from the electrode. Cells nearest the electrode continue to grow at their maximum rate in weeks-old biofilms 80-µm-thick, indicating nutrient or buffer diffusion into the biofilm is not rate-limiting. This pattern, where highest activity occurs at the electrode and declines with each cell layer, is present in thin biofilms (<5 µm) and fully grown biofilms (>20 µm), and at different anode redox potentials. These results suggest a growth penalty is associated with respiring insoluble electron acceptors at micron distances, which has important implications for improving microbial electrochemical devices as well as our understanding of syntrophic associations harnessing the phenomenon of microbial conductivity.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/99083, title ="ASM-Clust: classifying functionally diverse protein families using alignment score matrices", author = "Speth, Daan R. and Orphan, Victoria J.", month = "October", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20191004-102107177", note = "The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. \n\nbioRxiv preprint first posted online Oct. 3, 2019. \n\nThis work was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under award number DE-SC0016469 to Victoria J. Orphan. Daan R. Speth was supported by the Netherlands Organisation for Scientific Research, Rubicon award 019.153LW.039.", revision_no = "10", abstract = "Rapid advances in sequencing technology have resulted in the availability of genomes from organisms across the tree of life. Accurately interpreting the function of proteins in these genomes is a major challenge, as annotation transfer based on homology frequently results in misannotation and error propagation. This challenge is especially pressing for organisms whose genomes are directly obtained from environmental samples, as interpretation of their physiology and ecology is often based solely on the genome sequence. For complex protein (super)families containing a large number of sequences, classification can be used to determine whether annotation transfer is appropriate, or whether experimental evidence for function is lacking. Here we present a novel computational approach for de novo classification of large protein (super)families, based on clustering an alignment score matrix obtained by aligning all sequences in the family to a small subset of the data. We evaluate our approach on the enolase family in the Structure Function Linkage Database.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/96656, title ="Scientists’ warning to humanity: microorganisms and climate change", author = "Cavicchioli, Ricardo and Orphan, Victoria J.", journal = "Nature Reviews Microbiology", volume = "17", number = "9", pages = "569-586", month = "September", year = "2019", doi = "10.1038/s41579-019-0222-5", issn = "1740-1526", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190624-093344374", note = "© 2019 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nAccepted 22 May 2019; Published 18 June 2019. \n\nR.C. is indebted to T. Kolesnikow, K. Cavicchioli and X. Kolesnikow for assistance with figures and insightful comments on manuscript drafts. R.C.’s contribution was supported by the Australian Research Council. M.J.B.’s contribution was supported by the NASA North Atlantic Aerosol and Marine Ecosystem Study. Research by J.K.J was supported by the US Department of Energy, Office of Biological and Environmental Research, Soil Microbiome Scientific Focus Area ‘Phenotypic Response of the Soil Microbiome to Environmental Perturbations’ at the Pacific Northwest National Laboratory, under contract DE-AC05-76RLO 1830. M.B.S.'s contribution was supported by funds from the Gordon and Betty Moore Foundation (#3790) and National Science Foundation (OCE#1829831). V.I.R.'s contribution was supported by funds from the Department of Energy Genomic Sciences Program (#DE-SC0016440) and the National Aeronautics and Space Administration’s Interdisciplinary Research in Earth Science programme (#NNX17AK10G). \n\nAuthor Contributions: R.C., W.J.R. and K.N.T. conceived the article, R.C. wrote the article and all authors contributed to discussion of the content and reviewed or edited the manuscript before submission. \n\nThe authors declare no competing interests.", revision_no = "19", abstract = "In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial ‘unseen majority’. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/96205, title ="Age Mosaicism across Multiple Scales in Adult Tissues", author = "Arrojo e Drigo, Rafael and Lev-Ram, Varda", journal = "Cell Metabolism", volume = "30", number = "2", pages = "343-351", month = "August", year = "2019", doi = "10.1016/j.cmet.2019.05.010", issn = "1550-4131", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190607-131617878", note = "© 2019 Elsevier Inc. \n\nReceived 14 June 2018, Revised 18 October 2018, Accepted 11 May 2019, Available online 6 June 2019. \n\nThis work was supported by grants to M.W.H. from the NIH Transformative Research Award (R01 NS096786), the Keck Foundation, and the NOMIS Foundation. It was also supported by grants to M.H.E. and V.L.-R. from the NIH NINDS (NIH RO1 NS027177-30), which supported the creation of the ^(15)N/^(14)N mice; and the NIH NIGMS (5P41 GM103412-29), which supports the advanced technologies and instrument development activities of the NCMIR, most heavily used, and whose new methods developments were partly driven by this work. V.O. and M.H.E. are also supported to develop and carry out correlated light and electron microscopy with MIMS under an award from the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0016469). R.A.eD. is supported by an American Diabetes Association postdoctoral fellowship (1-18-PMF-007) and benefitted from assistance from Daniela Rhodes and the Nanyang Institute of Structural Biology (NISB) (at the Nanyang Technological University, Singapore) from a career development stipend. The authors are also thankful to Yunbin Guan, Ph.D., from the Caltech Microanalysis Center in the Division of Geological and Planetary Sciences, California Institute of Technology for technical support and assistance with MIMS image acquisition; to Dr. Ting-Di Wu and Dr. Jean-Luc Guerquin-Kern from the INSERM, Université Paris-Sud, Paris; and the Cell and Tissue Imaging Facility of the Institut Curie (PICT), a member of the France BioImaging National Infrastructure (ANR-10-INBS-04), for the use of Curie NanoSIMS instrument; Greg McMahon from the National Center of Excellence in Mass Spectrometry Imaging, National Physical Laboratory (NPL), UK; and to David O'Keefe (Salk Institute) for critical inputs on manuscript editing and revision. \n\nAuthor Contributions: R.A.eD. collected and analyzed the data and wrote the article. V.L.-E., S.T., R.R., T.D., E.B., S.P., and C.L. collected the data. V.O., M.H.E., and M.W.H. designed the study and wrote the article. \n\nThe authors declare no competing interests.", revision_no = "24", abstract = "Most neurons are not replaced during an animal’s lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using ^(15)N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/95945, title ="The Cell and the Sum of Its Parts: Patterns of Complexity in Biosignatures as Revealed by Deep UV Raman Spectroscopy", author = "Sapers, Haley M. and Razzell Hollis, Joseph", journal = "Frontiers in Microbiology", volume = "10", pages = "Art. No. 679", month = "May", year = "2019", doi = "10.3389/fmicb.2019.00679", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190530-074324675", note = "© 2019 Sapers, Razzell Hollis, Bhartia, Beegle, Orphan and Amend. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The U.S. Government retains a nonexclusive license to this work for non-commercial purposes. \n\nReceived: 18 September 2018; Accepted: 18 March 2019; Published: 14 May 2019. \n\nAuthor Contributions: HS designed the study with significant input from JH, LB, and RB. HS cultured the cells, prepared the cell samples, and collected the cellular Raman spectra. HS and JH designed the analytical pipeline and wrote the processing code. JH made the standard solutions, collected the standard Raman spectra, and finalized the Raman analyses. HS and JH wrote the manuscript with significant input from RB and LB. JA and VO contributed to discussion, feasibility, and text. \n\nThe work described in this paper was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. This work was funded by a NASA Astrobiology Institute–Life Underground (NAI-LU, NNA13AA92A) grant to JA, VO, and RB. Further support was provided by a Human Frontier Science Program postdoctoral fellowship to HS and a NASA Postdoctoral Program fellowship to JH. \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nWe thank Nicholas Laughton and Kyle Uckert for valuable assistance with Python programming and William Abbey, Evan Eshelman, Greg Wanger, Michael Malaska, Ranjani Murali, Daan Speth, and Sean Mullin for insightful conversations and discussions regarding data analysis and data visualization.", revision_no = "12", abstract = "The next NASA-led Mars mission (Mars 2020) will carry a suite of instrumentation dedicated to investigating Martian history and the in situ detection of potential biosignatures. SHERLOC, a deep UV Raman/Fluorescence spectrometer has the ability to detect and map the distribution of many organic compounds, including the aromatic molecules that are fundamental building blocks of life on Earth, at concentrations down to 1 ppm. The mere presence of organic compounds is not a biosignature: there is widespread distribution of reduced organic molecules in the Solar System. Life utilizes a select few of these molecules creating conspicuous enrichments of specific molecules that deviate from the distribution expected from purely abiotic processes. The detection of far from equilibrium concentrations of a specific subset of organic molecules, such as those uniquely enriched by biological processes, would comprise a universal biosignature independent of specific terrestrial biochemistry. The detectability and suitability of a small subset of organic molecules to adequately describe a living system is explored using the bacterium Escherichia coli as a model organism. The DUV Raman spectra of E. coli cells are dominated by the vibrational modes of the nucleobases adenine, guanine, cytosine, and thymine, and the aromatic amino acids tyrosine, tryptophan, and phenylalanine. We demonstrate that not only does the deep ultraviolet (DUV) Raman spectrum of E. coli reflect a distinct concentration of specific organic molecules, but that a sufficient molecular complexity is required to deconvolute the cellular spectrum. Furthermore, a linear combination of the DUV resonant compounds is insufficient to fully describe the cellular spectrum. The residual in the cellular spectrum indicates that DUV Raman spectroscopy enables differentiating between the presence of biomolecules and the complex uniquely biological organization and arrangements of these molecules in living systems. This study demonstrates the ability of DUV Raman spectroscopy to interrogate a complex biological system represented in a living cell, and differentiate between organic detection and a series of Raman features that derive from the molecular complexity inherent to life constituting a biosignature.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/89996, title ="Divergent methyl-coenzyme M reductase genes in a deep-subseafloor Archaeoglobi", author = "Boyd, Joel A. and Jungbluth, Sean P.", journal = "ISME Journal", volume = "13", number = "5", pages = "1269-1279", month = "May", year = "2019", doi = "10.1038/s41396-018-0343-2", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180927-114223337", note = "© 2019 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nReceived 07 October 2018; Revised 29 November 2018; Accepted\n11 December 2018; Published 16 January 2019. \n\nCode availability: All unpublished software used in this publication are available on github; sequence_windower (https://github.com/geronimp/window_sequence); EnrichM (github.com/geronimp/enrichM); GenomeTreeTK (github.com/dparks1134/GenomeTreeTk); CompareM v0.0.22 (https://github.com/dparks1134/CompareM); fastacxxch.count.py, (github.com/geronimp/HandyScripts/blob/master/99_random/fastacxxch.count.py); FinishM (v0.0.7 github.com/wwood/finishm). GTDB-Tk (github.com/Ecogenomics/GTDBTk). \n\nData availability: The datasets analysed during the current study are available in the NCBI SRA, accession number SRR3723048. The Candidatus “Polytropus marinifundus” genome is available under the NCBI bioproject SAMN10474933. All alignments and phylogenetic trees are available in the Supplementary Files 1–20. \n\nWe thank the captain and crew, A. Fisher, K. Becker, C. G. Wheat, and other members of the science teams on board R/V Atlantis cruise AT18-07. We also thank the pilots and crew of remote-operated vehicle Jason II. This research was supported by two grants from the National Science Foundation: Microbial Observatories (MCB06-04014 to MSR), and the Science and Technology Center for Dark Energy Biosphere Investigations (C-DEBI; OCE-0939564 to JPA). This study used samples and data provided by the Integrated Ocean Drilling Program. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research program under Award Number. Genomic Science Program of the United States Department of Energy Office of Biological and Environmental Research (DE-SC0016469, DE-SC0010580, DE-SC0016440); Australian Research Council (ARC) Future Fellowship (FT170100070 to G.W.T.); ARC Postgraduate Award (to JAB); ARC Discovery Early Career Researcher Award (DECRA; DE-160100248 to BJW); ARC DECRA (DE-170100428 to PNE). \n\nAuthor contributions: JAB and SPJ contributed equally to this work. MSR, JPA, and GWT designed the overall study and procured funding. JAB, SPJ, MSR, and GWT designed and carried out experiments and analyses around specific microbial hypotheses. JAB, SPJ, and GWT wrote the manuscript. All authors edited, reviewed and approved the final manuscript. \n\nThe authors declare that they have no conflict of interest.", revision_no = "43", abstract = "The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for β-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/98365, title ="Calorimetric determination of microbial activity in low-\u200benergy environments", author = "Amend, Jan and Feyhl-Buska, Jayme", pages = "GEOC-0044", month = "April", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190830-092706647", note = "© 2019 American Chemical Society.", revision_no = "9", abstract = "Calorimetric measurements of heat flow and total heat prodn. can inform on the energetics of microbial activity. Nanocalorimetry permits such investigations in environments (natural or lab.) where energy supplies are extremely limited and/or very few cells are metabolically active. We will present nanocalorimetry data obtained during incubations of unamended formation fluids from the oceanic crust (∼300 m deep at the Juan de Fuca Ridge flank) and the continental subsurface (∼1500 m deep at the Sanford Underground Research Facility in South Dakota). Enthalpic responses by the resident microbial communities to addn. of org. carbon, inorg. electron donors, and nutrients will also be discussed. We further used nanocalorimetry on lab. expts. with model organisms to det. the energetics assocd. with carbon, electron donor, and nutrient limitations. For example, with a sulfate reducing bacterium, we quantified the significant cost during the metabolic transition from ammonium assimilation to N_2 fixation. In another set of expts., with an evolving population of E. coli mutants that express the GASP (growth advantage in stationary phase) phenotype, we recorded distinct heat events assocd. with the emergence of nascent mutant populations.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/94306, title ="FIND: Identifying Functionally and Structurally Important Features in Protein Sequences with Deep Neural Networks", author = "Murali, Ranjani and Hemp, James", month = "March", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190401-084306692", note = "The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. \n\nThis work was funding by DARPA’s CwC program through ARO (W911NF-15-1-0543), the Moore Foundation(Award to VJO: GBMF3780), the Department of Energy (Award to VJO: DE-SC0016469) and the Center for Dark Biosphere Investigations (NSF Award to VJO: OCE-0939564). We are grateful to Daan Speth for reading the manuscript at multiple stages of completion and for his useful comments throughout the process. We would also like to thank Dr. Robert Gennis for reading the manuscript and offering helpful comments, Grayson Chadwick for useful discussion on bioinformatics strategies and Haley Sapers for her instructive critique of the figures. ", revision_no = "11", abstract = "The ability to correctly predict the functional role of proteins from their amino acid sequences would significantly advance biological studies at the molecular level by improving our ability to understand the biochemical capability of biological organisms from their genomic sequence. Existing methods that are geared towards protein function prediction or annotation mostly use alignment-based approaches and probabilistic models such as Hidden-Markov Models. In this work we introduce a deep learning architecture (Function Identification with Neural Descriptions or FIND) which performs protein annotation from primary sequence. The accuracy of our methods matches state of the art techniques, such as protein classifiers based on Hidden Markov Models. Further, our approach allows for model introspection via a neural attention mechanism, which weights parts of the amino acid sequence proportionally to their relevance for functional assignment. In this way, the attention weights automatically uncover structurally and functionally relevant features of the classified protein and find novel functional motifs in previously uncharacterized proteins. While this model is applicable to any database of proteins, we chose to apply this model to superfamilies of homologous proteins, with the aim of extracting features inherent to divergent protein families within a larger superfamily. This provided insight into the functional diversification of an enzyme superfamily and its adaptation to different physiological contexts. We tested our approach on three families (nitrogenases, cytochrome bd-type oxygen reductases and heme-copper oxygen reductases) and present a detailed analysis of the sequence characteristics identified in previously characterized proteins in the heme-copper oxygen reductase (HCO) superfamily. These are correlated with their catalytic relevance and evolutionary history. FIND was then applied to discover features in previously uncharacterized members of the HCO superfamily, providing insight into their unique sequence features. This modeling approach demonstrates the power of neural networks to recognize patterns in large datasets and can be utilized to discover biochemically and structurally important features in proteins from their amino acid sequences.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/92252, title ="Precise determination of equilibrium sulfur isotope effects during volatilization and deprotonation of dissolved H_2S", author = "Sim, Min Sub and Sessions, Alex L.", journal = "Geochimica et Cosmochimica Acta", volume = "248", pages = "242-251", month = "March", year = "2019", doi = "10.1016/j.gca.2019.01.016", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190114-132114245", note = "© 2019 Published by Elsevier Ltd. \n\nReceived 16 June 2018, Revised 29 December 2018, Accepted 9 January 2019, Available online 14 January 2019. \n\nThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No.2018R1D1A1B07050970) and an Agouron Geobiology Fellowship to MSS, Gordon and Betty Moore Foundation Grant GBMF 3306 to VJO and ALS, NSF award OCE-1436566 to ALS and NSF award OCE-1340174 to JFA. The authors are grateful to Guillaume Paris for assistance for isotope analysis. We also thank Daniel Eldridge, Boswell Wing, and an anonymous reviewer for constructive comments on an earlier version of this manuscript.", revision_no = "19", abstract = "Sulfide (H_2S, HS^−, and S^(2−)) is ubiquitous in marine porewaters as a result of microbial sulfate reduction, constituting the reductive end of the biogeochemical sulfur cycle. Stable isotopes have been widely used to constrain the sulfur cycle, because the redox transformations of sulfur compounds, such as microbial sulfate reduction, often exhibit sizable kinetic isotope effects. In contrast to sulfate ion (SO_4^(2−)), the most abundant form of dissolved sulfur in seawater, H2S is volatile and also deprotonated at near neutral pH. Equilibrium isotope partitioning between sulfide species can therefore overlap with kinetic isotope effects during reactions involving sulfide as either reactant or intermediate. Previous experimental attempts to measure equilibrium fractionation between H_2S and HS− have reached differing results, likely due to solutions of widely varying ionic strength. In this study, we measured the sulfur isotope fractionation between total dissolved sulfide and gaseous H2S at 20.6\u202f±\u202f0.5\u202f°C over the pH range from 2 to 8, and calculated the equilibrium isotope effects associated with deprotonation of dissolved H_2S. By using dilute solutions of Na2S, made possible by the improved sensitivity of mass spectrometric techniques, uncertainty in the first dissociation constant of H2S due to ionic strength could be better controlled. This in turn allowed us to close sulfur isotope mass balance for our experiments and increase the accuracy of the estimated fractionation factor. At equilibrium, aqueous H2S was enriched in ^(34)S by 0.7‰ and 3.1‰ relative to gaseous H_2S and aqueous HS−, respectively. The estimated fractionation between aqueous H_2S and HS^− lies between two earlier experimental reports, but agrees within the uncertainty of the measurements with a recent theoretical calculation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91996, title ="Microbial interactions in the anaerobic oxidation of methane: Model simulations constrained by process rates and activity patterns", author = "He, Xiaojia and Chadwick, Grayson", journal = "Environmental Microbiology", volume = "21", number = "2", pages = "631-647", month = "February", year = "2019", doi = "10.1111/1462-2920.14507", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190102-135257356", note = "© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd. \n\nReceived 2 July, 2018; revised 4 December, 2018; accepted 4 December, 2018. \n\nThis work was supported by the Genomic Sciences Program in the DOE Office of Science, Biological and Environmental Research DE‐SC0016469. The authors declare no conflict of interest.", revision_no = "22", abstract = "Proposed syntrophic interactions between the archaeal and bacterial cells mediating anaerobic oxidation of methane coupled with sulfate reduction include electron transfer through (1) the exchange of H2 or small organic molecules between methane‐oxidizing archaea and sulfate‐reducing bacteria, (2) the delivery of disulfide from methane‐oxidizing archaea to bacteria for disproportionation and (3) direct interspecies electron transfer. Each of these mechanisms was implemented in a reactive transport model. The simulated activities across different arrangements of archaeal and bacterial cells and aggregate sizes were compared to empirical data for AOM rates and intra‐aggregate spatial patterns of cell‐specific anabolic activity determined by FISH‐nanoSIMS. Simulation results showed that rates for chemical diffusion by mechanism (1) were limited by the build‐up of metabolites, while mechanisms (2) and (3) yielded cell specific rates and archaeal activity distributions that were consistent with observations from single cell resolved FISH‐nanoSIMS analyses. The novel integration of both intra‐aggregate and environmental data provided powerful constraints on the model results, but the similarities in model outcomes for mechanisms (2) and (3) highlight the need for additional observational data (e.g. genomic or physiological) on electron transfer and metabolic functioning of these globally important methanotrophic consortia.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/92254, title ="The next frontier for planetary and human exploration", author = "Stamenković, V. and Fischer, W. W.", journal = "Nature Astronomy", volume = "3", number = "2", pages = "116-120", month = "February", year = "2019", doi = "10.1038/s41550-018-0676-9", issn = "2397-3366", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190114-133208101", note = "© 2019 Springer Nature Publishing AG. \n\nPublished 14 January 2019. \n\nWe thank the Keck Institute for Space Studies (KISS) for kick-starting this work through a KISS Workshop held 12–16 February 2018 at the California Institute of Technology, Pasadena, CA, and the Canadian Institute for Advanced Studies (CIFAR) for allowing this discussion to expand with the Earth 4D workshop. Part of this work was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration.", revision_no = "18", abstract = "The surface of Mars has been well mapped and characterized, yet the subsurface — the most likely place to find signs of extant or extinct life and a repository of useful resources for human exploration — remains unexplored. In the near future this is set to change.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/92242, title ="Role of APS reductase in biogeochemical sulfur isotope fractionation", author = "Sim, Min Sub and Ogata, Hideaki", journal = "Nature Communications", volume = "10", pages = "Art. No. 44", month = "January", year = "2019", doi = "10.1038/s41467-018-07878-4", issn = "2041-1723", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190114-083925177", note = "© 2018 The Author(s).\nThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\n\nReceived 29 June 2018; Accepted 29 November 2018; Published\n09 January 2019.\n\nData availability:\nData supporting the findings of this study are available within the paper and in the supplementary information file or are available from the corresponding author upon reasonable request.\n\n\nThis work was supported by the Research Resettlement Fund for the new faculty of Seoul National University to M.S.S., the NASA Research Opportunities in Space and Earth Sciences grant award number NNX14AO48G to S.E.M. and V.J.O., JSPS KAKENHI Grant Number 10751084 to S.E.M., and the Gordon and Betty Moore Foundation Grant GBMF 3306 to V.J.O. and A.L.S. This research was a part of the project titled ‘Understanding the deepsea biosphere on seafloor hydrothermal vents in the Indian Ridge (20170411)’, funded by the Ministry of Oceans and Fisheries, Korea. We are grateful for insightful and helpful conversations with Boswell A. Wing, David T. Johnston, David A. Fike, and Itay Halevy. We are especially grateful to Tatsuhiko Yagi and Yoshiki Higuchi for helping with initiating collaboration.\n\nContributions:\nM.S.S. and S.E.M. devised the study. H.O. and W.L. purified APS reductase, and M.S.S. executed enzymatic assay and sulfur isotope measurements. M.S.S. and S.E.M. wrote the first draft of the manuscript, and J.F.A., A.L.S., and V.J.O. contributed to interpretation and writing.\n\nCompeting interests:\nThe authors declare no competing interests.\n", revision_no = "20", abstract = "Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91741, title ="Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea", author = "Yu, Hang and Susanti, Dwi", journal = "Frontiers in Microbiology", volume = "9", pages = "Art. No. 2917", month = "December", year = "2018", doi = "10.3389/fmicb.2018.02917", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181212-151637497", note = "© 2018 Yu, Susanti, McGlynn, Skennerton, Chourey, Iyer, Scheller, Tavormina, Hettich, Mukhopadhyay and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 01 September 2018; Accepted: 13 November 2018; Published: 03 December 2018. \n\nWe thank the Environmental Analysis Center at Caltech and its director Nathan Delaskas for the help and support with chemical sample analysis. We thank Roland Hatzenpichler, Danielle Goudeau, Rex R. Malmstrom, Tanja Woyke for their help with activity-based cell sorting and genome sequencing. Special thanks to Katherine Dawson, Grayson Chadwick and two reviewers for constructive comments on the manuscript. \n\nData Availability: The ANME genomes generated for this study have been deposited at NCBI GenBank database under the Whole Genome Shotgun project accession numbers QENH00000000, MZXQ00000000, and PYCL00000000 for ANME-1b (ANME sp. CONS3730B06UFb1), ANME-2b (ANME sp. HR1), and ANME-2c (ANME sp. S7142MS2) respectively. Protein sequences and alignments analyzed for this study can be found on FigShare: 10.6084/m9.figshare.7035917, 10.6084/m9.figshare.7036289, and 10.6084/m9.figshare.7037228. \n\nAuthor Contributions: HY, RH, BM, and VO designed research. HY, DS, SM, CS, KC, RI, SS, and PT performed research and data analysis. HY and VO wrote the paper with contribution from all authors. \n\nThis research and HY, SM, CS, SS, KC, RI, RH, and VO were supported by funding from the United States Department of Energy, Office of Science, Biological and Environmental Research Program under award number DE-SC0016469 and by a DOE Office of Science User Facility grant through the Joint Genome Institute and Environmental Molecular Science Laboratory (FICUS Grant 49001). HY, PT, and VO were additionally supported by the Gordon and Betty Moore Foundation through grant GBMF3780. This is Center for Dark Energy and Biosphere Investigations (C-DEBI) Contribution 449. SEM and VO was additionally supported by funding from the National Aeronautics and Space Administration Exobiology Grant NNX14AO48G. DS and BM were supported by the National Aeronautics and Space Administration Exobiology and Evolutionary Biology Grant NNX13AI05G. BM was also supported by Virginia Tech Agricultural Experiment Station Hatch Program (CRIS project VA-160021). \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.", revision_no = "14", abstract = "Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F_(420)-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/87541, title ="Widespread nitrogen fixation in sediments from diverse deep-sea sites of elevated carbon loading", author = "Dekas, Anne E. and Fike, David A.", journal = "Environmental Microbiology", volume = "20", number = "12", pages = "4281-4296", month = "December", year = "2018", doi = "10.1111/1462-2920.14342", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180703-145300132", note = "© 2018 John Wiley & Sons. \n\nManuscript received: 05 March 2018. Manuscript revised: 25 June 2018. Manuscript accepted: 25 June 2018. Accepted manuscript online: 02 July 2018. \n\nWe thank the crew and science parties of R/V Atlantis 15‐68, including Lisa Levin, Alexis Pasulka, Josh Steele, Jeffrey Marlow, Jennifer Glass and Elizabeth Trembath‐Reichert, and of R/V Western Flyer DR204‐208, including Shana Goffredi, Bob Vrijenhoek, Julio Harvey and Lonny Lundsten. We thank members of the Fike Laboratory (Washington University), including Dwight McKay, Stephanie Moore and Jennifer Houghton, as well as Fenfang Wu (California Institute of Technology) for assistance with the IRMS measurements. We thank Nathan Dalleska for assistance with IC measurements in the Caltech Environmental Analysis Centre. We thank Matthew Forbes and Karen Casciotti (Stanford University) for assistance with 15N2 purity measurements. We are grateful to MBARI for logistical support and ship time. Funding was provided by the Gordon and Betty Moore Foundation (GBMF no. 3780 to V. J. O.), the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE‐SC0003940 and DE‐SC0004949 to V. J. O.), and the National Science Foundation (MCB‐0348492 to V. J. O. and OCE‐1634297 to A. E. D.).", revision_no = "64", abstract = "Nitrogen fixation, the biological conversion of N_2 to NH_3, is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N_2 fixation in deep‐sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N_2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N_2 fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N_2 fixation was dependent on CH_4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate‐coupled methane oxidation. However, the pattern of diazotrophy was different in whale‐fall and associated reference sediments, where it was largely unaffected by CH_4, suggesting catabolically different diazotrophs at these sites.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/87789, title ="Convergent evolution of unusual complex I homologs with increased proton pumping capacity: energetic and ecological implications", author = "Chadwick, Grayson L. and Hemp, James", journal = "ISME Journal", volume = "12", number = "11", pages = "2668-2680", month = "November", year = "2018", doi = "10.1038/s41396-018-0210-1", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180711-165717508", note = "© International Society for Microbial Ecology 2018. \n\nReceived: 2 November 2017 / Revised: 17 January 2018 / Accepted: 20 March 2018. \n\nThis publication was funded by the Gordon and Betty Moore Foundation through Grant #GBMF3780 and through work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research [DE-SC0016469] and the NASA Astrobiology Institute, award # NNA13AA92A (to V.J.O.). G.L.C. was supported by the NIH/NRSA training grant, T32 GM007616. We would like to thank Michael Wagner and Holger Daims for their thoughtful reading and comments on the manuscript, as well as the careful attention of an anonymous reviewer. \n\nThe authors declare that they have no conflict of interest.", revision_no = "85", abstract = "Respiratory complex I is part of a large family of homologous enzymes that carry out the transfer of electrons between soluble cytoplasmic electron carriers and membrane-bound electron carriers. These complexes are vital bioenergetic enzymes that serve as the entry points into electron transport chains for a wide variety of microbial metabolisms, and electron transfer is coupled to proton translocation. The core complex of this enzyme is made up of 11 protein subunits, with three major proton pumping subunits. Here, we document a large number of modified complex I gene cassettes found in genome sequences from diverse cultured bacteria, shotgun metagenomics, and environmentally derived archaeal fosmids all of which encode a fourth proton pumping subunit. The incorporation of this extra subunit into a functional protein complex is supported by large amino acid insertions in the amphipathic helix that runs the length of the protein complex. Phylogenetic analyses reveal that these modified complexes appear to have arisen independently multiple times in a remarkable case of convergent molecular evolution. From an energetic perspective, we hypothesize that this modification on the canonical complex I architecture allows for the translocation of a fifth proton per reaction cycle—the physiological utility of this modified complex is discussed.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/89898, title ="Methane on Mars and Habitability: Challenges and Responses", author = "Yung, Yuk L. and Chen, Pin", journal = "Astrobiology", volume = "18", number = "10", pages = "1221-1242", month = "October", year = "2018", doi = "10.1089/ast.2018.1917", issn = "1557-8070", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180925-073151228", note = "© 2018 Yuk L. Yung et al., Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. \n\nPublished Online: 19 Sep 2018. \n\nWe dedicate this article to JPL scientist Mark Allen, whose spirit of generosity inspired many of the conversations and much of the work that brought us together. This work was initiated and supported by the W.M. Keck Institute for Space Studies. We thank the Director of the Keck Institute for Space Studies, Tom Prince, the Executive Director, Michele Judd, and the capable and dedicated KISS staff for hosting and supporting the workshops that led to this article. We thank Charles Carter for the cover illustration and Meg Rosenberg for her work on editing and formatting. We thank Danica Adams, Siteng Fan, Amanda Gao, Mimi Gerstell, Yancheng Luo, Aimee Oz, Andrew Sappey, Sindhoora Tallapragada, and Kyle Weng for their efforts in editing the article. We thank Daniel Stolper for his participation and input in the workshop. The research was partly carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. \n\nNo competing financial interests exist.", revision_no = "220", abstract = "Recent measurements of methane (CH_4) by the Mars Science Laboratory (MSL) now confront us with robust data that demand interpretation. Thus far, the MSL data have revealed a baseline level of CH_4 (∼0.4 parts per billion by volume [ppbv]), with seasonal variations, as well as greatly enhanced spikes of CH_4 with peak abundances of ∼7\u2009ppbv. What do these CH_4 revelations with drastically different abundances and temporal signatures represent in terms of interior geochemical processes, or is martian CH_4 a biosignature? Discerning how CH_4 generation occurs on Mars may shed light on the potential habitability of Mars. There is no evidence of life on the surface of Mars today, but microbes might reside beneath the surface. In this case, the carbon flux represented by CH_4 would serve as a link between a putative subterranean biosphere on Mars and what we can measure above the surface. Alternatively, CH_4 records modern geochemical activity. Here we ask the fundamental question: how active is Mars, geochemically and/or biologically? In this article, we examine geological, geochemical, and biogeochemical processes related to our overarching question. The martian atmosphere and surface are an overwhelmingly oxidizing environment, and life requires pairing of electron donors and electron acceptors, that is, redox gradients, as an essential source of energy. Therefore, a fundamental and critical question regarding the possibility of life on Mars is, “Where can we find redox gradients as energy sources for life on Mars?” Hence, regardless of the pathway that generates CH_4 on Mars, the presence of CH_4, a reduced species in an oxidant-rich environment, suggests the possibility of redox gradients supporting life and habitability on Mars. Recent missions such as ExoMars Trace Gas Orbiter may provide mapping of the global distribution of CH_4. To discriminate between abiotic and biotic sources of CH_4 on Mars, future studies should use a series of diagnostic geochemical analyses, preferably performed below the ground or at the ground/atmosphere interface, including measurements of CH_4 isotopes, methane/ethane ratios, H_2 gas concentration, and species such as acetic acid. Advances in the fields of Mars exploration and instrumentation will be driven, augmented, and supported by an improved understanding of atmospheric chemistry and dynamics, deep subsurface biogeochemistry, astrobiology, planetary geology, and geophysics. Future Mars exploration programs will have to expand the integration of complementary areas of expertise to generate synergistic and innovative ideas to realize breakthroughs in advancing our understanding of the potential of life and habitable conditions having existed on Mars. In this spirit, we conducted a set of interdisciplinary workshops. From this series has emerged a vision of technological, theoretical, and methodological innovations to explore the martian subsurface and to enhance spatial tracking of key volatiles, such as CH_4.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/89754, title ="The gut of the finch: uniqueness of the gut microbiome of the Galápagos vampire finch", author = "Michel, Alice J. and Ward, Lewis M.", journal = "Microbiome", volume = "6", pages = "Art. No. 167", month = "September", year = "2018", doi = "10.1186/s40168-018-0555-8", issn = "2049-2618", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180919-102939169", note = "© 2018 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. \n\nReceived: 29 May 2018; Accepted: 5 September 2018; Published: 19 September 2018. \n\nWe are grateful to Sofia Carvajal, Kurt Gielow, Sarah Knutie, Andres Leon, Simón Villamar, Angela Hansen, Sabrina McNew, Ashley Saulsberry, Carlos Vera, Ruben Heleno, Manuel Nogales, and Sandra Hervías for the invaluable assistance in the field; Alexis Pasulka, David Case, and Stephanie Connon for the assistance in lab and advice with the data analysis, as well as other members of the Caltech GE/Bi/ESE_246 molecular geobiology and Bi/GE_105 Evolution course including Rob Phillips, Courtney Chen, Anne Christian, Bianca Lepe, Kristin Anderson, Tristan Murphy, Matthew Smalley, and Tiffany Zhou; and to Parque Nacional Galápagos and Galápagos Science Center for their logistical support. All samples were collected under the Contrato Marco de Acceso a los Recursos Genéticos MAE-DNB-CM-2016-0041 and Galápagos National Park permit PC-0615. \n\nFunds were provided by GAIAS-USFQ Grant to JAC, the Gordon and Betty Moore Foundation through grant GMBF3780 to VJO, NSF-Postdoctoral Grant to DTB, Instrumental Crowdfunding, Le Fonds Québécois de la Recherche sur la Nature et les Technologies Postdoctoral Fellowship, a British Ornithologists’ Union Research Grant to KMG, and a NASA Earth and Space Science Fellowship to LMW. We also acknowledge support from Terence Barr and Caltech's Center for Environmental Microbial Interactions (CEMI). \n\nAvailability of data and materials: Raw reads were deposited and are available through the Sequence Read Archive under accession number SRP130314. \n\nAuthors’ contributions: AJM, LMW, SKG, and VJO analyzed the data and wrote the manuscript. KSD collected the stable isotope data and assisted with the interpretation. AJM, LMW, SKG, VJO, SM extracted DNA and prepared samples for sequencing. AB, JEM, SM, AO, GT, and KY helped analyze the data. DTB, KMG, ACU, and JAC collected fecal samples and observation data on finches on the Galápagos and assisted with the writing of the manuscript. All authors read and approved the final manuscript. \n\nAlice J. Michel and Lewis M. Ward contributed equally to this work. \n\nEthics approval and consent to participate: Not applicable. \n\nConsent for publication: Not applicable.\n\nThe authors declare that they have no competing interests.", revision_no = "39", abstract = "Background: Darwin’s finches are a clade of 19 species of passerine birds native to the Galápagos Islands, whose biogeography, specialized beak morphologies, and dietary choices—ranging from seeds to blood—make them a classic example of adaptive radiation. While these iconic birds have been intensely studied, the composition of their gut microbiome and the factors influencing it, including host species, diet, and biogeography, has not yet been explored. \n\nResults: We characterized the microbial community associated with 12 species of Darwin’s finches using high-throughput 16S rRNA sequencing of fecal samples from 114 individuals across nine islands, including the unusual blood-feeding vampire finch (Geospiza septentrionalis) from Darwin and Wolf Islands. The phylum-level core gut microbiome for Darwin’s finches included the Firmicutes, Gammaproteobacteria, and Actinobacteria, with members of the Bacteroidetes at conspicuously low abundance. The gut microbiome was surprisingly well conserved across the diversity of finch species, with one exception—the vampire finch—which harbored bacteria that were either absent or extremely rare in other finches, including Fusobacterium, Cetobacterium, Ureaplasma, Mucispirillum, Campylobacter, and various members of the Clostridia—bacteria known from the guts of carnivorous birds and reptiles. Complementary stable isotope analysis of feathers revealed exceptionally high δ15N isotope values in the vampire finch, resembling top marine predators. The Galápagos archipelago is also known for extreme wet and dry seasons, and we observed a significant seasonal shift in the gut microbial community of five additional finch species sampled during both seasons. \n\nConclusions: This study demonstrates the overall conservatism of the finch gut microbiome over short (<\u20091 Ma) divergence timescales, except in the most extreme case of dietary specialization, and elevates the evolutionary importance of seasonal shifts in driving not only species adaptation, but also gut microbiome composition.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/89965, title ="Metabolic marker gene mining provides insight in global mcrA diversity and, coupled with targeted genome reconstruction, sheds further light on metabolic potential of the Methanomassiliicoccales", author = "Speth, Daan R. and Orphan, Victoria J.", journal = "PeerJ", volume = "2018", number = "6", pages = "Art. No. e5614", month = "September", year = "2018", doi = "10.7717/peerj.5614", issn = "2167-8359", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180926-133207423", note = "© 2018 Speth and Orphan. Distributed under Creative Commons CC-BY 4.0. \n\nThe authors declare there are no competing interests. \n\nAuthor Contributions: Daan R. Speth conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. \n\nVictoria J. Orphan conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft. \n\nData Availability: The following information was supplied regarding data availability: https://github.com/dspeth/bioinfo_scripts/tree/master/metagenome_screening. \n\nThis manuscript is based upon work supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under award number DE-SC0016469 to Victoria J. Orphan. In addition, Daan R. Speth was supported by NWO Rubicon 019.153LW.039. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. \n\n We thank Woody Fischer and Connor Skennerton for helpful discussion and Grayson Chadwick for critically reading the manuscript.", revision_no = "29", abstract = "Over the past years, metagenomics has revolutionized our view of microbial diversity. Moreover, extracting near-complete genomes from metagenomes has led to the discovery of known metabolic traits in unsuspected lineages. Genome-resolved metagenomics relies on assembly of the sequencing reads and subsequent binning of assembled contigs, which might be hampered by strain heterogeneity or low abundance of a target organism. Here we present a complementary approach, metagenome marker gene mining, and use it to assess the global diversity of archaeal methane metabolism through the mcrA gene. To this end, we have screened 18,465 metagenomes for the presence of reads matching a database representative of all known mcrA proteins and reconstructed gene sequences from the matching reads. We use our mcrA dataset to assess the environmental distribution of the Methanomassiliicoccales and reconstruct and analyze a draft genome belonging to the ‘Lake Pavin cluster’, an uncultivated environmental clade of the Methanomassiliicoccales. Analysis of the ‘Lake Pavin cluster’ draft genome suggests that this organism has a more restricted capacity for hydrogenotrophic methylotrophic methanogenesis than previously studied Methanomassiliicoccales, with only genes for growth on methanol present. However, the presence of the soluble subunits of methyltetrahydromethanopterin:CoM methyltransferase (mtrAH) provide hypothetical pathways for methanol fermentation, and aceticlastic methanogenesis that await experimental verification. Thus, we show that marker gene mining can enhance the discovery power of metagenomics, by identifying novel lineages and aiding selection of targets for in-depth analyses. Marker gene mining is less sensitive to strain heterogeneity and has a lower abundance threshold than genome-resolved metagenomics, as it only requires short contigs and there is no binning step. Additionally, it is computationally cheaper than genome resolved metagenomics, since only a small subset of reads needs to be assembled. It is therefore a suitable approach to extract knowledge from the many publicly available sequencing projects.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/87091, title ="Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex", author = "Ijiri, Akira and Case, David H.", journal = "Science Advances", volume = "4", number = "6", pages = "Art. No. eaao4631", month = "June", year = "2018", doi = "10.1126/sciadv.aao4631", issn = "2375-2548", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180613-160656492", note = "© 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. \n\nReceived for publication July 24, 2017. Accepted for publication May 1, 2018. \n\nWe thank all crews, drilling team members, and technical staffs on the deep-sea drilling vessel Chikyu and the research vessel Hakuhomaru for support with core sampling from KMV#5 during JAMSTEC Chikyu Expeditions 903, 906, and KH06-04 cruises. We are also grateful to IODP for providing sediment core samples from Site C0002 during Expedition 315. We are grateful to S. Fukunaga, S. Hashimoto, K. Iijima, A. Imajo, H. Machiyama, Y. Nishio, S. Tanaka, H. Tomiyama, and N. Xiao for useful discussions and/or technical assistance. This is a contribution to the Deep Carbon Observatory. \n\nThis study was supported, in part, by the Japan Society for the Promotion of Science (JSPS) Strategic Fund for Strengthening Leading-Edge Research and Development (to JAMSTEC and F.I.), the JSPS Funding Program for Next Generation World-Leading Researchers (GR102 to F.I.), the Grant-in-Aid for Science Research (nos. 23681007, 26287128, and 17H01871 to A.I.; no. 26251041 to F.I.; no. 17H06105 to N.Y.), and the East Asia and Pacific Summer Institutes program through the NSF and JSPS (no. 1308171 to D.H.C.). Lipid analyses by M.Y.Y. and shipboard work by F.S. were financed by the European Research Council under the European Union’s Seventh Framework Programme—“Ideas” Specific Programme (no. 247153 to K.-U.H.). \n\nAuthor contributions: A.I. and F.I. designed the study. F.I. led the project as the chief scientist of JAMSTEC Chikyu Expeditions 903 and 906. Y.K. coordinated Expeditions 903 and 906 as the expedition project manager. A.I., F.I., R.R.A., T.H., Y.M., T. Toki, G.L.A., J.A., D.H.C., T.F., Y.I., H.I., J.K., H.K., K.-i.N., Y.N., M.N., H.R., S.S., F.S., A.T., W.T., T. Terada, H.T., and Y.T.Y. collected and analyzed the sediment core samples and data as shipboard scientists. S.H., S.K., Y.O., S.O., K.T., D.T.W., M.Y.Y., K.-U.H., M.I., M.A.L., S.M., V.J.O., T. Tuji, U.T., and N.Y. analyzed the samples and data as shore-based scientists. A.I. and F.I. cowrote the manuscript. All authors discussed the results and commented on the manuscript. \n\nThe authors declare that they have no competing interests. \n\nData and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The 16S rRNA gene sequences in this study were all deposited in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank nucleotide sequence databases under accession no. DRA001034. The 16S rRNA gene sequence of strain 1H1 was also available under accession no. LC170394. Strain 1H1 has been deposited in the Japan Collection of Microorganisms (JCM 19936). Additional data related to this paper may be requested from the authors.", revision_no = "23", abstract = "Microbial life inhabiting subseafloor sediments plays an important role in Earth’s carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (10^2 to 10^3 cells cm^(−3)) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85740, title ="Subgroup characteristics of marine methane-oxidizing ANME-2 archaea and their syntrophic partners revealed by integrated multimodal analytical microscopy", author = "McGlynn, Shawn E. and Chadwick, Grayson L.", journal = "Applied and Environmental Microbiology", volume = "84", number = "11", pages = "Art. No. e00399-18", month = "June", year = "2018", doi = "10.1128/AEM.00399-18", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180411-080037971", note = "© 2018 American Society for Microbiology. \n\nReceived 15 February 2018; Accepted 31 March 2018; Accepted manuscript posted online 6 April 2018. \n\nWe are grateful for the use of the facilities of the Beckman Resource Center for Transmission Electron Microscopy at Caltech (BRCem) and helpful advice from Alasdair McDowall and Mark Ladinsky. This work also benefited from the Applied Physics and Materials Science Department's Transmission Electron Microscopy Facility at Caltech, where EDS measurements were made with Carol M. Garland. \n\nThis work was funded by the U.S. Department of Energy, Office of Science, Office of Biological Environmental Research, under award numbers DE-SC0004949 and DE-SC0010574 and by a grant from the Gordon and Betty Moore Foundation Marine Microbiology Initiative (grant number 3780), both to V.J.O. S.E.M. was supported in part by an MEXT KAKENHI Grant-in-Aid for challenging exploratory research (grant award number 15K14608) and the Research Foundation for Opto-Science and Technology. New correlated microscopy and 3D EM method development, as well as access to advanced imaging resources housed at UCSD, was supported by a grant from the NIH National Institute of General Medical Sciences (number P41GM103412 to M.H.E.), an award which partially supports the National Center for Microscopy and Imaging Research.", revision_no = "26", abstract = "Phylogenetically diverse environmental ANME archaea and sulfate-reducing bacteria cooperatively catalyze the anaerobic oxidation of methane oxidation (AOM) in multicelled consortia within methane seep environments. To better understand these cells and their symbiotic associations, we applied a suite of electron microscopy approaches, including correlative fluorescence in situ hybridization-electron microscopy (FISH-EM), transmission electron microscopy (TEM), and serial block face scanning electron microscopy (SBEM) three-dimensional (3D) reconstructions. FISH-EM of methane seep-derived consortia revealed phylogenetic variability in terms of cell morphology, ultrastructure, and storage granules. Representatives of the ANME-2b clade, but not other ANME-2 groups, contained polyphosphate-like granules, while some bacteria associated with ANME-2a/2c contained two distinct phases of iron mineral chains resembling magnetosomes. 3D segmentation of two ANME-2 consortium types revealed cellular volumes of ANME and their symbiotic partners that were larger than previous estimates based on light microscopy. Polyphosphate-like granule-containing ANME (tentatively termed ANME-2b) were larger than both ANME with no granules and partner bacteria. This cell type was observed with up to 4 granules per cell, and the volume of the cell was larger in proportion to the number of granules inside it, but the percentage of the cell occupied by these granules did not vary with granule number. These results illuminate distinctions between ANME-2 archaeal lineages and partnering bacterial populations that are apparently unified in their ability to perform anaerobic methane oxidation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/81087, title ="Subnanogram proteomics: impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples", author = "Zhu, Ying and Zhao, Rui", journal = "International Journal of Mass Spectrometry", volume = "427", pages = "4-10", month = "April", year = "2018", doi = "10.1016/j.ijms.2017.08.016", issn = "1387-3806", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170901-140533444", note = "© 2017 Elsevier B.V. \n\nReceived 24 April 2017, Revised 3 August 2017, Accepted 22 August 2017, Available online 1 September 2017. \n\nA portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). This work was also supported by the NIH National Institute of General Medical Sciences (P41 GM103493) and the NIH National Institute of Biomedical Imaging and Bioengineering (1R21EB020976-01A1). Y.Z., R.Z., P.D.P., R.J.M., L.P.-T. W.-J. Q. and R.T.K would like to thank R.D.S. for many years of inspiration and friendship.", revision_no = "17", abstract = "One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ∼3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ∼ 95% for 0.5 ng samples and by ∼42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/83447, title ="Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods", author = "Pasulka, Alexis L. and Thamatrakoln, Kimberlee", journal = "Environmental Microbiology", volume = "20", number = "2", pages = "671-692", month = "February", year = "2018", doi = "10.1111/1462-2920.13996", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171127-134624207", note = "© 2017 Wiley. \n\nIssue online: 14 February 2018; Version of record online:\n14 December 2017; Accepted manuscript online: 21 November 2017; Manuscript Accepted: 12 November 2017; Manuscript Revised: 10 November 2017; Manuscript Received:\n8 August 2017. \n\nWe thank K. Dawson and G. Chadwick for many insightful conversations regarding data interpretation and analysis and R. Hatzenpichler and B. Babin for useful discussions regarding BONCAT and click chemistry. We also thank J. Nissomov, F. Natale and J. Latham for their technical assistance with the E. huxleyi-EhV experiments as well as Jennifer Brum and Sarah Schwenck for help exploring the use of TEM microscopy for mapping Syn1 viral particles. This work was funded by the Gordon and Betty Moore Foundation through Grant GBMF3780 to VJO, GBMF3789 to KDB and GBMF3305 and GBMF3790 to MBS as well as NSF Biological Oceanography awards to KT, KDB (OCE-1559179) and MBS (OCE-1536989). ALP was supported by an NSF OCE Postdoctoral Research Fellowship. The Caltech Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775, the Beckman Institute, and NIH (S10OD020013).", revision_no = "20", abstract = "While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1, and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labeling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.", } @techreport {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86060, title ="Methane on Mars and Habitability: Challenges and Responses", author = "Yung, Yuk L. and Chen, Pin", journal = "Astrobiology", volume = "18", pages = "1221-1242", month = "January", year = "2018", issn = "1557-8070", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180426-110252898", note = "Study Report prepared for the Keck Institute for Space Studies (KISS).\n\nWe dedicate this report to JPL scientist Mark Allen, whose spirit of generosity inspired many of the conversations and much of the work that brought us together. We thank the Director of the Keck Institute for Space Studies, Tom Prince, the Executive Director, Michele Judd, and the fantastic KISS staff for hosting our workshop and providing a wonderful environment for these discussions.", revision_no = "53", abstract = "Recent measurements of methane (CH_4) by the Mars Science Laboratory (MSL) now confront us with robust data that demand interpretation. Thus far, the MSL data have revealed a baseline level of CH_4 (∼0.4 parts per billion by volume [ppbv]), with seasonal variations, as well as greatly enhanced spikes of CH_4 with peak abundances of ∼7\u2009ppbv. What do these CH_4 revelations with drastically different abundances and temporal signatures represent in terms of interior geochemical processes, or is martian CH_4 a biosignature? Discerning how CH_4 generation occurs on Mars may shed light on the potential habitability of Mars. There is no evidence of life on the surface of Mars today, but microbes might reside beneath the surface. In this case, the carbon flux represented by CH_4 would serve as a link between a putative subterranean biosphere on Mars and what we can measure above the surface. Alternatively, CH_4 records modern geochemical activity. Here we ask the fundamental question: how active is Mars, geochemically and/or biologically? In this article, we examine geological, geochemical, and biogeochemical processes related to our overarching question. The martian atmosphere and surface are an overwhelmingly oxidizing environment, and life requires pairing of electron donors and electron acceptors, that is, redox gradients, as an essential source of energy. Therefore, a fundamental and critical question regarding the possibility of life on Mars is, “Where can we find redox gradients as energy sources for life on Mars?” Hence, regardless of the pathway that generates CH_4 on Mars, the presence of CH_4, a reduced species in an oxidant-rich environment, suggests the possibility of redox gradients supporting life and habitability on Mars. Recent missions such as ExoMars Trace Gas Orbiter may provide mapping of the global distribution of CH_4. To discriminate between abiotic and biotic sources of CH_4 on Mars, future studies should use a series of diagnostic geochemical analyses, preferably performed below the ground or at the ground/atmosphere interface, including measurements of CH_4 isotopes, methane/ethane ratios, H_2 gas concentration, and species such as acetic acid. Advances in the fields of Mars exploration and instrumentation will be driven, augmented, and supported by an improved understanding of atmospheric chemistry and dynamics, deep subsurface biogeochemistry, astrobiology, planetary geology, and geophysics. Future Mars exploration programs will have to expand the integration of complementary areas of expertise to generate synergistic and innovative ideas to realize breakthroughs in advancing our understanding of the potential of life and habitable conditions having existed on Mars. In this spirit, we conducted a set of interdisciplinary workshops. From this series has emerged a vision of technological, theoretical, and methodological innovations to explore the martian subsurface and to enhance spatial tracking of key volatiles, such as CH_4.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/82597, title ="Spatially resolved capture of hydrogen sulfide from the water column and sedimentary pore waters for abundance and stable isotopic analysis", author = "Fike, D. A. and Houghton, J. L.", journal = "Marine Chemistry", volume = "197", pages = "26-37", month = "December", year = "2017", doi = "10.1016/j.marchem.2017.10.004", issn = "0304-4203", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171023-152816244", note = "© 2017 Elsevier B.V. \n\nReceived 24 July 2017, Revised 15 October 2017, Accepted 17 October 2017, Available online 21 October 2017.", revision_no = "11", abstract = "Sulfur cycling is ubiquitous in sedimentary environments, where it plays a major role in mediating carbon remineralization and impacts both local and global redox budgets. Microbial sulfur cycling is dominated by metabolic activity that either produces (e.g., sulfate reduction, disproportionation) or consumes (sulfide oxidation) hydrogen sulfide (H_2S). As such, improved constraints on the production, distribution, and consumption of H_2S in the natural environment will increase our understanding of microbial sulfur cycling. These different microbial sulfur metabolisms are additionally associated with particular stable isotopic fractionations. Coupling measurements of the isotopic composition of the sulfide with its distribution can provide additional information about environmental conditions and microbial ecology. Here we investigate the kinetics of sulfide capture on photographic films as a way to document the spatial distribution of sulfide in complex natural environments as well as for in situ capture of H_2S for subsequent stable isotopic analysis. Laboratory experiments and timed field deployments demonstrate the ability to infer ambient sulfide abundances from the yield of sulfide on the films. This captured sulfide preserves the isotopic composition of the ambient sulfide, offset to slightly lower δ^(34)S values by ~ 1.2 ± 0.5‰ associated with the diffusion of sulfide into the film and subsequent reaction with silver to form Ag_2S precipitates. The resulting data enable the exploration of cm-scale lateral heterogeneity that complement most geochemical profiles using traditional techniques in natural environments. Because these films can easily be deployed over a large spatial area, they are also ideal for real-time assessment of the spatial and temporal dynamics of a site during initial reconnaissance and for integration over long timescales to capture ephemeral processes.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/84116, title ="Aerobic and Anaerobic Methanotrophic Communities Associated with Methane Hydrates Exposed on the Seafloor: A High-Pressure Sampling and Stable Isotope-Incubation Experiment", author = "Case, David H. and Ijiri, Akira", journal = "Frontiers in Microbiology", volume = "8", pages = "Art. No. 2569", month = "December", year = "2017", doi = "10.3389/fmicb.2017.02569", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180105-082606163", note = "© 2017 Case, Ijiri, Morono, Tavormina, Orphan and Inagaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 15 August 2017; Accepted: 11 December 2017; Published: 19 December 2017. \n\nAuthor Contributions: FI and VO: designed the research; FI: led the NT13–15 cruise; YM and FI: designed the high-pressure sampling and incubation system; DC, AI, YM, and FI: collected the deep-sea samples; DC, AI, and YM: performed a high-pressure incubation experiment; AI: performed geochemical analyses; DC, PT, and VO performed molecular analyses; DC and AI: wrote the manuscript with significant input from FI and VO; All authors contributed to interpretation of data. \n\nThis study was supported in part by the Japan Society for the Promotion of Science (JSPS) Strategic Fund for Strengthening Leading-Edge Research and Development (to JAMSTEC and FI), the JSPS Funding Program for Next Generation World-Leading Researchers (GR102 to FI), JSPS Grant-in-Aid for Scientific Research (24687004 to YM and 26251041 to FI), DC was supported by a National Science Foundation (NSF) Graduate Research Fellowship as well as the East Asian and Pacific Summer Institutes (EAPSI) Summer Fellowship, co-funded by the NSF and JSPS. \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nWe thank the crew of the R/V Natsushima cruise NT13-15, for their help at sea, without which this work would not have been possible. We also thank Makoto Nagasawa and Masamitsu Matsumoto (Syn Co. Inc.) for the construction of high-pressure devices and useful advices. Masazumi Tsutsumi, Yoko Ohtomo, Go-Ichiro Uramoto, Tatsuhiko Hoshino, Takeshi Terada, Nan Xiao helped with field and laboratory work. This is a contribution to the Deep Carbon Observatory.", revision_no = "12", abstract = "High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/82111, title ="Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds", author = "Trembath-Reichert, Elizabeth and Morono, Yuki", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "114", number = "44", pages = "E9206-E9215", month = "October", year = "2017", doi = "10.1073/pnas.1707525114", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171005-093343465", note = "© 2017 National Academy of Sciences. Freely available online through the PNAS open access option. NAS retains a nonexclusive License to Publish, and these articles are distributed under a CC BY-NC-ND license. \n\nEdited by David M. Karl, University of Hawaii, Honolulu, HI, and approved September 6, 2017 (received for review May 5, 2017). Published online before print October 3, 2017. \n\nWe thank the IODP for providing access and samples from the deep coalbed biosphere off Shimokita during Expedition 337. We thank the crew, drilling team members, laboratory technicians, and scientists on the drilling vessel Chikyu for supporting core sampling and onboard measurements. We also thank S. Fukunaga, S. Hashimoto, and A. Imajo [Japan Agency for Marine-Earth Science and Technology (JAMSTEC)] and T. Terada (Marine Works Japan, Ltd) for assistance in microbiological analyses; Y. Guan, F. Wu, C. Ma, and N. Dalleska (Caltech) for assistance with geochemical analyses; and A. L. Sessions, G. L. Chadwick, K. S. Metcalfe, M. K. Lloyd, and S. Kopf (Caltech) and H. Imachi (JAMSTEC) for feedback and valuable discussions. We appreciate the comments of two reviewers that also improved this manuscript. Funding for this work was provided by the Center for Dark Energy Biosphere (C-DEBI), NASA Astrobiology-Life Underground (NAI-LU; Award NNA13AA92A), the Gordon and Betty Moore Foundation Grant GBMF3780 (to V.J.O.), and Post Expedition Award (to E.T.-R. and V.J.O.), the Japan Society for the Promotion of Science (JSPS) Strategic Fund for Strengthening Leading-Edge Research and Development (F.I. and JAMSTEC), the JSPS Funding Program for Next Generation World-Leading Researchers (NEXT Program, Grant GR102 to F.I.), and JSPS Grants-in-Aid for Science Research (Grant 26251041 to F.I.; Grant 15K14907 to T.H.; and Grants 24687004, 15H05608, 24651018, 2665169, and 16K14817 to Y.M.). E.T.-R. was additionally supported, in part, by a Schlanger Ocean Drilling Fellowship, a C-DEBI travel grant for sample processing at the JAMSTEC Kochi Institute for Core Sample Research, and the Deep Life Cultivation Internship Program from the Deep Carbon Observatory (DCO). This is C-DEBI Grant contribution no. 389 and NAI-LU no. 314. \n\nAuthor contributions: E.T.-R., Y.M., F.I., and V.J.O. designed research; E.T.-R. performed research; Y.M., A.I., and T.H. contributed new reagents/analytic tools; E.T.-R., Y.M., A.I., T.H., K.S.D., and V.J.O. analyzed data; and E.T.-R., Y.M., K.S.D., F.I., and V.J.O. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nData deposition: Data are available at National Center for Biotechnology Information (NCBI) BioProject, https://www.ncbi.nlm.nih.gov/bioproject/ (PRJNA381552) and NCBI BioSample, https://www.ncbi.nlm.nih.gov/biosample/ (SAMN06676442-48). BioSamples are identified in Dataset S2. Nanometer-scale secondary ion mass spectrometry and geochemical data are available at Biological and Chemical Oceanography Data Management Office (https://www.bco-dmo.org/projects; Project 672592). \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707525114/-/DCSupplemental.", revision_no = "33", abstract = "The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of ^(13)C- or ^(15)N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/77616, title ="Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis", author = "Terrado, Ramon and Pasulka, Alexis L.", journal = "ISME Journal", volume = "11", number = "9", pages = "2022-2034", month = "September", year = "2017", doi = "10.1038/ismej.2017.68", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170522-080446020", note = "© 2017 The Authors. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/. \n\nReceived 21 October 2016; Revised 20 March 2017; Accepted 3 April 2017; Advance online publication 19 May 2017. \n\nThis collaborative research was supported by the Gordon and Betty Moore Foundation through Grant GBMF3299 to DAC and KBH at USC, and Grant (GBMF3780) to VJO at California Institute of Technology. \n\nThe authors declare no conflict of interest.", revision_no = "22", abstract = "Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modes between autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutrition differ among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritional flexibility, although most species generally fall towards the heterotrophic end of the mixotrophy spectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growing mixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence of either heat-killed bacteria enriched with ^(15)N and ^(13)C, or unlabeled heat-killed bacteria and labeled inorganic substrates (^(13)C-bicarbonate and ^(15)N-ammonium). The alga exhibited high growth rates (up to 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotope analyses revealed that Ochromonas obtained 84–99% of its carbon and 88–95% of its nitrogen from consumed bacteria. The chrysophyte assimilated inorganic ^(13)C-carbon and ^(15)N-nitrogen when bacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient to support net population growth of the alga. Our use of nanoSIMS represents its first application towards the study of a mixotrophic alga, enabling a better understanding and quantitative assessment of carbon and nutrient acquisition by this species.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/79841, title ="Methane-Fueled Syntrophy through Extracellular Electron Transfer: Uncovering the Genomic Traits Conserved within Diverse Bacterial Partners of Anaerobic Methanotrophic Archaea", author = "Skennerton, Connor T. and Chourey, Karuna", journal = "mBio", volume = "8", number = "4", pages = "Art. No. e00530-17", month = "August", year = "2017", doi = "10.1128/mBio.00530-17", issn = "2150-7511", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170807-095231730", note = "© 2017 Skennerton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. \n\nReceived 31 March 2017; Accepted 28 June 2017; Published 1 August 2017. \n\nWe thank C. Titus Brown and Lisa Cohen for assistance with sequencing the samples from the Santa Monica Mounds. G.W.T. acknowledges support by the University of Queensland Vice-Chancellor's Research Focused Fellowship. \n\nThis work was funded by the Gordon and Betty Moore Foundation through grant GBMF3780 (to V.J.O.); the US Department of Energy, Office of Science, Office of Biological Environmental Research, under award numbers DE-SC0003940 and DE-SC0010574 (to V.J.O.); and the National Science Foundation’s Center for Dark Energy Biosphere Investigations (C-DEBI) under award number OCE-0939564 (to V.J.O.). \n\nThis is contribution number 374. \n\nThis work was funded by the Gordon and Betty Moore Foundation through grant GBMF3780 (to V.J.O.); the US Department of Energy, Office of Science, Office of Biological Environmental Research, under award numbers DE-SC0003940 and DE-SC0010574 (to V.J.O.); and the National Science Foundation’s Center for Dark Energy Biosphere Investigations (C-DEBI) under award number OCE-0939564 (to V.J.O.). \n\nData availability: Raw sequencing data, metagenomic assemblies, and draft genome sequences are available under NCBI bioproject identifiers PRJNA326769 and PRJNA290197.", revision_no = "18", abstract = "The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/81447, title ="Monodeuterated Methane, an Isotopic Tool To Assess Biological Methane Metabolism Rates", author = "Marlow, Jeffrey J. and Steele, Joshua A.", journal = "mSphere", volume = "2", number = "4", pages = "Art. No. e00309", month = "July", year = "2017", doi = "10.1128/mSphereDirect.00309-17", issn = "2379-5042", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170914-133025440", note = "© 2017 Marlow et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. \n\nReceived 18 July 2017; Accepted 31 July 2017; Published 23 August 2017. \n\nWe thank the captains, crew, Alvin group, Jason group, and Science party members from the RV Atlantis on legs AT-15-68 and AT-18-10. Water analyzer measurements were conducted in the laboratory of Alex Sessions at the California Institute of Technology with technical support from Lichun Zhang. We are indebted to William Berelson at the University of Southern California and Nick Rollins for use of their pressure chambers and assistance with the incubation experiments. We thank Alex Sessions, Woodward Fischer, Dianne Newman, Tori Hoehler, Amy Rosenzweig, and Daniel Stolper for helpful conversations during the preparation of the manuscript. \n\nThis study was funded by grants from the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC001057), and the NASA Astrobiology Institute (award number NNA13AA92A) and by support from the Gordon and Betty Moore Foundation through grant GBMF3780 (to V.J.O.). J.J.M. was supported by a National Energy Technology Laboratory Methane Hydrate Research Fellowship funded by the National Research Council of the National Academies. This research used resources of the Oak Ridge Leadership Computing Facility. Oak Ridge National Laboratory is supported by the Office of Science of the U.S. Department of Energy. \n\nWe declare no conflict of interest.", revision_no = "11", abstract = "Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH_3D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH_3D procedure and the well-established ^(14)CH_4 radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH_3D method were directly and consistently proportional to ^(14)C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/74554, title ="Quantification and isotopic analysis of intracellular sulfur metabolites in the dissimilatory sulfate reduction pathway", author = "Sim, Min Sub and Paris, Guillaume", journal = "Geochimica et Cosmochimica Acta", volume = "206", pages = "57-72", month = "June", year = "2017", doi = "10.1016/j.gca.2017.02.024", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170227-143517457", note = "© 2017 Elsevier Ltd. \n\nReceived 30 July 2016, Accepted 18 February 2017, Available online 27 February 2017. \n\nThis work was supported by an Agouron Geobiology Fellowship to MSS and the Gordon and Betty Moore Foundation Grant GBMF 3306 to VJO and ALS.", revision_no = "18", abstract = "Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in ^(34)S and producing sulfide that is depleted in ^(34)S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfur isotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellular mechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopic ratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis. Intracellular sulfate and APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulates in the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in ^(34)S. The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfate and sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the net fractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This ‘reservoir effect’ diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations. Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the overall (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for the well-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while at the same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in this relationship. Our approach, together with ongoing investigations of the kinetic isotope fractionation by key enzymes in the sulfate reduction pathway, should provide an empirical basis for a quantitative model relating the magnitude of microbial isotope fractionation to their environmental and physiological controls.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85749, title ="Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging", author = "Glass, Jennifer B. and Chen, Si", journal = "Geomicrobiology Journal", volume = "35", number = "1", pages = "81-89", month = "May", year = "2017", doi = "10.1080/01490451.2017.1321068", issn = "0149-0451", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180411-113723264", note = "© 2018 Taylor & Francis. \n\nReceived 01 Nov 2016, Accepted 01 Apr 2017, Accepted author version posted online: 21 Apr 2017, Published online: 19 May 2017. \n\nThis work was supported by a NASA Astrobiology Postdoctoral Fellowship to J.B.G, NASA Exobiology award NNX14AJ87G to J.B.G., DOE Biological and Environmental Research award DE-SC0004949 to V.J.O, NSF award OCE-0939564 to V.J.O, NSF award OCE-1232814 to B.S.T., and NSF award OCE-1357375 to E.D.I. This work was also supported by the NASA Astrobiology Institute Alternative Earths Team (Science Mission Directorate award NNA15BB03A). Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. DOE Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). We thank two anonymous reviewers for helpful feedback on the previous version of this manuscript.", revision_no = "16", abstract = "Metal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/74756, title ="Rapid Quantification and Isotopic Analysis of Dissolved Sulfur Species", author = "Smith, Derek A. and Sessions, Alex L.", journal = "Rapid Communications in Mass Spectrometry", volume = "31", number = "9", pages = "791-803", month = "May", year = "2017", doi = "10.1002/rcm.7846", issn = "0951-4198", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170306-080715333", note = "© 2017 John Wiley & Sons, Inc. \n\nAccepted manuscript online: 1 March 2017; Manuscript Accepted: 23 February 2017; Manuscript Revised: 6 January 2017; Manuscript Received: 19 September 2016.", revision_no = "7", abstract = "Rationale: Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. \n\nMethods: Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5 minutes. Fluorescence detection (380 nm excitation, 480 nm emission) provides highly selective and sensitive quantitation, and Time of Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either ^(33)S or ^(34)S). \n\nResults: Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (μM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of ^(34)S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. \n\nConclusions: Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low μM detection limits. Using a high-resolution TOF-MS, accurate detection of as little as 0.01% ^(34)S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/71196, title ="Starvation and recovery in the deep-sea methanotroph Methyloprofundus sedimenti", author = "Tavormina, Patricia L. and Kellermann, Matthias Y.", journal = "Molecular Microbiology", volume = "103", number = "2", pages = "242-252", month = "January", year = "2017", doi = "10.1111/mmi.13553", issn = "0950-382X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20161017-155437081", note = "© 2016 John Wiley & Sons Ltd. \n\nIssue online: \n6 January 2017;\n\nVersion of record online: \n22 November 2016;\n\nAccepted manuscript online: \n14 October 2016;\n\nManuscript Accepted: \n8 October 2016.\n\n\nGC-FID measurements were performed in the Environmental Analysis Center at Caltech. We thank Paul Magyar for assistance with GC-FID, and Songye Chen for assistance segmenting crytomographic files. Special thanks for electron microscopy support from Dr. Alasdair McDowall, Howard Hughes Medical Institute. The Caltech electron microscopy facility is supported in part by the Gordon and Betty Moore Foundation, the Agouron Institute and the Beckman Foundation. Funding for this work was provided by the Gordon and Betty Moore Foundation (GBMF3780, VJO; GBMF3811, ND) and the National Science Foundation (OCE-1046144, DLV; EAR-0950600, MYK).\n", revision_no = "34", abstract = "In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane-derived carbon into the food web remain poorly described, due to a lack of cultured representative deep-sea methanotrophic prokaryotes. Here, the response of the deep-sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup-specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane-derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea.\n", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/66226, title ="Phylogenomic analysis of Candidatus ‘Izimaplasma’ species: free-living representatives from a Tenericutes clade found in methane seeps", author = "Skennerton, Connor T. and Haroon, Mohamed F.", journal = "ISME Journal", volume = "10", number = "11", pages = "2679-2692", month = "November", year = "2016", doi = "10.1038/ismej.2016.55", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160418-080655842", note = "© 2016 International Society for Microbial Ecology. \n\nReceived 13 January 2016; Revised 29 February 2016; Accepted 4 March 2016; Advance online publication 8 April 2016. \n\nThis work is funded by the Gordon and Betty Moore Foundation through Grant GBMF3780 and the US Department of Energy, Office of Science, Office of Biological Environmental Research under award numbers DE-SC0003940 and DE-SC0010574 (to VJO). Additional support was provided by Caltech’s Center for Environmental Microbial Interactions and the Howard Hughes Medical Institute. We thank D Smith, S Scheller, S Zinder, J Hemp and D Newman for helpful discussions and M Imelfort for bioinformatics assistance. GWT was supported by an ARC Queen Elizabeth II fellowship (ARC-DP1093175). \n\nThe authors declare no conflict of interest.", revision_no = "39", abstract = "Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as ‘NB1-n’ (SILVA taxonomy) or ‘RF3’ (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these ‘NB1-n’ representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10\u2009°C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus ‘Izimaplasma sp. HR1’ and Candidatus ‘Izimaplasma sp. HR2’ for the two genome representatives.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/69506, title ="Measurement of rare isotopologues of nitrous oxide by high-resolution multi-collector mass spectrometry", author = "Magyar, Paul M. and Orphan, Victoria J.", journal = "Rapid Communications in Mass Spectrometry", volume = "30", number = "17", pages = "1923-1940", month = "September", year = "2016", doi = "10.1002/rcm.7671", issn = "0951-4198", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160808-234129625", note = "© 2016 John Wiley & Sons, Ltd. \n\nVersion of record online: 5 August 2016; Manuscript Accepted: 18 June 2016; Manuscript Revised: 16 June 2016; Manuscript Received: 16 March 2016. \n\nThis work was supported by a grant from the Gordon and Betty Moore Foundation Marine Microbiology Initiative (Grant #3306) and by NSF-EAR. We thank Dianne Newman for providing the P. aeruginosa ∆nosZ mutant and for the use of her laboratory; Sebastian Kopf for aid in microbial culturing; Nami Kitchen for assistance in the laboratory; and Daniel Stolper, Alison Piasecki, and Matthieu Clog for helpful discussions. We thank Karen Casciotti, Shuhei Ono, and Nathaniel Ostrom for providing calibrated reference samples and Sakae Toyoda and Naohiro Yoshida for measuring the isotopic composition of our reference gas. Finally, we thank Nathaniel Ostrom for helpful comments on an an earlier draft of this manuscript, as well as two anonymous reviewers for their comments.", revision_no = "6", abstract = "Rationale: Bulk and position-specific stable isotope characterization of nitrous oxide represents one of the most powerful tools for identifying its environmental sources and sinks. Constraining ^(14)N^(15)N^(18)O and ^(15)N^(14)N^(18)O will add two new dimensions to our ability to uniquely fingerprint N_2O sources. \n\nMethods: We describe a technique to measure six singly and doubly substituted isotopic variants of N2O, constraining the values of δ^(15)N, δ^(18)O, ∆^(17)O, ^(15)N site preference, and the clumped isotopomers ^(14)N^(15)N^(18)O and ^(15)N^(14)N^(18)O. The technique uses a Thermo MAT 253 Ultra, a high-resolution multi-collector gas source isotope ratio mass spectrometer. It requires 8–10 hours per sample and ~10 micromoles or more of pure N_2O. \n\nResults: We demonstrate the precision and accuracy of these measurements by analyzing N_2O brought to equilibrium in its position-specific and clumped isotopic composition by heating in the presence of a catalyst. Finally, an illustrative analysis of biogenic N_2O from a denitrifying bacterium suggests that its clumped isotopic composition is controlled by kinetic isotope effects in N_2O production. \n\nConclusions: We developed a method for measuring six isotopic variants of N_2O and tested it with analyses of biogenic N_2O. The added isotopic constraints provided by these measurements will enhance our ability to apportion N_2O sources.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63109, title ="Microbial eukaryotic distributions and diversity patterns in a deep-sea methane seep ecosystem", author = "Pasulka, Alexis L. and Levin, Lisa A.", journal = "Environmental Microbiology", volume = "18", number = "9", pages = "3022-3043", month = "September", year = "2016", doi = "10.1111/1462-2920.13185", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151221-150217687", note = "© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.\n\nReceived 7 October, 2015; revised 3 December, 2015; accepted 8 December, 2015. First published: 25 January 2016. \n\nSpecial Issue on Ecophysiology of Anaerobes and their Habitat. \n\nWe are grateful to the captain and crew of the R/V Atlantis and the JASON pilots who helped make sampling possible. We offer thanks to J. Marlow, A. Dekas, A. Green-Saxena, S. Connon and many others for help at sea and/or in the laboratory. In particular, we would like to thank R. Leon for help developing at-sea methods, J. Glass for providing sulfide data, G. Chadwick, E. Wilbanks and C. Skennerton for programming assistance, G. Mendoza for helpful input regarding the statistical analyses, and E. Allen for insightful discussions about sample processing and analyses. Support for this research was provided by grant OCE 0825791, OCE 0826254 and OCE 0939557 from the US National Science Foundation (NSF). VJO is supported by a grant from the Gordon and Betty Moore Foundation Marine Microbial Initiative (GBMF3780). NSF Graduate Research Fellowships supported ALP and DHC, and a P.E.O Scholar Award additionally supported ALP.", revision_no = "32", abstract = "Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/69624, title ="Characterization of Chemosynthetic Microbial Mats Associated with Intertidal Hydrothermal Sulfur Vents in White Point, San Pedro, CA, USA", author = "Miranda, Priscilla J. and McLain, Nathan K.", journal = "Frontiers in Microbiology", volume = "7", pages = "Art. No. 1163", month = "July", year = "2016", doi = "10.3389/fmicb.2016.01163", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160815-131417667", note = "© 2016 Miranda, McLain, Hatzenpichler, Orphan and Dillon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 01 April 2016; Accepted: 12 July 2016; Published: 27 July 2016. \n\nAuthor Contributions: Conception or design of the work: PM, RH, VO, and JD. Data acquisition, analysis and interpretation: PM, NM, RH, and JD. Drafting the article: PM, and JD. Critical revision of the article: PM, NM, RH, VO, and JD. All authors have read and approved this submission. \n\nThis study was performed with support from the National Science Foundation (EAR-1124398 to JD, EAR-112391 to VO) and CSU-COAST student research grant to PM. Partial support was additionally provided by the Gordon and Betty Moore Foundation through grant GBMF3306 to VO. \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\nWe thank Christine Whitcraft and Lora Stevens for project advising and Katherine Dawson for providing the WP sulfide measurements.", revision_no = "8", abstract = "The shallow-sea hydrothermal vents at White Point (WP) in Palos Verdes on the southern California coast support microbial mats and provide easily accessed settings in which to study chemolithoautotrophic sulfur cycling. Previous studies have cultured sulfur-oxidizing bacteria from the WP mats; however, almost nothing is known about the in situ diversity and activity of the microorganisms in these habitats. We studied the diversity, micron-scale spatial associations and metabolic activity of the mat community via sequence analysis of 16S rRNA and aprA genes, fluorescence in situ hybridization (FISH) microscopy and sulfate reduction rate (SRR) measurements. Sequence analysis revealed a diverse group of bacteria, dominated by sulfur cycling gamma-, epsilon-, and deltaproteobacterial lineages such as Marithrix, Sulfurovum, and Desulfuromusa. FISH microscopy suggests a close physical association between sulfur-oxidizing and sulfur-reducing genotypes, while radiotracer studies showed low, but detectable, SRR. Comparative 16S rRNA gene sequence analyses indicate the WP sulfur vent microbial mat community is similar, but distinct from other hydrothermal vent communities representing a range of biotopes and lithologic settings. These findings suggest a complete biological sulfur cycle is operating in the WP mat ecosystem mediated by diverse bacterial lineages, with some similarity with deep-sea hydrothermal vent communities.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/69572, title ="Members of the methanotrophic genus Methylomarinum inhabit inland mud pots", author = "Fradet, Danielle T. and Tavormina, Patricia L.", journal = "PeerJ", volume = "4", pages = "Art. No. e2116", month = "July", year = "2016", doi = "10.7717/peerj.2116", issn = "2167-8359", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160811-151334696", note = "© 2016 Fradet et al. Distributed under Creative Commons CC-BY 4.0. \n\nSubmitted 25 February 2016; Accepted 18 May 2016; Published 12 July 2016. \n\nWe thank Roland Hatzenpichler and Derek Smith for sampling the Salton Sea mudpots in 2012 and providing critical methodological, graphic, and scientific input.We thank Alyssa Boedigheimer for assistance in sample collection in January 2016. We thank Stephanie Connon for assistance with the 16S rRNA gene phylogeny. \n\nFunding for this work was provided by the Gordon and Betty Moore Foundation, in a grant to Victoria J. Orphan (grant no. GBMF3780). This research was additionally supported by a grant from the NASA Astrobiology Institute (Award # NNA13AA92A). This is NAI-Life Underground Publication Number 083. The funders had no role in\nstudy design, data collection and analysis, decision to publish, or preparation of the manuscript. \n\nGrant Disclosures: The following grant information was disclosed by the authors: Gordon and Betty Moore Foundation, in a grant to Victoria J. Orphan: GBMF3780. NASA Astrobiology Institute, in a grant to Victoria J. Orphan: NNA13AA92A. \n\nThe authors declare that they have no competing interests. \n\nAuthor Contributions: Danielle T. Fradet conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper. Patricia L. Tavormina conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. Victoria J. Orphan conceived and designed the experiments, wrote the paper, reviewed drafts of the paper, intellectual Support, encouragement, and funding.", revision_no = "22", abstract = "Proteobacteria capable of converting the greenhouse gas methane to biomass, energy, and carbon dioxide represent a small but important sink in global methane inventories. Currently, 23 genera of methane oxidizing (methanotrophic) proteobacteria have been described, although many are represented by only a single validly described species. Here we describe a new methanotrophic isolate that shares phenotypic characteristics and phylogenetic relatedness with the marine methanotroph Methylomarinum vadi. However, the new isolate derives from a terrestrial saline mud pot at the northern terminus of the Eastern Pacific Rise (EPR). This new cultivar expands our knowledge of the ecology of Methylomarinum, ultimately towards a fuller understanding of the role of this genus in global methane cycling.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/68706, title ="Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia", author = "Hatzenpichler, Roland and Connon, Stephanie A.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "113", number = "28", pages = "E4069-E4078", month = "July", year = "2016", doi = "10.1073/pnas.1603757113", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160628-092522305", note = "© 2016 National Academy of Sciences.\n\nEdited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved May 25, 2016 (received for review March 7, 2016). Published online before print June 28, 2016. \n\nWe thank Alexis Pasulka and Kat Dawson for shipboard sample processing, Silvan Scheller and Kat Dawson for measurements of AOM rates and methane concentrations, Hang Yu for performing cline assays, Connor Skennerton for help during sampling of sediment incubations, David Case for discussions on tag sequence analyses, and Shawn McGlynn for discussions on storage compounds. David Case, Kat Dawson, and Elizabeth Wilbanks are acknowledged for critical comments on the manuscript. We thank The Biological Imaging Facility of California Institute of Technology for access to their confocal microscope. We thank the crew and pilots of R/V Atlantis Cruises AT-15-68 and AT-18-10 to Hydrate Ridge (supported by National Science Foundation Grant OCE-0825791) and the R/V Western Flyer Cruise to Santa Monica Basin run by the Monterey Bay Aquarium Research Institute. R.H. was supported by an Erwin Schrödinger Postdoctoral Fellowship from the Austrian Science Fund (FWF) (project no. J 3162-B20), and a postdoctoral fellowship from the Center for Dark Energy Biosphere Investigations (C-DEBI). Funding for this project was provided by Gordon and Betty Moore Foundation Grant GBMF3780 (to V.J.O.), Department of Energy (DOE) Grant DE-PS02-09ER09-25 (to V.J.O.), and a JGI Director Discretionary Project Award (to R.H. and V.J.O.). The work conducted by the DOE Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract DE-AC02-05CH11231. This is C-DEBI Contribution 330. \n\nAuthor contributions: R.H. and V.J.O. designed research; R.H., S.A.C., and D.G. performed research; R.R.M., T.W., and V.J.O. contributed new reagents/analytic tools; R.H. and D.G. analyzed data; and R.H. and V.J.O. wrote the paper with input from all authors. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nData deposition: The sequences reported in this paper have been deposited in the National Center for Biotechnology Information GenBank database (accession nos. KT945170–KT945234 and KU564217–KU564240) and Sequence Read Archive (accession no. SRP066109). \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1603757113/-/DCSupplemental.", revision_no = "23", abstract = "To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/68526, title ="Stable Isotope Phenotyping via Cluster Analysis of NanoSIMS Data As a Method for Characterizing Distinct Microbial Ecophysiologies and Sulfur-Cycling in the Environment", author = "Dawson, Katherine S. and Scheller, Silvan", journal = "Frontiers in Microbiology", volume = "7", pages = "Art. No. 774", month = "May", year = "2016", doi = "10.3389/fmicb.2016.00774", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160620-104423818", note = "© 2016 Dawson, Scheller, Dillon and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.\n\nReceived: 11 January 2016; Accepted: 09 May 2016; Published: 26 May 2016. \n\nWe thank Y. Guan in Caltech's Center for Microanalysis for technical assistance with the NanoSIMS 50L measurements, P. Miranda for the introduction and assistance with field work at White Point, and A. Saxena-Green, A. Pasulka, and R. Hatzenpichler for helpful discussions. This research is funded by the Gordon and Betty Moore Foundation through Grant GBMF 3306, a grant from the National Science Foundation (EAR-1123391), as well as the NASA Astrobiology Institute (Award # NNA13AA92A; to VO). This is NAI-Life Underground Publication Number 80. \n\nAuthor Contributions: KD designed and carried out experiments and wrote the manuscript. VO designed experiments and wrote the manuscript. SS developed the labeled sulfur oxidation protocol and provided feedback on the manuscript. JD provided valuable contextual information for experimental design and feedback on the manuscript. \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.", revision_no = "14", abstract = "Stable isotope probing (SIP) is a valuable tool for gaining insights into ecophysiology and biogeochemical cycling of environmental microbial communities by tracking isotopically labeled compounds into cellular macromolecules as well as into byproducts of respiration. SIP, in conjunction with nanoscale secondary ion mass spectrometry (NanoSIMS), allows for the visualization of isotope incorporation at the single cell level. In this manner, both active cells within a diverse population as well as heterogeneity in metabolism within a homogeneous population can be observed. The ecophysiological implications of these single cell stable isotope measurements are often limited to the taxonomic resolution of paired fluorescence in situ hybridization (FISH) microscopy. Here we introduce a taxonomy-independent method using multi-isotope SIP and NanoSIMS for identifying and grouping phenotypically similar microbial cells by their chemical and isotopic fingerprint. This method was applied to SIP experiments in a sulfur-cycling biofilm collected from sulfidic intertidal vents amended with ^(13)C-acetate, ^(15)N-ammonium, and 33S-sulfate. Using a cluster analysis technique based on fuzzy c-means to group cells according to their isotope (^(13)C/^(12)C, ^(15)N/^(14)N, and ^(33)S/^(32)S) and elemental ratio (C/CN and S/CN) profiles, our analysis partitioned ~2200 cellular regions of interest (ROIs) into five distinct groups. These isotope phenotype groupings are reflective of the variation in labeled substrate uptake by cells in a multispecies metabolic network dominated by Gamma- and Deltaproteobacteria. Populations independently grouped by isotope phenotype were subsequently compared with paired FISH data, demonstrating a single coherent deltaproteobacterial cluster and multiple gammaproteobacterial groups, highlighting the distinct ecophysiologies of spatially-associated microbes within the sulfur-cycling biofilm from White Point Beach, CA.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/67556, title ="Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities", author = "Marlow, Jeffrey J. and Skennerton, Connor T.", journal = "Frontiers in Microbiology", volume = "7", pages = "Art. No. 563", month = "April", year = "2016", doi = "10.3389/fmicb.2016.00563", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160601-152032127", note = "Copyright © 2016 Marlow, Skennerton, Li, Chourey, Hettich, Pan and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 21 February 2016; Accepted: 04 April 2016; Published: 29 April 2016. \n\nTHIS ARTICLE IS PART OF THE RESEARCH TOPIC\nStudies on Life at the Energetic Edge – from Laboratory Experiments to Field-Based Investigations. \n\nAuthor Contributions:\n\nJM, CS, RH, and VO designed the study; JM performed incubation set-up, microbiological and geochemical analyses; CS constructed the metagenomic databases and developed the computational architecture for protein analysis; KC developed protein extraction procedures; ZL and CP performed proteomic SIP computational analysis; JM analyzed proteomic findings from an environmental microbiological perspective and wrote the manuscript with input from all authors.\n\nFunding:\n\nThis work was supported by the US Department of Energy, Office of Science, Office of Biological Environmental Research, under award numbers DE-SC0004949 and DE-SC0010574, and the Life Underground NASA Astrobiology Institute (NNA13AA92A) (to VO). JM was supported by a National Energy Technology Laboratory Methane Hydrate Research Fellowship funded by the National Research Council of the National Academies. The funders had no role in study design, data collection and interpretation, or the manuscript preparation and submission process.\n\nConflict of Interest Statement:\n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\nAcknowledgments:\n\nWe thank the Captains, Crew, Alvin group, Jason group, and Science party members from RV Atlantis legs AT-15-68, and AT-18-10. David Case and Stephanie Connon assisted with iTAG data collection and interpretation; Shawn McGlynn performed the nanoSIMS analysis, and Joshua Steele, Rachel Poretsky, and Shulei Sun collected and processed earlier iterations of the metagenomic database. We thank Peter Girguis and the ARPA-E REMOTE team for helpful comments during the preparation of this manuscript. This research used resources of the Oak Ridge Leadership Computing Facility. Oak Ridge National Laboratory is supported by the Office of Science of the U.S. Department of Energy.\n", revision_no = "41", abstract = "Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to ^(15)NH_4^+ and CH_4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were ^(15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis—including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)—were all observed to be actively synthesized (^(15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of ^(15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/66572, title ="Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments", author = "Trembath-Reichert, Elizabeth and Case, David H.", journal = "PeerJ", volume = "4", pages = "Art. No. e1913", month = "April", year = "2016", doi = "10.7717/peerj.1913", issn = "2167-8359", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160502-081644901", note = "© 2016 Trembath-Reichert et al. Distributed under Creative Commons CC-BY 4.0. \n\nSubmitted 12 January 2016; Accepted 18 March 2016; Published 18 April 2016. \n\nWe thank the crew of the R/V Atlantis and DSV JASON II, as well as Abigail Green-Saxena and Joshua Steele for assistance with optimization of the Magneto-FISH protocol and Stephanie Connon for assistance with sequencing. We also are grateful to Katherine Dawson, Emil Ruff, and two anonymous reviewers for providing comments on this manuscript. \n\nFunding: This research is funded by the Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0003940) and the Gordon and Betty Moore Foundation through Grant GBMF 3780 (both to VJO). ETR was supported by a NIH/NRSA Training Grant (5 T32 GM07616). Funding for DHC was provided in part by an NSF Graduate Research Fellowship. Samples were collected with funding from the National Science Foundation (BIO-OCE 0825791; to VJO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. \n\nAuthor Contributions: Elizabeth Trembath-Reichert conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. \n\nDavid H. Case conceived and designed the experiments, performed the experiments, reviewed drafts of the paper. \n\nVictoria J. Orphan contributed reagents/materials/analysis tools, designed the experiments, reviewed drafts of the paper. \n\nData availability: \n\nThe following information was supplied regarding data availability: \n\nGenBank sequences: \n\nAprA ( KT280505– KT280517), DsrA ( KT280518– KT280533), McrA ( KT280534– KT280581), Archaeal 16S rRNA ( KT280582– KT280632), Bacterial 16S rRNA ( KT280633– KT280909), SoxB ( KT280910– KT280928) \n\nSRA: accession SAMN03879962, BioSample: SAMN03879962, Sample name: PC47 (5133–5137) mixed slurry. \n\nThe authors declare there are no competing interests.", revision_no = "27", abstract = "Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60573, title ="Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses", author = "Dekas, Anne E. and Connon, Stephanie A.", journal = "ISME Journal", volume = "10", number = "3", pages = "678-692", month = "March", year = "2016", doi = "10.1038/ismej.2015.145", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150928-110118499", note = "© 2015 International Society for Microbial Ecology. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ \n\nReceived 31 January 2015; Revised 29 May 2015; Accepted 5 July 2015; Advance online publication 22 September 2015. \n\nWe thank the pilots, crew and science party of AT15-11 and AT15-59, especially Lisa Levin, Jake Bailey and Shana Goffredi; Abigail Green-Saxena and Joshua Steele for valuable discussions; Bethany Jenkins for consultation with RNA extraction protocol development; John Eiler and Yunbin Guan for assistance with NanoSIMS measurements. We additionally thank three anonymous reviewers for their careful critique of the manuscript. Funding was provided by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0003940 and DE-SC0004949 to VJO), the National Science Foundation (MCB-0348492 to VJO and a Graduate Research Fellowship to AED) and the Gordon and Betty Moore Foundation (GBMF no. 3780 to VJO, and via support for The Caltech Center for Microanalysis housing the CAMECA NanoSIMS 50 L). The writing of this manuscript by AED was partially performed while funded by a Lawrence Postdoctoral Fellowship under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. \n\nThe authors declare no conflict of interest.", revision_no = "40", abstract = "To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no ^(15)NH^+_4 assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N_2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. ^(15)N_2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, ^(15)N_2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced ^(15)N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/64490, title ="Artificial electron acceptors decouple archaeal methane oxidation from sulfate reduction", author = "Scheller, Silvan and Yu, Hang", journal = "Science", volume = "351", number = "6274", pages = "703-707", month = "February", year = "2016", doi = "10.1126/science.aad7154", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160216-090201597", note = "© 2016 American Association for the Advancement of Science. \n\nReceived 26 October 2015; accepted 20 January 2016. \n\nWe thank Y. Guan for assistance with the nanoSIMS, the Beckman\nResource Center (BRCem) for sectioning, M. Aoki for FISH analysis of ANME-2a and ANME-2c consortia, and S. Goffredi and C. Skennerton for editorial comments. We are grateful to P. Brewer from the Monterey Bay Aquarium Research Institute for providing the opportunity to participate in the 2013 research expedition and A. Pasulka and K. Dawson for their contributions in shipboard sample processing. This work was supported by the U.S. Department of Energy Biological and Environmental Research program (grants DE-SC0010574 and DE-SC0004940) and funding by the Gordon and Betty Moore Foundation through grants GBMF3306 and GBMF3780 (to V.J.O.). S.S. was supported in part by the Swiss National Science Foundation (grant no. PBEZP2_142903). All data are available in the supplementary materials. Archaeal 16S rRNA, mcrA genes, and bacterial 16S rRNA genes were deposited with the National Center for Biotechnology Information under accession numbers KU324182 to KU324260, KU324346 to KU324428, and KU324261 to KU324345, respectively. S.S., H.Y., and V.J.O. devised the study, and S.S., H.Y., G.L.C., and S.M. conducted the experiments and analyses. S.S. and V.J.O. wrote the manuscript, with contributions from all authors to data analysis, figure generation, and the final manuscript.", revision_no = "15", abstract = "The oxidation of methane with sulfate is an important microbial metabolism in the global carbon cycle. In marine methane seeps, this process is mediated by consortia of anaerobic methanotrophic archaea (ANME) that live in syntrophy with sulfate-reducing bacteria (SRB). The underlying interdependencies within this uncultured symbiotic partnership are poorly understood. We used a combination of rate measurements and single-cell stable isotope probing to demonstrate that ANME in deep-sea sediments can be catabolically and anabolically decoupled from their syntrophic SRB partners using soluble artificial oxidants. The ANME still sustain high rates of methane oxidation in the absence of sulfate as the terminal oxidant, lending support to the hypothesis that interspecies extracellular electron transfer is the syntrophic mechanism for the anaerobic oxidation of methane.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63308, title ="Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum", author = "Kopf, Sebastian H. and Sessions, Alex L.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "113", number = "2", pages = "E110-E116", month = "January", year = "2016", doi = "10.1073/pnas.1512057112", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160104-065151473", note = "© 2015 National Academy of Sciences. \n\nPublished online before print December 29, 2015. \n\nWe are grateful to Shawn McGlynn, Ryan Hunter, Abigail Green-Saxena, Yunbin Guan, Alejandro LaRiviere, Ian Booth, Nathan Dalleska, Fadi Asfour, Douglas Li, Kyle McCallin, Sally Ward, Thomas Keens, and patients of the Pulmonary CF Clinic at CHLA for supporting this study. We thank the editor and reviewers for constructive criticism that improved the manuscript. This research was supported by grants from the NIH (Grant 5R01HL117328-03) and the Howard Hughes Medical Institute (HHMI) (to D.K.N.). S.H.K. was an HHMI International Research Scholar, and D.K.N. is an HHMI Investigator. \n\nAuthor contributions: S.H.K., A.L.S., and D.K.N. designed research; S.H.K., E.S.C., and Y.H. performed research; S.H.K., A.L.S., E.S.C., C.R., L.V., V.J.O., R.K., and D.K.N. analyzed data; S.H.K. and D.K.N. wrote the paper; and C.R., L.V., and R.K. provided clinical advice and assistance. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1512057112/-/DCSupplemental.", revision_no = "21", abstract = "Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63496, title ="Genomic Reconstruction of an Uncultured Hydrothermal Vent Gammaproteobacterial Methanotroph (Family Methylothermaceae) Indicates Multiple Adaptations to Oxygen Limitation", author = "Skennerton, Connor T. and Ward, Lewis M.", journal = "Frontiers in Microbiology", volume = "6", pages = "Art. No. 1425", month = "December", year = "2015", doi = "10.3389/fmicb.2015.01425", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160108-103050704", note = "© 2015 Skennerton, Ward, Michel, Metcalfe, Valiente, Mullin, Chan, Gradinaru and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 02 October 2015; Accepted: 30 November 2015; Published: 23 December 2015. \n\nThe metagenomic analysis and annotation for B42 was done in part by the students of the GeBI 246 Molecular Geobiology course at Caltech. We thank Ben Harrison for XRD analysis on sample #2044C and Patty Tavormina for assistance during the course and critical reading of this manuscript. We also thank Chief Scientist Robert Vrijenhoek (Monterey Bay Aquarium Research Institute) for providing the opportunity to collect samples during the 2005 tuim06mv cruise. We thank Igor Antoshechkin of the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech for his services and input during sequencing of the genomic library. \n\nThis research was supported in part by a grant from the NASA Astrobiology Institute (Award # NNA13AA92A) and the Gordon and Betty Moore Foundation Marine Microbiology Initiative (GBMF3780) to VO. This is NAI-Life Underground Publication Number 070. Part of this work was supported by grants to VG: NIH 1R21MH103824-01; the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative and by the Beckman Institute for Optogenetics and CLARITY. KC is supported by the NIH Predoctoral Training in Biology and Chemistry grant (2T32GM007616-36). LW was supported by an NSF Graduate Research Fellowship. Sample collection from the Tu’i Malila vent field was funded by the National Science Foundation (NSF), grant number NSF OCE-0241613, awarded to Robert Vrijenhoek. \n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.", revision_no = "13", abstract = "Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at ∼80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x–12x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other Proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63334, title ="Methane Seep Carbonates Host Distinct, Diverse, and Dynamic Microbial Assemblages", author = "Case, David H. and Pasulka, Alexis L.", journal = "mBio", volume = "6", number = "6", pages = "Art. No. e01348-15", month = "December", year = "2015", doi = "10.1128/mBio.01348-15", issn = "2150-7511", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160104-131039931", note = "© 2015 Case et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. \n\nReceived 12 August 2015; Accepted 9 November 2015; Published 22 December 2015. \n\nWe thank Connor Skennerton and Elizabeth Trembath-Reichert for helpful discussions on data processing. We acknowledge Stephanie Connon, Patricia Tavormina, Josh Steele, Heather Grotzinger, and shipboard teams from the Orphan, Levin, Rathburn, and Rouse labs for assistance with sample collection and processing. We are indebted to the captain, crew, and pilots of the DSV Alvin and ROV Jason II from cruises AT15-11, AT15-44, AT15-68, who made this work possible. We thank the two anonymous reviewers, whose comments strengthened the study. \n\nThis research was supported by a grant to V.O. from the NASA Astrobiology Institute (award NNA13AA92A). This work was also support by a National Science Foundation (NSF) grant (OCE-0825791) and a Gordon and Betty Moore Foundation Marine Microbiology Initiative grant (3780) to V.O. D.C. was supported by an NSF Graduate Research Fellowship. Levin lab research was supported by NSF grants OCE-0826254 and OCE-0939557.", revision_no = "27", abstract = "Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63309, title ="A unified initiative to harness Earth's microbiomes", author = "Alivisatos, A. P. and Blaser, M. J.", journal = "Science", volume = "350", number = "6260", pages = "507-508", month = "October", year = "2015", doi = "10.1126/science.aac8480 ", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160104-070726621", note = "© 2015 American Association for the Advancement of Science. \n\nPublished Online October 28 2015.", revision_no = "17", abstract = "Despite their centrality to life on Earth, we know little about how microbes (1) interact with each other, their hosts, or their environment. Although DNA sequencing technologies have enabled a new view of the ubiquity and diversity of microorganisms, this has mainly yielded snapshots that shed limited light on microbial functions or community dynamics. Given that nearly every habitat and organism hosts a diverse constellation of microorganisms—its “microbiome”—such knowledge could transform our understanding of the world and launch innovations in agriculture, energy, health, the environment, and more (see the photo). We propose an interdisciplinary Unified Microbiome Initiative (UMI) to discover and advance tools to understand and harness the capabilities of Earth's microbial ecosystems. The impacts of oceans and soil microbes on atmospheric CO_2 are critical for understanding climate change (2). By manipulating interactions at the root-soil-microbe interface, we may reduce agricultural pesticide, fertilizer, and water use enrich marginal land and rehabilitate degraded soils. Microbes can degrade plant cell walls (for biofuels), and synthesize myriad small molecules for new bioproducts, including antibiotics (3). Restoring normal human microbial ecosystems can save lives [e.g., fecal microbiome transplantation for Clostridium difficile infections (4)]. Rational management of microbial communities in and around us has implications for asthma, diabetes, obesity, infectious diseases, psychiatric illnesses, and other afflictions (5, 6). The human microbiome is a target and a source for new drugs (7) and an essential tool for precision medicine (8). ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/61783, title ="Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics", author = "Evans, Paul N. and Parks, Donovan H.", journal = "Science", volume = "350", number = "6259", pages = "434-438", month = "October", year = "2015", doi = "10.1126/science.aac7745", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151103-081330300", note = "© 2015 American Association for the Advancement of Science. \n\nReceived 11 June 2015; accepted 14 September 2015. \n\nWe thank E. Gagen and P. Hugenholtz for valuable comments and suggestions; K. Baublys, SGS-Leeder, and Australian Laboratory Services staff for sample collection; and M. Butler and S. Low for library preparation and sequencing. This study was supported by the Australian Research Council (ARC) Linkage Project (grant LP100200730) and the U.S. Department of Energy’s Office of Biological Environmental Research (award no. DE-SC0010574). D.H.P. is supported by the Natural Sciences and Engineering Research Council of Canada. S.J.R. is supported by an Australian Postgraduate Award Industry scholarship. G.W.T. is supported by an ARC Queen Elizabeth II Fellowship (grant DP1093175). The authors declare no conflicts of interest. Our Whole Genome Shotgun projects have been deposited in the DNA DataBank of Japan, the European Molecular Biology Laboratory repository, and NIH’s GenBank under the accession numbers LIHJ00000000 (BA1) and LIHK00000000 (BA2). The versions described in this paper are LIHJ01000000 (BA1) and LIHK01000000 (BA2). Non-euryarchaeotal Surat Basin mcrA sequences have been deposited under the accession numbers KT387805 to KT387832, and unprocessed reads have been deposited under the accession number SRX1122679.", revision_no = "16", abstract = "Methanogenic and methanotrophic archaea play important roles in the global flux of methane. Culture-independent approaches are providing deeper insight into the diversity and evolution of methane-metabolizing microorganisms, but, until now, no compelling evidence has existed for methane metabolism in archaea outside the phylum Euryarchaeota. We performed metagenomic sequencing of a deep aquifer, recovering two near-complete genomes belonging to the archaeal phylum Bathyarchaeota (formerly known as the Miscellaneous Crenarchaeotal Group). These genomes contain divergent homologs of the genes necessary for methane metabolism, including those that encode the methyl–coenzyme M reductase (MCR) complex. Additional non-euryarchaeotal MCR-encoding genes identified in a range of environments suggest that unrecognized archaeal lineages may also contribute to global methane cycling. These findings indicate that methane metabolism arose before the last common ancestor of the Euryarchaeota and Bathyarchaeota.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/59070, title ="Single cell activity reveals direct electron transfer in methanotrophic consortia", author = "McGlynn, Shawn E. and Chadwick, Grayson L.", journal = "Nature", volume = "526", number = "7574", pages = "531-535", month = "October", year = "2015", doi = "10.1038/nature15512", issn = "0028-0836", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150729-141906854", note = "© 2015 Macmillan Publishers Limited.\n\nReceived 06 March 2015 Accepted 10 August 2015 Published online 16 September 2015.\n\nWe are grateful for the use of the facilities of the Beckman Resource Center for Transmission Electron Microscopy at Caltech (BRCem) and advice provided by A. McDowall, our collaborators T. Deerinck and M. Ellisman from the National Center for Microscopy and Imaging Research (NCMIR), C. Miele (UGA) and M. El-Naggar at USC. Metagenomic binning of ANME-2b was conducted by C. Skennerton and M. Haroon in collaboration with G. Tyson and M. Imelfort (University of Queensland). This work was supported by the US Department of Energy, Office of Science, Office of Biological Environmental Research under award numbers (DE-SC0004949 and DE-SC0010574) and a grant from the Gordon and Betty Moore foundation Marine Microbiology Initiative (grant number 3780). V.J.O. is supported by a DOE-BER early career grant (DE-SC0003940). S.E.M. acknowledges support from an Agouron Geobiology Option post-doctoral fellowship in the Division of Geological and Planetary Sciences at Caltech and C.P.K. was supported by the NASA Astrobiology Institute (award number NNA13AA92A). This is NAI-Life Underground Publication 049.\n\nAuthor Contributions:\nV.J.O., S.M. and G.L.C. devised the study, S.M. and G.L.C.\nconducted the experiments and analyses and C.P.K. conducted the diffusion and\nelectrical conductivity modelling, and all authors contributed to data interpretation and\nwriting of the manuscript.", revision_no = "48", abstract = "Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/62084, title ="A novel sister clade to the enterobacteria microviruses (family Microviridae) identified in methane seep sediments", author = "Bryson, Samuel Joseph and Thurber, Andrew R.", journal = "Environmental Microbiology", volume = "17", number = "10", pages = "3708-3721", month = "October", year = "2015", doi = "10.1111/1462-2920.12758", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151112-154353069", note = "© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd. \n\nReceived 10 June, 2014; revised 15 December, 2014; accepted 15 December, 2014. \n\nThe Gordon and Betty Moore Foundation (GBMF) provided funding to RVT for the generation of these metagenomes through the viral sequencing program. Funding also was provided by a National Science Foundation Grant #BIO-OCE 0825791 to VJO and #BIO-OCE 096037 to RVT, in addition to #OPP-1103428 that supported ART during the writing of this manuscript. Special thanks to Drs Jerome Payet and Jesse Zaneveld for helpful discussions of viral tree generation and analysis. The Oregon State University Center for Genome Research and Biocomputing (CGRB) provided computational infrastructure for genome assembly. The authors of this manuscript declare that they have no conflicts of interest in regard to the work presented here.", revision_no = "14", abstract = "Methane seep microbial communities perform a key ecosystem service by consuming the greenhouse gas methane prior to its release into the hydrosphere, minimizing the impact of marine methane sources on our climate. Although previous studies have examined the ecology and biochemistry of these communities, none has examined viral assemblages associated with these habitats. We employed virus particle purification, genome amplification, pyrosequencing and gene/genome reconstruction and annotation on two metagenomic libraries, one prepared for ssDNA and the other for all DNA, to identify the viral community in a methane seep. Similarity analysis of these libraries (raw and assembled) revealed a community dominated by phages, with a significant proportion of similarities to the Microviridae family of ssDNA phages. We define these viruses as the Eel River Basin Microviridae (ERBM). Assembly and comparison of 21 ERBM closed circular genomes identified five as members of a novel sister clade to the Microvirus genus of Enterobacteria phages. Comparisons among other metagenomes and these Microviridae major-capsid sequences indicated that this clade of phages is currently unique to the Eel River Basin sediments. Given this ERBM clade's relationship to the Microviridae genus Microvirus, we define this sister clade as the candidate genus Pequeñovirus.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/61456, title ="Comparison of Archaeal and Bacterial Diversity in Methane Seep Carbonate Nodules and Host Sediments, Eel River Basin and Hydrate Ridge, USA", author = "Mason, Olivia U. and Case, David H.", journal = "Microbial Ecology", volume = "70", number = "3", pages = "766-784", month = "October", year = "2015", doi = "10.1007/s00248-015-0615-6", issn = "0095-3628", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151023-073758726", note = "© 2015 Springer Science+Business Media New York. \n\nReceived: 29 November 2014; Accepted: 10 April 2015; Published online: 7 May 2015. \n\nOlivia U. Mason and David H. Case share co-first authorship. \n\nVO conceived of the study and collected the samples at sea. OM processed the samples and optimized DNA extraction, as well as TRFLP and clone library analyses. DC performed iTAG processing and analyses, beta diversity analyses, and was the coordinating author of the manuscript. TN provided XRD data, RL performed the isotopic composition analyses, JB provided thin section images, and RT performed the pore water geochemical measurements. DC, OM, and VO principally contributed to writing the manuscript. Three anonymous reviews provided constructive suggestions to improve the manuscript. Elizabeth Trembath-Reichert, Stephanie Connon, and Jeff Marlow helped with customization of the Silva115_NR99 database. Alexis Pasulka provided helpful discussion regarding ordination and statistical probing of microbial communities. Josh Steele also provided discussion on ecological statistics and aided with bench-top lab work. Benjamin Harrison helped with TRFLP data interpretation. Jeff Marlow provided useful feedback on the manuscript. The crew of the R/V Atlantis cruise AT-15-11, as well as the pilots of DSV Alvin dives AD4249 and 4256, aided in sample recovery at sea. Funding for this work was provided by a National Science Foundation grant (BIO-OCE 0825791) to VO and an early career grant by the United States Department of Energy, Office of Biological and Environmental Research (DE-SC0003940) to VO. This research was also supported by a grant from the NASA Astrobiology Institute (Award #NNA13AA92A) to VO. This is NAI-Life Underground Publication 009. DC was funded by a National Science Foundation Graduate Research Fellowship.", revision_no = "22", abstract = "Anaerobic oxidation of methane (AOM) impacts carbon cycling by acting as a methane sink and by sequestering inorganic carbon via AOM-induced carbonate precipitation. These precipitates commonly take the form of carbonate nodules that form within methane seep sediments. The timing and sequence of nodule formation within methane seep sediments are not well understood. Further, the microbial diversity associated with sediment-hosted nodules has not been well characterized and the degree to which nodules reflect the microbial assemblage in surrounding sediments is unknown. Here, we conducted a comparative study of microbial assemblages in methane-derived authigenic carbonate nodules and their host sediments using molecular, mineralogical, and geochemical methods. Analysis of 16S rRNA gene diversity from paired carbonate nodules and sediments revealed that both sample types contained methanotrophic archaea (ANME-1 and ANME-2) and syntrophic sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae), as well as other microbial community members. The combination of geochemical and molecular data from Eel River Basin and Hydrate Ridge suggested that some nodules formed in situ and captured the local sediment-hosted microbial community, while other nodules may have been translocated or may represent a record of conditions prior to the contemporary environment. Taken together, this comparative analysis offers clues to the formation regimes and mechanisms of sediment-hosted carbonate nodules.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/57229, title ="Metabolic associations with archaea drive shifts in hydrogen isotope fractionation in sulfate-reducing bacterial lipids in cocultures and methane seeps", author = "Dawson, K. S. and Osburn, M. R.", journal = "Geobiology", volume = "13", number = "5", pages = "462-477", month = "September", year = "2015", doi = "10.1111/gbi.12140", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150505-105030332", note = "© 2015 John Wiley & Sons Ltd. \n\nReceived 4 November 2014; accepted 30 March 2015. Article first published online: 28 Apr 2015. \n\nWe thank the shipboard, scientific crew and pilots of the R/V Atlantis and DSV Jason. Stephanie Connon, Fenfang Wu, and Lichun Zhang provided technical assistance. Laurence Bird and Katherine Freeman provided the ^(13)C_(lipid) measurements. We would also like to thank four anonymous reviewers for their comments toward improving this manuscript. This study was supported by a grant from the Penn State Astrobiology Research Centre (through the NASA Astrobiology Institute (NNA09DA76A)) to VJO, a PSARC post-doctoral fellowship to KSD and an NSF Graduate Fellowship to MRO. Additional support was provided by the Gordon and Betty Moore Foundation Marine Microbiology Initiative (grant #3780) and an early career grant from the U.S. Department of Energy, Office of Biological and Environmental Research to VJO.", revision_no = "23", abstract = "Correlation between hydrogen isotope fractionation in fatty acids and carbon metabolism in pure cultures of bacteria indicates the potential of biomarker D/H analysis as a tool for diagnosing carbon substrate usage in environmental samples. However, most environments, in particular anaerobic habitats, are built from metabolic networks of micro-organisms rather than a single organism. The effect of these networks on D/H of lipids has not been explored and may complicate the interpretation of these analyses. Syntrophy represents an extreme example of metabolic interdependence. Here, we analyzed the effect of metabolic interactions on the D/H biosignatures of sulfate-reducing bacteria (SRB) using both laboratory maintained cocultures of the methanogen Methanosarcina acetivorans and the SRB Desulfococcus multivorans in addition to environmental samples harboring uncultured syntrophic consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing Deltaproteobacteria (SRB) recovered from deep-sea methane seeps. Consistent with previously reported trends, we observed a ~80‰ range in hydrogen isotope fractionation (ε_(lipid–water)) for D. multivorans grown under different carbon assimilation conditions, with more D-enriched values associated with heterotrophic growth. In contrast, for cocultures of D. multivorans with M. acetivorans, we observed a reduced range of ε_(lipid–water) values (~36‰) across substrates with shifts of up to 61‰ compared to monocultures. Sediment cores from methane seep settings in Hydrate Ridge (offshore Oregon, USA) showed similar D-enrichment in diagnostic SRB fatty acids coinciding with peaks in ANME/SRB consortia concentration suggesting that metabolic associations are connected to the observed shifts in ε_(lipid–water) values.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/56756, title ="Autoendoliths: a distinct type of rock-hosted microbial life", author = "Marlow, J. and Peckmann, J.", journal = "Geobiology", volume = "13", number = "4", pages = "303-307", month = "July", year = "2015", doi = "10.1111/gbi.12131", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150420-090013681", note = "© 2015 John Wiley & Sons Ltd.\n\nReceived 26 December 2014; accepted 21 February 2015.\nArticle first published online: 16 Apr. 2015.\n\nThis research was supported by a grant from the NASA Astrobiology Institute (Award # NNA13AA92A, to V.J.O.) and is NAI Life Underground Publication 008. J.J.M. was supported by a National Energy Technology Laboratory Methane Hydrate Research Fellowship funded by the National Research Council of the National Academies. We thank the three anonymous referees for their comments, which helped improve the manuscript.", revision_no = "16", abstract = "The continued exploration of Earth’s biological potential\nhas revealed a range of unexpected microbial habitats. The\ndiscovery of organisms inhabiting rock interiors, known as\nendoliths, was one such revelation that has altered our perspective\nof habitability, bioenergetics, and the relationship\nbetween biology and geology (Walker & Pace, 2007).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/55062, title ="Heavy water and ¹⁵N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats", author = "Kopf, Sebastian H. and McGlynn, Shawn E.", journal = "Environmental Microbiology", volume = "17", number = "7", pages = "2542-2556", month = "July", year = "2015", doi = "10.1111/1462-2920.12752", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150220-131055817", note = "© 2015 John Wiley & Sons, Inc.\n\nAccepted manuscript online: 5 FEB 2015; Manuscript Accepted: 12 DEC 2014.\n\nWe thank Nathan Dalleska and the Caltech Environmental Analysis Center for instrumentation that benefited this project, Grayson Chadwick and Kat Dawson for helpful discussions, and members of the Newman and Orphan labs, as well as John Cliff and two anonymous reviewers for constructive criticism that improved the manuscript. This work was supported by grants from the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health (Grant No. 5R01HL117328-03, to D.K.N.), and from the Gordon and Betty Moore Foundation (Grant No. GBMF3780 to V.J.O.). D.K.N. is an HHMI Investigator. S.H.K. is an HHMI International Student Research Fellow.", revision_no = "37", abstract = "To measure single cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labeling of microbial populations with heavy water (a passive tracer) and ¹⁵N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (~0.1 atom % ²H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (~6 hours, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium vs. amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/79111, title ="Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)", author = "Hatzenpichler, Roland and Orphan, Victoria J.", pages = "145-157", month = "April", year = "2015", doi = "10.1007/8623_2015_61", issn = "1949-2448", isbn = "978-3-662-49129-4", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170714-110726557", note = "© 2015 Springer-Verlag Berlin Heidelberg. \n\nFirst Online: 04 April 2015.", revision_no = "10", abstract = "Bioorthogonal noncanonical amino acid tagging (BONCAT) is a recently developed method for studying microbial in situ activity. This technique is based on the in vivo incorporation of artificial amino acids that carry modifiable chemical tags into newly synthesized proteins. BONCAT has been demonstrated to be effective in labeling the proteomes of a wide range of taxonomically and physiologically distinct Archaea and bacteria without resulting in preferential synthesis or degradation of proteins. After chemical fixation of cells, surrogate-containing proteins can be detected by whole-cell fluorescence staining using azide-alkyne click chemistry. When used in conjunction with rRNA-targeted fluorescence in situ hybridization (FISH), BONCAT allows the simultaneous taxonomic identification of a microbial cell and its translational activity. Rather than studying the bulk proteome, BONCAT is able to specifically target proteins that have been expressed in reaction to an experimental condition. BONCAT-FISH thus provides researchers with a selective, sensitive, fast, and inexpensive fluorescence microscopy technique for studying microbial in situ activity on an individual cell level.\n\nThis protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, l-azidohomoalanine (AHA) and l-homopropargylglycine (HPG), which are both surrogates of l-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT studies and how they can be overcome.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/51567, title ="Methyloprofundus sedimenti gen. nov., sp. nov., an obligate methanotroph from ocean sediment belonging to the ‘deep sea-1’ clade of marine methanotrophs", author = "Tavormina, Patricia L. and Hatzenpichler, Roland", journal = "International Journal of Systematic and Evolutionary Microbiology", volume = "65", number = "1", pages = "251-259", month = "January", year = "2015", doi = "10.1099/ijs.0.062927-0", issn = "1466-5026", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141111-104738855", note = "© 2015 IUMS. \n\nWe would like to thank Shana Goffredi, Shannon Johnson, and the captain and crew of the Western Flyer (Monterey Bay Aquarium Research Institute) for source materials for this study. We also thank Dr. Gary Martin (Occidental College) for invaluable assistance with transmission electron microscopy, and two anonymous reviewers for critical review of the work contained herein. Funding for this work was provided by grants from NASA ASTEP (NNG06GB34G, VJO) the National Science Foundation OCE (MCB-0348492, VJO), and an Erwin Schrodinger Postdoctoral Fellowship of the Austrian Science Fund (FWF), J 3162-B20 (RH). ", revision_no = "23", abstract = "We report the isolation and growth characteristics of a gammaproteobacterial methane-oxidizing bacterium (Methylococcaceae strain WF1, \"whale fall 1\" that shares 98% 16S rRNA identity with uncultivated free-living methanotrophs and the methanotrophic endosymbionts of deep sea mussels, 94.6% 16S rRNA identity with Methylobacter species, and 93.6% 16S rRNA identity with Methylomonas and Methylosarcina species. Strain WF1 represents the first cultivar from the 'Deep Sea 1' clade of marine methanotrophs, which includes members that participate in methane oxidation in sediments and the water column in addition to mussel endosymbionts. WF1 cells were elongated cocci approximately 1.5 µm in diameter, and occurred singly, in pairs and clumps. The cell wall was Gram negative, and stacked intracytoplasmic membranes and storage granules were evident. The genomic GC content of WF1 was 40.5%, significantly lower than currently described cultivars, and the major fatty acids were 16:0, 16:1 ω9c, 16:1 ω9t, 16:1 ω8c and 16:2 ω9, 14. Growth occurred in liquid media at an optimal temperature of 23oC, and was dependent on the presence of methane or methanol. Atmospheric nitrogen could serve as the sole nitrogen source for WF1, a capacity that had not been functionally demonstrated in members of Methylobacter. On the basis of unique morphological, physiological, and phylogenetic properties this strain represents the type species within a new genus, and we propose the name Methyloprofundus sedimenti (type strain WF1 = BCCM LMG 28393 = ATCC BAA-2619).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53247, title ="Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh", author = "Wilbanks, Elizabeth G. and Jaekel, Ulrike", journal = "Environmental Microbiology", volume = "16", number = "11", pages = "3398-3415", month = "November", year = "2014", doi = "10.1111/1462-2920.12388", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150107-070238329", note = "© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.\nThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received 7 November, 2013; revised 30 December, 2013; accepted 5 January, 2014. \n\nWe would like to acknowledge the excellent work of many\nstudents, teaching fellows, and instructors who contributed\nto this research over the many years of the Microbial Diversity course at the Marine Biological Laboratory; in particular we thank Cristina Moraru and Rebekah J. Ward for assistance in developing embedding and CARD-FISH protocols,\nJarrod J. Scott for providing 16S rRNA gene sequence data\nfrom 2007 and Alexander P. Petroff for helpful discussions.\nMany thanks to Douglas C. Nelson and Susan E. Alford for\ntheir work with the radiocarbon fixation assay, Abigail Green-Saxena and Yunbin Guan for assistance with nanoSIMS\ndata acquisition, Fotios C. Kafantaris for microvoltammetry\nwork, Jennifer Houghton Julie Huber for use of her lab and Claire Beaudoin for sulfide microsensors measurements, and to Nanelle R. Barash and Annette R. Rowe for their critical reading of the manuscript. This work was supported by NSF grants DEB-1310168, EAR-1124389, and EAR-1123391, a grant from the Gordon and Betty Moore Foundation (#3306), and awards to Elizabeth G. Wilbanks from the NSF Graduate Research Fellowship, UC Davis Dissertation Year Fellowship, P.E.O. Scholar Award, and the NAI/APS Lewis and Clark Fund in Astrobiology. This research was performed by participants in the MBL Microbial Diversity course and was supported in part by the Howard Hughes Medical Foundation, the Gordon and Betty Moore Foundation (#2493), the NSF (DEB-0917499), the US DOE (DEFG02-10ER13361), and the NASA Astrobiology Institute.", revision_no = "19", abstract = "Microbial metabolism is the engine that drives global biogeochemical cycles, yet many key transformations are carried out by microbial consortia over short spatiotemporal scales that elude detection by traditional analytical approaches. We investigate syntrophic sulfur cycling in the ‘pink berry’ consortia of the Sippewissett Salt Marsh through an integrative study at the microbial scale. The pink berries are macroscopic, photosynthetic microbial aggregates composed primarily of two closely associated species: sulfide-oxidizing purple sulfur bacteria (PB-PSB1) and sulfate-reducing bacteria (PB-SRB1). Using metagenomic sequencing and ^(34)S-enriched sulfate stable isotope probing coupled with nanoSIMS, we demonstrate interspecies transfer of reduced sulfur metabolites from PB-SRB1 to PB-PSB1. The pink berries catalyse net sulfide oxidation and maintain internal sulfide concentrations of 0–500\u2009μm. Sulfide within the berries, captured on silver wires and analysed using secondary ion mass spectrometer, increased in abundance towards the berry interior, while δ^(34)S-sulfide decreased from 6‰ to −31‰ from the exterior to interior of the berry. These values correspond to sulfate–sulfide isotopic fractionations (15–53‰) consistent with either sulfate reduction or a mixture of reductive and oxidative metabolisms. Together this combined metagenomic and high-resolution isotopic analysis demonstrates active sulfur cycling at the microscale within well-structured macroscopic consortia consisting of sulfide-oxidizing anoxygenic phototrophs and sulfate-reducing bacteria.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/52204, title ="Spatial variability in photosynthetic and heterotrophic activity drives localized δ^(13)C_(org) fluctuations and carbonate precipitation in hypersaline microbial mats", author = "Houghton, J. and Fike, D.", journal = "Geobiology", volume = "12", number = "6", pages = "557-574", month = "November", year = "2014", doi = "10.1111/gbi.12113", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141201-092909674", note = "© 2014 John Wiley & Sons Ltd.\n\nReceived 18 March 2014; accepted 30 August 2014.\nArticle first published online: 13 OCT 2014.\n\nWe would like to thank NASA AMES NAI team, particularly Mike Kubo, Linda Jahnke, and Abigail Green-Saxena for discussion and assistance in the laboratory and field, and Exportadora del Sal, S. A. for access to the field site. Funding for this work was supported by NSF EAR-1124389 as well as a Packard Fellowship and a Hansewissenschaftskolleg Fellowship to D.A.F., NSF EAR-1123391 to V.J.O, and NSF EAR-1304352 and EAR-1261423 to G.D.", revision_no = "14", abstract = "Modern laminated photosynthetic microbial mats are ideal environments to study how microbial activity creates and modifies carbon and sulfur isotopic signatures prior to lithification. Laminated microbial mats from a hypersaline lagoon (Guerrero Negro, Baja California, Mexico) maintained in a flume in a greenhouse at NASA Ames Research Center were sampled for δ^(13)C of organic material and carbonate to assess the impact of carbon fixation (e.g., photosynthesis) and decomposition (e.g., bacterial respiration) on δ^(13)C signatures. In the photic zone, the δ^(13)C_(org) signature records a complex relationship between the activities of cyanobacteria under variable conditions of CO_2 limitation with a significant contribution from green sulfur bacteria using the reductive TCA cycle for carbon fixation. Carbonate is present in some layers of the mat, associated with high concentrations of bacteriochlorophyll e (characteristic of green sulfur bacteria) and exhibits δ^(13)C signatures similar to DIC in the overlying water column (−2.0‰), with small but variable decreases consistent with localized heterotrophic activity from sulfate-reducing bacteria (SRB). Model results indicate respiration rates in the upper 12 mm of the mat alter in situ pH and HCO_3^-\nconcentrations to create both phototrophic CO_2 limitation and carbonate supersaturation, leading to local precipitation of carbonate minerals. The measured activity of SRB with depth suggests they variably contribute to decomposition in the mat dependent on organic substrate concentrations. Millimeter-scale variability in the δ^(13)C_(org) signature beneath the photic zone in the mat is a result of shifting dominance between cyanobacteria and green sulfur bacteria with the aggregate signature overprinted by heterotrophic reworking by SRB and methanogens. These observations highlight the impact of sedimentary microbial processes on δ^(13)C_(org) signatures; these processes need to be considered when attempting to relate observed isotopic signatures in ancient sedimentary strata to conditions in the overlying water column at the time of deposition and associated inferences about carbon cycling.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/49939, title ="Iron oxides stimulate sulfate-driven anaerobic methane oxidation in seeps", author = "Sivan, Orit and Antler, Gilad", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "111", number = "40", pages = "E4139-E4147", month = "October", year = "2014", doi = "10.1073/pnas.1412269111 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140923-111251974", note = "Copyright © 2014 National Academy of Sciences. \n\nEdited by Mark H. Thiemens, University of California, San Diego, La Jolla, CA, and approved August 28, 2014 (received for review June 30, 2014) Published online before print September 22, 2014, doi: 10.1073/pnas.1412269111.\n\nWe thank Stephanie Connon for the help in the laboratory, George Rossman for the hematite powder, and Jiwchar Ganor and his laboratory members for the help with the sulfate measurements. Thanks to Matthias Kellermann and Itay Bar-Or for the help and fruitful discussions. This research was supported by Israel Science Foundation Grant 643/12 (to O.S.), Department of Energy Biological Environmental Research Grant DE-SC0004949, and Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant 3306 (to V.J.O.). Funding for sample collection was provided by National Science Foundation Biological Oceanography Grant 0825791.\n\nAuthor contributions: O.S. and V.J.O. designed research; O.S., G.A., and J.J.M. performed research; O.S., G.A., A.V.T., J.J.M., and V.J.O. analyzed data; and O.S., G.A., A.V.T., J.J.M., and V.J.O. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412269111/-/DCSupplemental.", revision_no = "23", abstract = "Seep sediments are dominated by intensive microbial sulfate reduction coupled to the anaerobic oxidation of methane (AOM). Through geochemical measurements of incubation experiments with methane seep sediments collected from Hydrate Ridge, we provide insight into the role of iron oxides in sulfate-driven AOM. Seep sediments incubated with ^(13)C-labeled methane showed co-occurring sulfate reduction, AOM, and methanogenesis. The isotope fractionation factors for sulfur and oxygen isotopes in sulfate were about 40‰ and 22‰, respectively, reinforcing the difference between microbial sulfate reduction in methane seeps versus other sedimentary environments (for example, sulfur isotope fractionation above 60‰ in sulfate reduction coupled to organic carbon oxidation or in diffusive sedimentary sulfate–methane transition zone). The addition of hematite to these microcosm experiments resulted in significant microbial iron reduction as well as enhancing sulfate-driven AOM. The magnitude of the isotope fractionation of sulfur and oxygen isotopes in sulfate from these incubations was lowered by about 50%, indicating the involvement of iron oxides during sulfate reduction in methane seeps. The similar relative change between the oxygen versus sulfur isotopes of sulfate in all experiments (with and without hematite addition) suggests that oxidized forms of iron, naturally present in the sediment incubations, were involved in sulfate reduction, with hematite addition increasing the sulfate recycling or the activity of sulfur-cycling microorganisms by about 40%. These results highlight a role for natural iron oxides during bacterial sulfate reduction in methane seeps not only as nutrient but also as stimulator of sulfur recycling.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/50167, title ="Carbonate-hosted methanotrophy represents an unrecognized methane sink in the deep sea", author = "Marlow, Jeffrey J. and Steele, Joshua A.", journal = "Nature Communications", volume = "5", number = "10", pages = "Art. No. 6094", month = "October", year = "2014", doi = "10.1038/ncomms6094", issn = "2041-1723", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141002-104139439", note = "© 2014 Macmillan Publishers Limited. \n\nReceived 24 March 2014; Accepted 28 August 2014; Published 14 October 2014. \n\nWe thank the Captains, Crew, Alvin group, Jason group and Science party members from RV Atlantis legs AT-15-11, AT-15-44, AT-15-59, AT-15-68 and AT-18-10. Patricia Tavormina and Stephanie Connon kindly helped with pmo analysis and molecular cloning procedures, and Olivia Mason prepared the 16S rRNA gene clone library sequences for A.Nod-2518. Anne Dekas, Abigail Green-Saxena and Yunbin Guan aided with nanoSIMS operation, and Alex Parker assisted with porosity and permeability measurements. Shawn McGlynn, Roland Hatzenpichler, Jennifer Glass, Kat Dawson, Hank Yu, David Case and Hiroyuki Imachi provided useful comments throughout the experimentation and writing process. This study was funded by grants from the National Science Foundation (OCE-0825791 and OCE-0939559), the National Aeronautics and Space Administration (NASA) Astrobiology Institute (award #NNA04CC06A and #NNA13AA92A) and the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant #3780, to V.J.O. J.J.M. was partially supported by a National Energy Technology Laboratory Methane Hydrate Research Fellowship funded by the National Research Council of the National Academies.\n\nNCBI Reference Sequence: KF616507; KF616827", revision_no = "34", abstract = "The atmospheric flux of methane from the oceans is largely mitigated through microbially mediated sulphate-coupled methane oxidation, resulting in the precipitation of authigenic carbonates. Deep-sea carbonates are common around active and palaeo-methane seepage, and have primarily been viewed as passive recorders of methane oxidation; their role as active and unique microbial habitats capable of continued methane consumption has not been examined. Here we show that seep-associated carbonates harbour active microbial communities, serving as dynamic methane sinks. Microbial aggregate abundance within the carbonate interior exceeds that of seep sediments, and molecular diversity surveys reveal methanotrophic communities within protolithic nodules and well-lithified carbonate pavements. Aggregations of microbial cells within the carbonate matrix actively oxidize methane as indicated by stable isotope FISH–nanoSIMS experiments and ^(14)CH_4 radiotracer rate measurements. Carbonate-hosted methanotrophy extends the known ecological niche of these important methane consumers and represents a previously unrecognized methane sink that warrants consideration in global methane budgets.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/52235, title ="Spatial distribution of nitrogen fixation in methane seep sediment and the role of the ANME archaea", author = "Dekas, Anne E. and Chadwick, Grayson L.", journal = "Environmental Microbiology", volume = "16", number = "10", pages = "3012-3029", month = "October", year = "2014", doi = "10.1111/1462-2920.12247 ", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141201-133733752", note = "© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.\n\nReceived 11 February, 2013; revised 24 May, 2013; accepted\n9 August, 2013.\nArticle first published online: 24 SEP 2013.\n\nWe would like to thank Lisa Levin, Burt Thomas, David\nFike, Jake Bailey, Rachel Poretsky, Shana Goffredi and\nthe crew and science parties of AT 15–44 and AT 15–59\nfor assistance in sample collection. Yunbin Guan and John\nEiler are acknowledged for assistance with NanoSIMS\noperations, Bill Ussler (Monterey Bay Aquarium Research\nInstitute) and Nathan Dalleska (Caltech Environmental\nAnalysis Center) for assistance with methane and ammonium\nmeasurements, Steve Skinner for map construction,\nand Stephanie Connon and Abigail Green-Saxena for assistance\nwith the sulfide measurements and CARD-FISH\nrespectively. We are also grateful to Abigail Green-Saxena\nand Roland Hatzenpichler for detailed reviews of this\nmanuscript. We would also like to thank three anonymous\nreviewers for their careful consideration and suggested\nimprovements to the manuscript. Funding was provided by\nthe Department of Energy Biological & Environmental\nResearch (BER) (Early Career Award DE-SC0003940 and\nDE-SC0004949 to V.J.O) and the National Science Foundation\n(MCB-0348492 to V.J.O and EF-0801741 to S.B.J.)\nand an NSF Graduate Research Fellowship to A.E.D. The\nediting of this work by A.E.D. was partially performed under\nthe auspices of the US Department of Energy by Lawrence\nLivermore National Laboratory under Contract DE-AC52-07NA27344. The Caltech Center for Microanalysis, housing\nthe CAMECA NanoSIMS 50L, is funded in part by the\nGordon and Betty Moore Foundation.", revision_no = "22", abstract = "Nitrogen (N_2) fixation was investigated at Mound 12, Costa Rica, to determine its spatial distribution and biogeochemical controls in deep-sea methane seep sediment. Using ^(15)N_2 tracer experiments and isotope ratio mass spectrometry analysis, we observed that seep N_2 fixation is methane-dependent, and that N_2 fixation rates peak in a narrow sediment depth horizon corresponding to increased abundance of aggregates of anaerobic methanotrophic archaea (ANME-2) and sulfate-reducing bacteria (SRB). Using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS), we directly measured ^(15)N_2 uptake by ANME-2/SRB aggregates (n\u2009=\u200926) and observed maximum ^(15)N incorporation within ANME-2-dominated areas of the aggregates, consistent with previous analyses. NanoSIMS analysis of single cells (n\u2009=\u200934) from the same microcosm experiment revealed no ^(15)N_2 uptake. Together, these observations suggest that ANME-2, and possibly physically associated SRB, mediate the majority of new nitrogen production within the seep ecosystem. ANME-2 diazotrophy was observed while in association with members of two distinct orders of SRB: Desulfobacteraceae and Desulfobulbaceae. The rate of N_2 fixation per unit volume biomass was independent of the identity of the associated SRB, aggregate size and morphology. Our results show that the distribution of seep N_2 fixation is heterogeneous, laterally and with depth in the sediment, and is likely influenced by chemical gradients affecting the abundance and activity of ANME-2/SRB aggregates.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44082, title ="In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry", author = "Hatzenpichler, Roland and Scheller, Silvan", journal = "Environmental Microbiology", volume = "16", number = "8", pages = "2568-2590", month = "August", year = "2014", doi = "10.1111/1462-2920.12436", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140303-103306009", note = "© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.\nThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.\n\nAccepted manuscript online: 26 Feb 2014; Manuscript Accepted: 18 Feb 2014; Manuscript Received: 20 Dec 2013. \n\nThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12436. \n\nWe thank Katherine Dawson and Hiroyuki Imachi for advice on anaerobic culturing, Yunbin Guan for assistance with nanoSIMS analyses, John D. Bagert for discussions on click chemistry, Grayson Chadwick for calculating genomic Met contents, and Jennifer Glass for helpful comments on an early version of this manuscript. We acknowledge the Caltech Proteome Exploration Laboratory (PEL) staff for analyzing mass spectrometry samples and their technical assistance with sample preparation and interpretation of results. The PEL is supported by the Beckman Institute and the Gordon & Betty Moore Foundation. Roland Hatzenpichler was supported via an O.K. Earl Postdoctoral Scholarship awarded by Caltech’s Division of Geological and Planetary Sciences as well as an Erwin Schrӧdinger Postdoctoral Fellowship of the Austrian Science Fund (FWF), J 3162-B20. Silvan Scheller was supported by the Swiss National Science Foundation (grant PBEZP2_142903). Funding for this project was provided by the Gordon and Betty Moore Foundation through Grant GBMF3780 to VJO, grant from the Department of Energy (DE-PS02-09ER09-25) to VJO, and by a National Institutes of Health grant NIH R01 GM062523 to DAT.\n\nPlease note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.", revision_no = "58", abstract = "Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for L-methionine, followed by fluorescent labeling of AHA containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater, and anoxic sediment. We also developed combined assays that couple BONCAT with rRNA-targeted FISH, enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labeling by nanoSIMS (^(15)NH_4^+ assimilation) for individual cells within a sediment sourced enrichment culture showed concordance between AHA positive cells and ^(15)N enrichment. BONCAT-FISH offers a fast, inexpensive, and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single cell level.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47287, title ="Geochemical, metagenomic and metaproteomic insights into trace metal utilization by methane-oxidizing microbial consortia in sulphidic marine sediments", author = "Glass, Jennifer B. and Yu, Hang", journal = "Environmental Microbiology", volume = "16", number = "6", pages = "1592-1633", month = "June", year = "2014", doi = "10.1111/1462-2920.12314", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140717-091139174", note = "© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.\n\nReceived 25 August, 2013; accepted 13 October, 2013.\n\nWe thank Stephanie Connon, Nathan Dalleska, Varun Gadh,\nAlexis Pasulka, Annelie Pernthaler, Rachel Poretsky and\nPatricia Tavormina for technical assistance; Jess Adkins, Ken Farley, Lindsay Hedges, Guillaume Paris, and Alex Rider for assistance with ICP-MS analysis; Steve Bates, Anthony\nChappaz, George Helz, Sebastian Kopf, Timothy Lyons,\nJames Morgan, Silvan Scheller, Silke Severmann and Laura\nWasylenki for helpful discussions; and Roland Hatzenpichler\nfor manuscript comments. We are grateful to the captain,\npilots, crew and shipboard research parties of the R/V\nWestern Flyer and R/V Atlantis (AT-15–68 and AT-18-10) for\ntheir invaluable support. We also thank Bill Ussler III, Charlie Paull and Husen Zhang for assistance with sample collection from Santa Monica Basin in 2005. We also acknowledge the Gordon and Betty Moore Foundation, and Stephan Schuster for financial and technical support with sequencing BC3 and BC4 at Penn State. This work was supported by grants from the Department of Energy Division of Biological and Environmental Research (DE-SC0004949), the National Aeronautics and Space Administration Astrobiology Institute (Penn State Astrobiology Research Center), and the Gordon and Betty Moore Foundation and the National Science Foundation (OCE-0825791) to V.J.O. Samples from Eel River Basin and Hydrate Ridge were collected as part of NSF funded projects (MCB-0348492; OCE-0825791) to V.J.O. J.B.G. was supported by a National Aeronautics and Space Administration Astrobiology Postdoctoral Fellowship.", revision_no = "29", abstract = "Microbes have obligate requirements for trace metals in metalloenzymes that catalyse important biogeochemical reactions. In anoxic methane- and sulphide-rich environments, microbes may have unique adaptations for metal acquisition and utilization because of decreased bioavailability as a result of metal sulphide precipitation. However, micronutrient cycling is largely unexplored in cold (≤\u200910°C) and sulphidic (>\u20091\u2009mM ΣH_(2)S) deep-sea methane seep ecosystems. We investigated trace metal geochemistry and microbial metal utilization in methane seeps offshore Oregon and California, USA, and report dissolved concentrations of nickel (0.5–270\u2009nM), cobalt (0.5–6\u2009nM), molybdenum (10–5600\u2009nM) and tungsten (0.3–8\u2009nM) in Hydrate Ridge sediment porewaters. Despite low levels of cobalt and tungsten, metagenomic and metaproteomic data suggest that microbial consortia catalysing anaerobic oxidation of methane (AOM) utilize both scarce micronutrients in addition to nickel and molybdenum. Genetic machinery for cobalt-containing vitamin B_(12) biosynthesis was present in both anaerobic methanotrophic archaea (ANME) and sulphate-reducing bacteria. Proteins affiliated with the tungsten-containing form of formylmethanofuran dehydrogenase were expressed in ANME from two seep ecosystems, the first evidence for expression of a tungstoenzyme in psychrophilic microorganisms. Overall, our data suggest that AOM consortia use specialized biochemical strategies to overcome the challenges of metal availability in sulphidic environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44532, title ="The rise and fall of methanotrophy following a deepwater oil-well blowout", author = "Crespo-Medina, M. and Meile, C. D.", journal = "Nature Geoscience", volume = "7", number = "6", pages = "423-427", month = "June", year = "2014", doi = "10.1038/ngeo2156", issn = "1752-0894", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140326-130213946", note = "© 2014 Macmillan Publishers Limited. \n\nReceived 14 January 2014; Accepted 04 April 2014; Published online 11 May 2014. \n\nWe thank C. Mann, A. Vossmeyer, J. Slaughter, C. Comerford, L. Potter, V. Samarkin and\nS. Cummings for assistance at sea and/or in the laboratory; M. Chistoserdova for\nproviding advice on constructing qPCR primers and for providing pure cultures of\nmethanotrophs; I. MacDonald, T. Treude and M. Chistoserdova provided constructive\nfeedback on a previous version of this manuscript. Finally we thank the science parties\nand ship's crews of RV Pelican, RV Nancy Foster, RVWalton Smith, RV Oceanus, RV Cape\nHatteras, MY Arctic Sunrise and RV Atlantis. This work was supported by the NOAA\nAward NA07AR4300464 to the National Institute for Undersea Science and Technology\n(V.L.A., A.R.D. and S.B.J.), the Department of Energy (Gulf of Mexico Gas Hydrate\nResearch Consortium, 07-11-036 to J.P.C.), the National Science Foundation\n(OCE-1043225 to S.B.J., OCE-0926699 to T.A.V. and OCE-1042934 to A.M.S.), the Gulf\nof Mexico Research Initiative (ECOGIG (S.B.J., V.L.A., A.B., J.P.C., A.R.D, J.P.M., C.D.M.\nand T.A.V.) and DEEP-C (J.P.C.)) and the Northern Gulf Institute (A.M.S.). Greenpeace\nand Texas A&M at Galveston facilitated the Arctic Sunrise expedition. This is ECOGIG\ncontribution #192 and the data fall under GRIIDC accession number\n(R1.x132.134:0057).", revision_no = "60", abstract = "The blowout of the Macondo oil well in the Gulf of Mexico in April 2010 injected up to 500,000 tonnes of natural gas, mainly methane, into the deep sea1. Most of the methane released was thought to have been consumed by marine microbes between July and August 20102, 3. Here, we report spatially extensive measurements of methane concentrations and oxidation rates in the nine months following the spill. We show that although gas-rich deepwater plumes were a short-lived feature, water column concentrations of methane remained above background levels throughout the rest of the year. Rates of microbial methane oxidation peaked in the deepwater plumes in May and early June, coincident with a rapid rise in the abundance of known and new methane-oxidizing microbes. At this time, rates of methane oxidation reached up to 5,900 nmol l−1 d−1—the highest rates documented in the global pelagic ocean before the blowout4. Rates of methane oxidation fell to less than 50 nmol l−1 d−1 in late June, and continued to decline throughout the remainder of the year. We suggest the precipitous drop in methane consumption in late June, despite the persistence of methane in the water column, underscores the important role that physiological and environmental factors play in constraining the activity of methane-oxidizing bacteria in the Gulf of Mexico.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44740, title ="The Potential for Biologically Catalyzed Anaerobic Methane Oxidation on Ancient Mars", author = "Marlow, Jeffrey J. and LaRowe, Douglas E.", journal = "Astrobiology", volume = "14", number = "4", pages = "292-307", month = "April", year = "2014", doi = "10.1089/ast.2013.1078", issn = "1557-8070", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140408-093723442", note = "© 2014 Mary Ann Liebert, Inc.\n\nSubmitted 22 July 2013; Accepted 16 February 2014; Online Ahead of Print: March 31, 2014.\n\nJ.J.M. would like to thank Dawn Cardace, Andrew Dale,\nand Megan Newcombe for helpful discussion and consultation\nand the NETL-National Academy of Sciences Methane Hydrate Research Fellowship for financial support. D.E.L. and J.P.A. would like to acknowledge financial support from the Life Underground NASA Astrobiology Institute (NAI) based at USC. V.J.O. acknowledges the Penn State Astrobiology Research Center NAI.", revision_no = "15", abstract = "This study examines the potential for the biologically mediated anaerobic oxidation of methane (AOM) coupled to sulfate reduction on ancient Mars. Seven distinct fluids representative of putative martian groundwater were used to calculate Gibbs energy values in the presence of dissolved methane under a range of atmospheric CO_2 partial pressures. In all scenarios, AOM is exergonic, ranging from −31 to −135\u2009kJ/mol CH_4. A reaction transport model was constructed to examine how environmentally relevant parameters such as advection velocity, reactant concentrations, and biomass production rate affect the spatial and temporal dependences of AOM reaction rates. Two geologically supported models for ancient martian AOM are presented: a sulfate-rich groundwater with methane produced from serpentinization by-products, and acid-sulfate fluids with methane from basalt alteration. The simulations presented in this study indicate that AOM could have been a feasible metabolism on ancient Mars, and fossil or isotopic evidence of this metabolic pathway may persist beneath the surface and in surface exposures of eroded ancient terrains.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44218, title ="Microsporidia-nematode associations in methane seeps reveal basal fungal parasitism in the deep sea", author = "Sapir, Amir and Dillman, Adler R.", journal = "Frontiers in Microbiology", volume = "5", pages = "Art. No. 43", month = "February", year = "2014", doi = "10.3389/fmicb.2014.00043", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140310-102130554", note = "© 2014 Sapir, Dillman, Connon, Grupe, Ingels, Mundo-Ocampo, Levin, Baldwin, Orphan and Sternberg. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. \n\nReceived: 29 November 2013; Paper pending published: 14 December 2013; Accepted: 21 January 2014; Published online: 10 February 2014.\n\nWe thank captain, crew, Alvin and Jason II pilots, and cruise participants of Atlantis legs 15-68 and 18-10 for assistance with sample collection. We thank Alasdair McDowall for excellent TEM assistance, Nathalie De Hauwere and the Flanders Marine Institute for drawing the HR map, Stephen Meisenhelter for worm picking, Emily Troemel for sharing reagents, John Curington for Latin grammar advice, Katja Guilini for sharing samples, slides, published data about HR nematodes, James Becnel for comments on the manuscript, and Greg Rouse for E4 rock photo. This work was supported by the Howard Hughes Medical Institute, with which PWS is an investigator, sample collection was supported by NSF OCE 0826254 to LAL and NSF\nOCE-0825791 to VJO, and an NIH USPHS Training Grant (T32GM07616) to A.R.D. JI is supported by a Marie Curie Intra-European Fellowship within the 7th European Community\nFramework Programme (Grant Agreement FP7-PEOPLE-2011-IEF No 300879).\nRunning title: Microsporidia parasitism in deep-sea methane seeps", revision_no = "35", abstract = "The deep sea is Earth's largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over 2 years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes' intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host's body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep-sea biosphere. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/43402, title ="Nitrate-based niche differentiation by distinct sulfate-reducing bacteria involved in the anaerobic oxidation of methane", author = "Green-Saxena, A. and Dekas, A. E.", journal = "ISME Journal", volume = "8", number = "1", pages = "150-163", month = "January", year = "2014", doi = "10.1038/ismej.2013.147", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140116-093516040", note = "© 2014 International Society for Microbial Ecology.\n\nReceived 12 March 2013; revised 19 July 2013; accepted 22 July\n2013; published online 5 September 2013.\n\nWe would like to thank Grayson Chadwick for his help with image analysis, Elizabeth Trembath-Reichert for contributing clone sequences, Ankur Saxena for figure design, Yunbin Guan NanoSIMS technical assistance and\nJoshua Steele for statistical analyses. We would like to\nalso acknowledge Tsege Embaye for methane and sulfate\nmeasurements from AT 15–11 and the science party of\ncruises AT 15–11, AT 15–44, AT 15–68 and AT 18–10 and\npilots of the DSRV Alvin and ROV Jason for their\nassistance with various aspects of this work. Funding for\nthis work was provided by the Department of Energy\nDivision of Biological Research (DE-SC0004949; to VJO),\nand a National Science Foundation Graduate Research\nFellowship (to AG-S). The editing of this work by AD was\npartially performed under the auspices of the US\nDepartment of Energy by Lawrence Livermore National\nLaboratory under Contract DE-AC52-07NA27344. Samples\nwere collected with funding from the National Science\nFoundation (BIO-OCE 0825791; to VJO).", revision_no = "29", abstract = "Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps; however, the ecophysiology of these different symbiotic associations has not been examined. Here, we applied a combination of molecular, geochemical and Fluorescence in situ hybridization (FISH) coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations (Eel River Basin, Hydrate Ridge and Costa Rican Margin seeps) to investigate the distribution and physiology of a newly identified subgroup of the Desulfobulbaceae (seepDBB) found in consortia with ANME-2c archaea, and compared these with the more commonly observed associations between the same ANME partner and the Desulfobacteraceae (DSS). FISH analyses revealed aggregates of seepDBB cells in association with ANME-2 from both environmental samples and laboratory incubations that are distinct in their structure relative to co-occurring ANME/DSS consortia. ANME/seepDBB aggregates were most abundant in shallow sediment depths below sulfide-oxidizing microbial mats. Depth profiles of ANME/seepDBB aggregate abundance revealed a positive correlation with elevated porewater nitrate relative to ANME/DSS aggregates in all seep sites examined. This relationship with nitrate was supported by sediment microcosm experiments, in which the abundance of ANME/seepDBB was greater in nitrate-amended incubations relative to the unamended control. FISH-NanoSIMS additionally revealed significantly higher 15N-nitrate incorporation levels in individual aggregates of ANME/seepDBB relative to ANME/DSS aggregates from the same incubation. These combined results suggest that nitrate is a geochemical effector of ANME/seepDBB aggregate distribution, and provides a unique niche for these consortia through their utilization of a greater range of nitrogen substrates than the ANME/DSS.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41361, title ="Filamentous sulfur bacteria preserved in modern and ancient phosphatic sediments: implications for the role of oxygen and bacteria in phosphogenesis", author = "Bailey, J. V. and Corsetti, F. A.", journal = "Geobiology", volume = "11", number = "5", pages = "397-405", month = "September", year = "2013", doi = "10.1111/gbi.12046", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130917-101809185", note = "© 2013 John Wiley & Sons Ltd.\n\nReceived 18 January 2013; accepted 24 May 2013.\n\nArticle first published online: 20 Jun. 2013.\n\nWe thank Nick Seaton, Ellery Frahm, Anette von der\nHandt, Will Berelson, Chi Ma, and Bing Luo for assistance\nwith the instrument analysis and sample preparation. We\nthank Greg Rouse for providing the photography used in\nFig. 1B. We also thank Lisa Levin, Tony Rathburn and the\nparticipants and crew of cruise AT 15-44 (supported by\nthe National Science Foundation OCE-0826254) for\nassisting with the collection of the samples from Costa\nRica. We also thank three anonymous reviewers whose\ncomments improved the manuscript.\n\nPortions of this work were supported by an Agouron\nGeobiology Postdoctoral Fellowship and National Science\nFoundation grant EAR-1057119 to JVB, and by the\nNational Natural Science Foundation of China (41172035)\nand China Geology Survey (1212011120140). Additional\nfunding was provided by an AAPG Kenneth H. Crandall\nMemorial Grant-in-Aid to SEG. Parts of this work were\ncarried out in the Characterization Facility at the University\nof Minnesota, which receives partial support from NSF\nthrough the MRSEC program.", revision_no = "25", abstract = "Marine phosphate-rich sedimentary deposits (phosphorites) are important geological reservoirs for the biologically essential nutrient phosphorous. Phosphorites first appear in abundance approximately 600 million years ago, but their proliferation at that time is poorly understood. Recent marine phosphorites spatially correlate with the habitats of vacuolated sulfide-oxidizing bacteria that store polyphosphates under oxic conditions to be utilized under sulfidic conditions. Hydrolysis of the stored polyphosphate results in the rapid precipitation of the phosphate-rich mineral apatite—providing a mechanism to explain the association between modern phosphorites and these bacteria. Whether sulfur bacteria were important to the formation of ancient phosphorites has been unresolved. Here, we present the remains of modern sulfide-oxidizing bacteria that are partially encrusted in apatite, providing evidence that bacterially mediated phosphogenesis can rapidly permineralize sulfide-oxidizing bacteria and perhaps other types of organic remains. We also describe filamentous microfossils that resemble modern sulfide-oxidizing bacteria from two major phosphogenic episodes in the geologic record. These microfossils contain sulfur-rich inclusions that may represent relict sulfur globules, a diagnostic feature of modern sulfide-oxidizing bacteria. These findings suggest that sulfur bacteria, which are known to mediate the precipitation of apatite in modern sediments, were also present in certain phosphogenic settings for at least the last 600 million years. If polyphosphate-utilizing sulfide-oxidizing bacteria also played a role in the formation of ancient phosphorites, their requirements for oxygen, or oxygen-requiring metabolites such as nitrate, might explain the temporal correlation between the first appearance of globally distributed marine phosphorites and increasing oxygenation of Neoproterozoic oceans.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41193, title ="Polyphosphate Storage during Sporulation in the Gram-Negative Bacterium Acetonema longum", author = "Tocheva, Elitza I. and Dekas, Anne E.", journal = "Journal of Bacteriology", volume = "195", number = "17", pages = "3940-3946", month = "September", year = "2013", doi = "10.1128/JB.00712-13 ", issn = "0021-9193", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130909-141442695", note = "© 2013 American Society for Microbiology.\n\nReceived 16 June 2013 Accepted 20 June 2013;\nPublished ahead of print 28 June 2013.\n\nWe acknowledge the use of electron microscopy facilities at the UCLA\nElectron Imaging Center for NanoMachines at the California NanoSystems\nInstitute (CNSI) and thank Ivo Atanasov and Dan Taso for technical\nassistance with EDX data collection and analysis. We thank Yunbin Guan\nand John Eiler for assistance with the NanoSIMS measurements. We\nthank Adrian Ponce for providing the C. sporogenes spores.\nThe NanoSIMS apparatus is housed within the Caltech Microanalysis\nCenter and is partially funded by the Gordon and Betty Moore Foundation.\nThis work was funded in part by the Howard Hughes Medical Institute,\nthe Caltech Center for Environmental Microbial Interactions, and\ngifts to Caltech from the Gordon and Betty Moore Foundation.\nThis work was partially performed under the auspices of the U.S. Department\nof Energy by Lawrence Livermore National Laboratory under\ncontract DE-AC52-07NA27344.", revision_no = "19", abstract = "Using electron cryotomography, we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore membrane and ultimately accounts for ∼7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX) analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of polyphosphate (poly-P). Unlike the Gram-positive Bacilli and Clostridia, A. longum spores retain their outer spore membrane upon germination. To explore the possibility that the granules in A. longum may be involved in this unique process, we imaged purified Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Clostridium sporogenes spores. Even though B. cereus and B. thuringiensis contain the ppk and ppx genes, none of the spores from Gram-positive bacteria had granules. We speculate that poly-P in A. longum may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer membrane.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47452, title ="Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor", author = "Ussler, William, III and Preston, Christina", journal = "Environmental Science and Technology", volume = "47", number = "16", pages = "9339-9346", month = "August", year = "2013", doi = "10.1021/es4023199", issn = "0013-936X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327897", note = "Copyright © 2013 American Chemical Society. \n\nReceived: May 24, 2013; Revised: July 24, 2013; Accepted: July 24, 2013; Published: July 24, 2013. \n\nDevelopment and application of ESP technology has been funded in part by grants from the David and Lucile Packard Foundation through funds allocated by the Monterey Bay Aquarium Research Institute (MBARI), NSF (OCE-0314222 and EF-0424599), NASA Astrobiology (NNG06GB34G, NNX09AB78G), Keck Foundation (by subcontract from University of Washington, Seattle), and Gordon and Betty Moore Foundation (ERG731). Elif Demir-Hilton, Kevan Yamahara, and three anonymous reviewers provided constructive comments and feedback on earlier versions of this manuscript. We thank the engineering technicians and machinists at MBARI for their invaluable help and dedication toward instrument development and the crews and ROV pilots of the R/Vs Point Lobos and Western Flyer for their support and expertise during field operations. \n\n\nThe authors declare no competing financial interest.\n\nSupporting Information:\n\nDetailed descriptions of qPCR assay development, nucleic acid\nextraction, ISMS operation and calibration, and dissolved\nmethane concentration analysis. Figure S1-S2 shows characteristic\nfeatures of the seafloor at the MARS deployment site.\nFigure S2-S11 illustrates water column methane concentration\nprofiles from above the Santa Monica mound. Table S1-S5 lists\nprimer and probe sequences used for the qPCR 5′-nuclease\nassays. Table S2-S8 summarizes qPCR standard curve\nparameters for in situ and postdeployment gene expression\nassays. This material is available free of charge via the Internet\nat http://pubs.acs.org.\n", revision_no = "20", abstract = "Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The DESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38983, title ="Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone", author = "Tavormina, Patricia L. and Ussler, William, III", journal = "Environmental Microbiology Reports", volume = "5", number = "3", pages = "414-423", month = "June", year = "2013", doi = "10.1111/1758-2229.12025", issn = "1758-2229", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130619-100723976", note = "© 2012 John Wiley & Sons Ltd and Society for Applied Microbiology. \n\nReceived 6 September, 2012; accepted 3 December, 2012. Article first published online: 29 Jan 2013. \n\nWe would like to thank the captain and crew of the RV Atlantis for the 2009 and 2010 Costa Rica expeditions, Shana Goffredi and Rachel Poretsky for assistance in collecting and processing samples, and numerous lab members and three anonymous reviewers for critical review and improving the quality of this study. Support was provided by grants from NASA ASTEP (NNG06GB34G to V. J. O.) and the National Science Foundation OCE (MCB-0348492 to V. J. O.) and MCB (EF-0541797 and MCB-0948202 to M. G. K.) programmes\nand through a grant to the Monterey Bay Aquarium\nResearch Institute from the David and Lucile Packard Foundation (W. U.).", revision_no = "55", abstract = "Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine environment, suggesting widespread potential for oxidation of reduced substrates. Here, we used the well-defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating significantly with reduced environmental oxygen concentrations and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene-utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO-encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affiliated phylogenetically with the recently described Cu-MMO ‘pXMO’, and Group Z clustered with Cu-MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced molecules and advance our understanding of the microorganisms involved in methane cycling in the OMZ environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44207, title ="Global molecular analyses of methane metabolism in methanotrophic Alphaproteobacterium, Methylosinus trichosporium OB3b. Part II. metabolomics and 13C-labeling study", author = "Yang, Song and Matsen, Janet B.", journal = "Frontiers in Microbiology", volume = "4", pages = "Art. No. 70", month = "April", year = "2013", doi = "10.3389/fmicb.2013.00070", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140310-084832957", note = "© 2013 Yang, Matsen, Konopka, Green-Saxena, Clubb, Sadilek, Orphan, Beck and Kalyuzhnaya. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. \n\nReceived: 01 January 2013; paper pending published: 28 January 2013; accepted:\n12 March 2013; published online: 03 April 2013.\n\nWe are very grateful to Dr. Mary E. Lidstrom, Dr. Ivan Bergand\nDr. Ludmila Chistoserdova for insightful suggestions on the manuscript. This work was supported by the DOE (DE-SC0005154).", revision_no = "13", abstract = "In this work we use metabolomics and ^(13)C-labeling data to refine central metabolic pathways for methane utilization in Methylosinus trichosporium OB3b, a model alphaproteobacterial methanotrophic bacterium. We demonstrate here that similar to non-methane utilizing methylotrophic alphaproteobacteria the core metabolism of the microbe is represented by several tightly connected metabolic cycles, such as the serine pathway, the ethylmalonyl-CoA (EMC) pathway, and the citric acid (TCA) cycle. Both in silico estimations and stable isotope labeling experiments combined with single cell (NanoSIMS) and bulk biomass analyses indicate that a significantly larger portion of the cell carbon (over 60%) is derived from CO_2 in this methanotroph. Our ^(13) C-labeling studies revealed an unusual topology of the assimilatory network in which phosph(enol) pyruvate/pyruvate interconversions are key metabolic switches. A set of additional pathways for carbon fixation are identified and discussed.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/42181, title ="High resolution profiling of sulfur and oxygen speciation in microbial mats: Implications for coupling of hydrogen peroxide with sulfur species", author = "Druschel, Greg K. and Kafantaris, Fotios", pages = "GEOC-38", month = "April", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131101-092236803", note = "© 2013 American Chemical Society.", revision_no = "13", abstract = "Hydrogen sulfide is produced in microbial mats by sulfate reducing bacteria, and diffuses towards the surface\nuntil it is consumed. Cyanobacteria in these mats are able to produce oxygen photochem., and when\nsubjected to high UV light intensity, can also produce hydrogen peroxide. Microelectrodes have proven to have\nsufficient resoln. to resolve the sulfur and oxygen chem. in these mats, demonstrating the links between\nthese populations of bacteria in modern mats. In sulfur-dominated cyanobacterial mats in several settings, we\nhave obsd. both high hydrogen sulfide fluxes and the periodic prodn. of significant amts. of hydrogen peroxide\nat times of high UV stress using voltammetric Au-amalgam microelectrodes. We review the use of voltammetric\nand amperometric microelectrodes in approaching this chem., and how the relative rates of sulfur species\noxidn. with oxygen or peroxide may play a significant role in shaping the chem., isotopic, and biol. compn. of\nthese systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37576, title ="Classifying the metal dependence of uncharacterized nitrogenases", author = "McGlynn, Shawn E. and Boyd, Eric S.", journal = "Frontiers in Microbiology", volume = "3", pages = "Art. No. 419", month = "January", year = "2013", doi = "10.3389/fmicb.2012.00419 ", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130320-133744148", note = "© 2013 McGlynn, Boyd, Peters and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.\n\nReceived: 09 October 2012; Paper pending published: 02 November 2012; Accepted: 20 November 2012; Published online: 30 January 2013.\n\nShawn E. McGlynn is an Agouron Geobiology Option postdoctoral fellow in the Division of Geological and Planetary Sciences at Caltech and is grateful for support provided by the Agouron Institute. Additional support for this work was provided by the Department of Energy Grant DE-SC0004949 (to Victoria J Orphan) and the NASA Astrobiology Institute (NAI) NNA08C-N85A (to John W. Peters and Eric S. Boyd). Eric S. Boyd also wishes to acknowledge support from the National Science Foundation (EAR-1123689 and PIRE-0968421). The authors are grateful for comments from Aaron D. Goldman, Joshua A. Steele, Wolfgang Nitschke, and members of the laboratory of Victoria Orphan.", revision_no = "10", abstract = "Nitrogenase enzymes have evolved complex iron–sulfur (Fe–S) containing cofactors that most commonly contain molybdenum (MoFe, Nif) as a heterometal but also exist as vanadium (VFe, Vnf) and heterometal-independent (Fe-only, Anf) forms. All three varieties are capable of the reduction of dinitrogen (N_2) to ammonia (NH_3) but exhibit differences in catalytic rates and substrate specificity unique to metal type. Recently, N_2 reduction activity was observed in archaeal methanotrophs and methanogens that encode for nitrogenase homologs which do not cluster phylogenetically with previously characterized nitrogenases. To gain insight into the metal cofactors of these uncharacterized nitrogenase homologs, predicted three-dimensional structures of the nitrogenase active site metal-cofactor binding subunits NifD, VnfD, and AnfD were generated and compared. Dendrograms based on structural similarity indicate nitrogenase homologs cluster based on heterometal content and that uncharacterized nitrogenase D homologs cluster with NifD, providing evidence that the structure of the enzyme has evolved in response to metal utilization. Characterization of the structural environment of the nitrogenase active site revealed amino acid variations that are unique to each class of nitrogenase as defined by heterometal cofactor content; uncharacterized nitrogenases contain amino acids near the active site most similar to NifD. Together, these results suggest that uncharacterized nitrogenase homologs present in numerous anaerobic methanogens, archaeal methanotrophs, and firmicutes bind FeMo-co in their active site, and add to growing evidence that diversification of metal utilization likely occurred in an anoxic habitat.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41620, title ="Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH", author = "Trembath-Reichert, Elizabeth and Green-Saxena, Abigail", number = "531", pages = "21-44", month = "January", year = "2013", doi = "10.1016/B978-0-12-407863-5.00002-2", isbn = "9780124078635", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131002-134144694", note = "© 2013 Elsevier Inc. \n\nWe acknowledge Annelie (Pernthaler) Wendeberg, Rachel Poretsky, Joshua Steele for their\ncontributions towards the development and optimization of Magneto-FISH. Stephanie\nCannon, Jen Glass, Kat Dawson, Hiroyuki Imachi, and Caltech Genomics Center are also\nacknowledged for their assistance with various aspects of this project. Funding for this\nwork was provided by the Gordon and Betty Moore foundation and a DOE early career\ngrant (to V. J. O.) and NIH/NRSA training grant 5 T32 GM07616 (to E. T. R.).", revision_no = "13", abstract = "Magneto-FISH, in combination with metagenomic techniques, explores the middle\nground between single-cell analysis and complex community characterization in bulk\nsamples to better understand microbial partnerships and their roles in ecosystems. The\nMagneto-FISH method combines the selectivity of catalyzed reporter deposition–\nfluorescence in situ hybridization (CARD–FISH) with immunomagnetic capture to provide\ntargeted molecular and metagenomic analysis of co-associated microorganisms in\nthe environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups\nassociated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as\nwell as provided insight into their physiological potential using metagenomics. Here,\nwe demonstrate the utility of this method for capturing aggregated consortia using\na series of nested oligonucleotide probes of differing specificity designed to target\neither the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA\nand mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for largeand\nsmall-volume samples and evaluates the strengths and limitations of this method\npredominantly focusing on (1) the relationship between FISH probe specificity and sample\nselectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples,\nand (3) suggestions for adapting the Magneto-FISH method for other microbial systems,\nincluding potential for single-cell recovery.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/36242, title ="Active sulfur cycling by diverse mesophilic and thermophilic microorganisms in terrestrial mud volcanoes of Azerbaijan", author = "Green-Saxena, A. and Feyzullayev, A.", journal = "Environmental Microbiology", volume = "14", number = "12", pages = "3271-3286", month = "December", year = "2012", doi = "10.1111/1462-2920.12015 ", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130108-135620155", note = "© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 5 March, 2012; revised 9 July, 2012; accepted 28 September, 2012. Article first published online: 1 Nov. 2012. \n\nWe would like to acknowledge Dr Chingiz Aliyev and Rauf\nBagirli for assistance during field trip and laboratory investigations\nin Baku; Daniela Zoch and Holger Probst for technical\nwork at BGR, and Andrea Vieth-Hillebrandt and Kristin\nGünther for water analysis at Helmholtz Centre Potsdam\nGFZ. Funding for this work was provided by a DOE Career\ngrant (to V.J.O.), a NSF GRFP (to A.G.-S.) and through the\nForschungsverbund GeoEnergie of the German Ministry for\nEducation and Research (BMBF, to J.K. and P.S.). We would\nalso like to thank two anonymous reviewers for their valuable\ninput.", revision_no = "34", abstract = "Terrestrial mud volcanoes (TMVs) represent geochemically diverse habitats with varying sulfur sources and yet sulfur cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in four TMVs in Azerbaijan. A combination of geochemical analyses, biological rate measurements and molecular diversity surveys (targeting metabolic genes aprA and dsrA and SSU ribosomal RNA) supported the presence of active sulfur-oxidizing and sulfate-reducing guilds in all four TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. The TMVs varied in potential sulfate reduction rates (SRR) by up to four orders of magnitude with highest SRR observed in sediments where in situ sulfate concentrations were highest. Maximum temperatures at which SRR were measured was 60°C in two TMVs. Corresponding with these trends in SRR, members of the potentially thermophilic, spore-forming, Desulfotomaculum were detected in these TMVs by targeted 16S rRNA analysis. Additional sulfate-reducing bacterial lineages included members of the Desulfobacteraceae and Desulfobulbaceae detected by aprA and dsrA analyses and likely contributing to the mesophilic SRR measured. Phylotypes affiliated with sulfide-oxidizing Gamma- and Betaproteobacteria were abundant in aprA libraries from low sulfate TMVs, while the highest sulfate TMV harboured 16S rRNA phylotypes associated with sulfur-oxidizing Epsilonproteobacteria. Altogether, the biogeochemical and microbiological data indicate these unique terrestrial habitats support diverse active sulfur-cycling microorganisms reflecting the in situ geochemical environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/39808, title ="Measurement of nitrous oxide isotopologues and isotopomers by the MAT 253 Ultra", author = "Magyar, Paul and Kopf, Sebastian", journal = "Mineralogical Magazine", volume = "76", number = "6", pages = "2054-2054", month = "November", year = "2012", issn = "0026-461X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130807-132330822", note = "© 2012 by the Mineralogical Society of Great Britain and Ireland.\n\nOpen Access Article.\n\nPublished online 19 November 2012.", revision_no = "10", abstract = "The global budget of nitrous oxide is dominated by terrestrial and marine biological sources and atmospheric sinks. Details of the\nbudget remain unclear, including the cause of increasing atmospheric N_2O concentrations. Marine sources of N_2O include\ndenitrification and nitrification. Our understanding of the major microbial players in the nitrogen cycle has changed in recent years\n(for example, the nitrifying Archaea), and the overall contributions of these organisms to N_2O production and their isotopic signatures\nare poorly constrained [1].", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47451, title ="Environmental Microbiology: evolution of your premier journal", author = "Polz, Martin and Orphan, Victoria", journal = "Environmental Microbiology", volume = "14", number = "10", pages = "2617-2619", month = "October", year = "2012", doi = "10.1111/j.1462-2920.2012.02869.x", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327760", note = "© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.\n\nArticle first published online: 3 OCT 2012.", revision_no = "10", abstract = "[no abstract]", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33305, title ="Archaea in metazoan diets: implications for food webs and biogeochemical cycling", author = "Thurber, Andrew R. and Levin, Lisa A.", journal = "ISME Journal", volume = "6", number = "8", pages = "1602-1612", month = "August", year = "2012", doi = "10.1038/ismej.2012.16", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120817-140759648", note = "© 2012 International Society for Microbial Ecology.\n\nReceived 29 July 2011; revised 2 February 2012; accepted 2\nFebruary 2012; published online 8 March 2012.\n\nWe are indebted to the Captains, Crew, Alvin group and\nScience parties from RV Atlantis legs 15-9; 15-11; 15-44;\nand 15-59. Guillermo Mendoza, Jennifer Gonzalez and Drs\nBrigitte Ebbe, Ken Halanych, Ray Lee and Greg Rouse all\nhelped tremendously at sea with sorting and identification\nof the dorvilleid species. Dr Shana Goffredi kindly\nchecked for archaeal symbionts within the Costa Rican\nDorvilleid. Dr William Gerwick provided access to the\nanalytical instrumentation necessary to carry out this\nresearch and Cameron Coates, Jo Nunnery and Tak Suyama were always present to help trouble shoot analytical problems. Two anonymous reviewers, and Drs G Rouse and Lihini Aluwihare provided helpful comments on an earlier version of this manuscript. This research was supported by NSF Grants OCE 0425317, OCE 0826254 and OCE-0939557, and grant UAF-050141 from the West Coast National Undersea Research Center to LA Levin, OCE-0939559 and OCE-0825791 grants to VJ\nOrphan and a UC Marine Council award to W Gerwick and LA Levin, in addition to the Michael M Mullin Memorial fellowship, Sidney E Frank Foundation Fellowship, UC Marine Council CEQI Fellowship and the graduate office of Scripps Institution of Oceanography’s support of AR Thurber.", revision_no = "19", abstract = "Although the importance of trophic linkages, including ‘top-down forcing’, on energy flow and ecosystem productivity is recognized, the influence of metazoan grazing on Archaea and the biogeochemical processes that they mediate is unknown. Here, we test if: (1) Archaea provide a food source sufficient to allow metazoan fauna to complete their life cycle; (2) neutral lipid biomarkers (including crocetane) can be used to identify Archaea consumers; and (3) archaeal aggregates are a dietary source for methane seep metazoans. In the laboratory, we demonstrated that a dorvilleid polychaete, Ophryotrocha labronica, can complete its life cycle on two strains of Euryarchaeota with the same growth rate as when fed bacterial and eukaryotic food. Archaea were therefore confirmed as a digestible and nutritious food source sufficient to sustain metazoan populations. Both strains of Euryarchaeota used as food sources had unique lipids that were not incorporated into O. labronica tissues. At methane seeps, sulfate-reducing bacteria that form aggregations and live syntrophically with anaerobic-methane oxidizing Archaea contain isotopically and structurally unique fatty acids (FAs). These biomarkers were incorporated into tissues of an endolithofaunal dorvilleid polychaete species from Costa Rica (mean bulk δ^(13)C=−92±4‰; polar lipids −116‰) documenting consumption of archaeal-bacterial aggregates. FA composition of additional soft-sediment methane seep species from Oregon and California provided evidence that consumption of archaeal-bacterial aggregates is widespread at methane seeps. This work is the first to show that Archaea are consumed by heterotrophic metazoans, a trophic process we coin as ‘archivory’.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/32041, title ="A hydrothermal seep on the Costa Rica margin: middle ground in a continuum of reducing ecosystems", author = "Levin, Lisa A. and Orphan, Victoria J.", journal = "Proceedings of the Royal Society of London. Series B, Biological Sciences", volume = "279", number = "1738", pages = "2580-2588", month = "July", year = "2012", doi = "10.1098/rspb.2012.0205 ", issn = "0962-8452", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120622-114155202", note = "© 2012 The Royal Society.\n\nReceived January 27, 2012; Accepted February 13, 2012.\n\nWe thank the Captain and crew of the RV Atlantis, the\nAlvin Pilots, as well as scientific participants of AT\n15_59, especially A. Thurber, G. Mendoza and A. Dekas\nfor their assistance at sea. We also thank H. Sahling and\nM. Tryon for their assistance in providing maps and other\nlocation information, H. Carson and R. Vargas for their\nobservations during AD 4513, and P. Tavormina, S. Connon and M. Muscovova for their assistance and contributions to the molecular community analysis. Two anonymous reviewers provided constructive comments on an earlier version of the manuscript. The research was supported by the US National Science Foundation grants OCE 0826254, 0825436, 0825791, 0939559 and 0939557.", revision_no = "21", abstract = "Upon their initial discovery, hydrothermal vents and methane seeps were considered to be related but distinct ecosystems, with different distributions, geomorphology, temperatures, geochemical properties and mostly different species. However, subsequently discovered vents and seep systems have blurred this distinction. Here, we report on a composite, hydrothermal seep ecosystem at a subducting seamount on the convergent Costa Rica margin that represents an intermediate between vent and seep ecosystems. Diffuse flow of shimmering, warm fluids with high methane concentrations supports a mixture of microbes, animal species, assemblages and trophic pathways with vent and seep affinities. Their coexistence reinforces the continuity of reducing environments and exemplifies a setting conducive to interactive evolution of vent and seep biota. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/31541, title ="Method for Assessing Mineral Composition-Dependent Patterns in Microbial Diversity Using Magnetic and Density Separation", author = "Harrison, Benjamin K. and Orphan, Victoria J.", journal = "Geomicrobiology Journal", volume = "29", number = "5", pages = "435-449", month = "March", year = "2012", doi = "10.1080/01490451.2011.581327 ", issn = "0149-0451", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120518-133946986", note = "© 2012 California Institute of Technology.\n\nReceived 27 October 2010; accepted 4 April 2011. Available online: 05 Mar 2012. \n\nWe thank the crew and shipboard parties and chief scientist B.\nVrijenhoek of the R/V Melville (May, 2005) and R/V Atlantis (October,\n2006) for support in sample collection. We also are grateful to S.\nGoffredi, A. Dekas, A. Green, J. Bailey, O. Mason, P. Tavormina, D.\nFike, S. Connon, J. Steele and two anonymous reviewers for helpful\ncomments and editorial advice. This work was supported by a grant\nfrom the Gordon and Betty Moore Foundation (to VJO), a Schlanger\nOcean Drilling Graduate Fellowship (to BKH) and funding from the\nNASA Astrobiology Institute (Grant award # 3903-CIT-NASA.A76A)\nas part of the Penn State Astrobiology Research Center (PSARC).\nCollection of samples from the Eel River Basin and Lau Basin was\nsupported by funding from the National Science Foundation (MCB-0348492 to VJO) and (OCE-0241613 to B. Vrijenhoek).", revision_no = "15", abstract = "This study introduces a new method for characterizing mineral-associated microbial diversity in sedimentary environments, a habitat that has been intrinsically challenging to study in regard to microbe-mineral interactions. Mineral components were enriched from bulk environmental samples by magnetic susceptibility or density separation techniques and used in subsequent molecular and microscopic analyses. Testing and optimization of the method was performed on geochemically-distinct sediment horizons from Eel River Basin methane seeps and pyrite and sphalerite-rich hydrothermal vent samples from the Lau Basin. Initial results show reproducible variations in microbial diversity between mineral fractions from marine sedimentary environments enriched in authigenic pyrite and/or transition metal-bearing clay minerals. Specifically, different archaeal clades associated with the anaerobic oxidation of methane and putative sulfate-reducing deltaproteobbacteria show preferential colonization patterns, suggesting potential ecophysiological differences between closely-related taxa. These results indicate that mineral colonization may influence the extent and distribution of microbial diversity throughout unconsolidated sediments of the marine subsurface. The combination of mineral separation and molecular analyses introduced here provide a new approach for revealing previously concealed patterns of mineral-associated microbial diversity across a wide range of environments, from hard rock habitats to fine-grained lithologies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/29686, title ="Trace metal requirements for microbial enzymes involved in the production and consumption of methane and nitrous oxide", author = "Glass, Jennifer B. and Orphan, Victoria J.", journal = "Frontiers in Microbiology", volume = "3", pages = "Art. No. 61", month = "February", year = "2012", doi = "10.3389/fmicb.2012.00061", issn = "1664-302X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120312-095005613", note = "© 2012 Glass and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited. \n\nReceived: 05 December 2011; Paper pending published: 21 December 2011; Accepted: 05 February 2012; Published online: 21 February 2012. \n\nThe authors acknowledge funding from the National Aeronautics and Space Administration (NASA) Astrobiology Postdoctoral Fellowship (to Jennifer B. Glass), the NASA Astrobiology Institute (NNA04CC06A), and the U.S. Department of Energy’s Office of Biological and Environmental Research Early Career Award (to Victoria J. Orphan). Thanks to Roland Hatzenpichler, Patricia Tavormina, Shawn McGlynn, Anne Dekas, Joshua Steele, Hiroyuki Imachi, Lisa Stein and Aubrey Zerkle for helpful comments on drafts of this manuscript.", revision_no = "12", abstract = "Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO_2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH_4), and nitrous oxide (N_2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH_4 and N_2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH_4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N_2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N_2O reductase, the only known enzyme capable of microbial N_2O conversion to N_2, have only been found in classical denitrifiers. Accumulation of N_2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N_2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide-metal scavenging.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/35307, title ="Molecular biology's contributions to geobiology", author = "Newman, Dianne K. and Orphan, Victoria J.", pages = "228-249", month = "January", year = "2012", isbn = "9781118280812", url = "https://resolver.caltech.edu/CaltechAUTHORS:20121106-134254144", note = "© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd. \n\nThe authors gratefully acknowledge the Howard Hughes Medical Institute (D.KN.), Gordon and Betty Moore Foundation and DOE Early Career Grant (V.J.O.), and NSF OCE-0937404 (A.L.R) for support. We also thank our students, postdocs, and colleagues for shaping our thinking on these topics over the years.", revision_no = "15", abstract = "On August 7, 1996, US President Bill Clinton held a press conference to announce the possibility that the Allan Hills 84001 meteorite might provide insight into ancient life on Mars. With soaring rhetoric, he declared: 'Today, rock 84001 speaks to us across all those billions of years and millions of miles. It speaks of the possibility of life. If this discovery is confirmed, it will surely be one of the most stunning insights into our universe that science has ever uncovered. Its implications are as far-reaching and awe-inspiring as can be imagined. Even as it promises answers to some of our oldest questions, it poses still others even more fundamental.' Shortly thereafter, NASA expanded its support for astro-and geobiological research, which marked the beginning of a renaissance in geobiology. Seemingly overnight, geobiology was transformed from a somewhat arcane discipline to a glamorous field that promised to reveal the secrets of life. While today, most geobiologists would agree that the evidence for past life in AH84001 is inconclusive at best, and find the hype surrounding its discovery to be comical, nonetheless, the excitement it engendered has had a long-lasting and positive impact on our science. The enduring consequence of Clinton's press conference was that it called attention to the fact that life has been leaving signatures in its environment (be it earthly or extraterrestrial) for billions of years. In the years following the meteorite's discovery, it has become clear that to understand life's traces and-more importantly---effects on its environment, it is necessary to understand how life leaves its imprint and whether this can be distinguished from similar imprints left by abiotic processes. This is a central challenge in geobiology.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/29146, title ="Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria", author = "Bailey, Jake V. and Salman, Verena", journal = "ISME Journal", volume = "5", number = "12", pages = "1926-1935", month = "December", year = "2011", doi = "10.1038/ismej.2011.66", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120206-101449345", note = "© 2011 International Society for Microbial Ecology. \n\nReceived 20 September 2010; revised 28 March 2011; accepted 3 May 2011; published online 23 June 2011. \n\nThis study was funded by the Agouron Institute, the\nUniversity of Minnesota Department of Geology and\nGeophysics, the Max Planck Society, and the National\nScience Foundation (OCE-0826254 and OCE 09-39557).\nParts of this work were carried out in the Characterization\nFacility, University of Minnesota, a member of the NSF funded\nMaterials Research Facilities Network (http://www.mrfn.org) via the MRSEC program with assistance\nfrom Chris Frethem and Fang Zhou. Scanning electron\nmicroscopy was performed in the University of Minnesota,\nTwin Cities, Limnological Research Center with the\nsupport of Amy Myrbo. Furthermore, we thank Tony\nRathburn, Shana Goffredi, Anders Waren, Danwei Huang,\nAnne Dekas, Olivia Mason, Ben Harrison, the crew of the\nR/V Atlantis and pilots of the DSV Alvin (15–44 and\n15–59) for assistance at sea, and Ben Grupe for providing\nlive gastropods from Hydrate Ridge. We thank the\nreviewers of this manuscript for valuable comments that\nhelped improve the manuscript. The authors declare no conflict of interest.", revision_no = "17", abstract = "We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/51702, title ="The Apparent Involvement of ANMEs in Mineral Dependent Methane Oxidation, as an Analog for Possible Martian Methanotrophy", author = "House, Christopher H. and Beal, Emily J.", journal = "Life", volume = "1", number = "1", pages = "19-33", month = "November", year = "2011", doi = "10.3390/life1010019", issn = "2075-1729", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141113-095602878", note = "© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). \n\nReceived: 25 August 2011; in revised form: 14 September 2011; Accepted: 11 November 2011; Published: 18 November 2011. \n\nWe thank three anonymous reviewers for very useful comments, suggestions, and critics, as well as\nA. Dekas, A. Green-Saxena, A. Schmitt and Z. Zhang for technical assistance. We also thank the\nshipboard scientists, crew and pilots of R ⁄ V Atlantis. Funding for this project has come from the\nNational Science Foundation (MCB-0348492), National Aeronautics and Space Administration\n(NASA) Astrobiology Institute under NASA–Ames Cooperative Agreement NNA04CC06A,\nDepartment of Energy (DE-SC0004949), and the Penn State Biogeochemical Research Initiative for\nEducation (BRIE) funded by NSF (IGERT) Grant DGE-9972759.The UCLA ion Microprobe\nis partially supported by a grant from the National Science Foundation Instrumentation and\nFacilities Program.", revision_no = "13", abstract = "On Earth, marine anaerobic methane oxidation (AOM) can be driven by the\nmicrobial reduction of sulfate, iron, and manganese. Here, we have further characterized\nmarine sediment incubations to determine if the mineral dependent methane oxidation\ninvolves similar microorganisms to those found for sulfate-dependent methane oxidation.\nThrough FISH and FISH-SIMS analyses using ^(13)C and ^(15)N labeled substrates, we find that\nthe most active cells during manganese dependent AOM are primarily mixed and\nmixed-cluster aggregates of archaea and bacteria. Overall, our control experiment using\nsulfate showed two active bacterial clusters, two active shell aggregates, one active mixed\naggregate, and an active archaeal sarcina, the last of which appeared to take up methane in\nthe absence of a closely-associated bacterial partner. A single example of a shell aggregate\nappeared to be active in the manganese incubation, along with three mixed aggregates and\nan archaeal sarcina. These results suggest that the microorganisms (e.g., ANME-2) found\nactive in the manganese-dependent incubations are likely capable of sulfate-dependent\nAOM. Similar metabolic flexibility for Martian methanotrophs would mean that the same\nmicrobial groups could inhabit a diverse set of Martian mineralogical crustal environments.\nThe recently discovered seasonal Martian plumes of methane outgassing could be coupled\nto the reduction of abundant surface sulfates and extensive metal oxides, providing a feasible metabolism for present and past Mars. In an optimistic scenario Martian\nmethanotrophy consumes much of the periodic methane released supporting on the order\nof 10,000 microbial cells per cm2 of Martian surface. Alternatively, most of the methane\nreleased each year could be oxidized through an abiotic process requiring biological\nmethane oxidation to be more limited. If under this scenario, 1% of this methane flux were\noxidized by biology in surface soils or in subsurface aquifers (prior to release), a total of\nabout 10^(20) microbial cells could be supported through methanotrophy with the cells\nconcentrated in regions of methane release.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/25114, title ="Microbiology: Hydrogen for dinner", author = "Orphan, Victoria J. and Hoehler, Tori M.", journal = "Nature", volume = "476", number = "7359", pages = "154-155", month = "August", year = "2011", doi = "10.1038/476154a", issn = "0028-0836", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110826-090605868", note = "© 2011 Macmillan Publishers Limited. \n\nPublished online 10 August 2011.", revision_no = "13", abstract = "The vast array of bacterium–animal symbioses at deep-sea hydrothermal vents was thought to be fuelled by just two chemicals. A study of one such symbiosis in its environmental context reveals a third energy source.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23239, title ="Multicellular photo-magnetotactic bacteria", author = "Shapiro, Orr H. and Hatzenpichler, Roland", journal = "Environmental Microbiology Reports", volume = "3", number = "2", pages = "233-238", month = "April", year = "2011", doi = "10.1111/j.1758-2229.2010.00215.x", issn = "1758-2229", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110404-113053920", note = "© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.\n\nReceived 1 February, 2010; accepted 17 August, 2010.\nArticle first published online: 30 Sep. 2010.\n\nThis study was performed during the 2009 Microbial \nDiversity course at the Marine Biological Laboratory, Woods Hole, MA, USA. The Microbial Diversity course was supported by funding from the US National Science Foundation, the US\nDepartment of Energy, The Gordon and Betty Moore Foundation,\nthe Howard Hughes Medical Foundation and the Marine Biological Laboratories. OHS and RH acknowledge financial support by The Gordon and Betty Moore Foundation, the University of Vienna, the Moshe Shilo Memorial Fund, and the Ben Gurion University of the Negev. OHS is recipient of a Levi-Eshkol PhD-fellowship from the Israeli Ministry of Science. RH is recipient of a PhD-fellowship (DOC) of the Austrian Academy of Sciences. OHS and RH thank all faculty, lecturers, students (a.k.a. the micronauts) and teaching assistants (most of all Cristina Moraru) for a great summer. Heather Fullerton is acknowledged for help with sampling. We thank Alexander Petroff for helpful discussions, and Zeiss for providing microscopic equipment and technical support.", revision_no = "23", abstract = "Multicellular magnetotactic bacteria (MMB) are unique microorganisms typically comprised of 10–40 bacterial cells arranged around a central acellular compartment. Their life cycle has no known unicellular stage and division occurs by separation of a single MMB aggregate into two identical offspring. In this study, South-seeking multicellular magnetotactic bacteria (ssMMB) were enriched from a New England salt marsh. When exposed to light, ssMMB reversed their magnetotactic behaviour to become North-seeking. The exposure time needed to generate the reversal response varied with light wavelength and intensity. Extensive exposure to light appeared to be lethal. This is the first report of a Northern hemisphere MMB displaying South-seeking behaviour and the first time a MMB is found to exhibit photo-magnetotaxis. We suggest that this mechanism enables ssMMB to optimize their location with regard to chemical gradients and light intensities, and propose a model to explain the peculiar balance between photo- and magnetotaxis.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47450, title ="Fish-Sims: Characterizing the Metabolic Potential and Interspecies Interactions between Uncultured Environmental Microorganisms", author = "Orphan, Victoria", journal = "Biophysical Journal", volume = "100", number = "3, S1", pages = "37a", month = "February", year = "2011", issn = "0006-3495", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327659", note = "Copyright © 2011 Biophysical Society.", revision_no = "12", abstract = "The ability to link phylogenetic identity with metabolic function is one of the longstanding goals in the field of microbial ecology. With greater than 99% of the global microbial diversity missing from culture collections, there is a critical need for alternative, culture-independent approaches that can extend beyond diversity surveys and directly illuminate the ecological and biogeochemical roles of microorganisms in nature. To this end, research incorporating the use of secondary ion mass spectrometry (SIMS and nanoSIMS) combined with stable isotope tracers and molecular methodologies such as fluorescence in situ hybridization (FISH) have opened new and exciting avenues of research to study the metabolic potential of individual microorganisms, microbial consortia, and symbiotic associations. Here, we will highlight the application of FISH combined with nanoSIMS ion imaging to unravel complex microbial associations and trophic relationships in marine ecosystems fueled by methane.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/22708, title ="A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gammaproteobacterial methanotrophs", author = "Tavormina, Patricia L. and Orphan, Victoria J.", journal = "Environmental Microbiology Reports", volume = "3", number = "1", pages = "91-100", month = "February", year = "2011", doi = "10.1111/j.1758-2229.2010.00192.x ", issn = "1758-2229", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110308-093513808", note = "© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 20 February, 2010; accepted 1 June, 2010. Article first published online: 20 Jul. 2010. \n\nWe thank Dan Arp for providing genomic DNA from\nNitrosomonas europaea, and Burt Thomas for peat bog\nsamples. We also thank three anonymous reviewers for critical reading of this manuscript. Funding for this project was provided by the Gordon and Betty Moore Foundation, and from National Aeronautics Space Administration grant NNF06GB34G (VJO), UW Royalty Research Fund grant 4064 and NSF Grant MCB-0604269 (M.G. Kalyuzhnaya), ERC Advanced Grant 232937 (MSMJ) and the Office of the EVP Research of UofL (M.G. Klotz).", revision_no = "21", abstract = "Genomes of alphaproteobacterial and verrucomicrobial methane-oxidizing bacteria (MOB) encode sequence-divergent copies of particulate methane monooxygenase [pMMO = (PmoABC); pmoCAB]. In contrast, sequenced gammaproteobacterial MOB (Gamma-MOB) genomes contain single or multiple near-identical copies of pmoCAB operons. In betaproteobacterial ammonia-oxidizing bacteria (Beta-AOB), near-identical amoCAB operons encode ammonia monooxygenase (AMO), a homologue of pMMO. Here, we report that Gamma-MOB in the genera Methylomonas, Methylobacter and Methylomicrobium also encode a sequence-divergent particulate monooxygenase (pXMO). Whereas all known genes encoding pMMO or AMO cluster in the order ‘CAB’, the genes encoding pXMO are uniquely organized in the non-canonical form ‘pxmABC.’ Steady state pxm mRNA was detected in cultures of Methylomonas sp. as well as in freshwater creek sediment samples, demonstrating that pxm genes are expressed in culture and in situ. Inclusion of PxmA and PxmB proteins in phylogenetic analyses of the Pmo/Amo protein superfamilies created trifurcated trees with three major clades: (i) Pmo of Alpha- and Gamma-MOB and Amo of Gamma-AOB; (ii) Amo of Beta-AOB, Pmo of putative ethane-oxidizing Gamma-MOB and Pxm of Gamma-MOB; and (iii) verrucomicrobial Pmo and Amo of ammonia-oxidizing Archaea. These data support but do not prove the hypothesis that oxygen-dependent methane and ammonia monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer while being integrated into the catabolic contexts of their extant hosts.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/22687, title ="Getting cozy: hidden microbial interactions in nature", author = "Orphan, Victoria J.", journal = "Environmental Microbiology Reports", volume = "3", number = "1", pages = "16-18", month = "February", year = "2011", doi = "10.1111/j.1758-2229.2010.00236.x", issn = "1758-2229", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110307-110634731", note = "© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nArticle first published online: 8 Feb. 2011.", revision_no = "15", abstract = "Understanding microbial interactions is integral to microbial\necology and yet this fundamental component has\nproven to be one of the most challenging to define and\nstudy in nature. While trophic structure, competition and\nfitness are often discussed in the context of microbial\ncommunities, the description of microbe-microbe symbiotic\nassociations are rare (Overmann and Schubert,\n2002), and if identified, are often poorly characterized.\nBroadly defined, symbiosis covers a wide spectrum of\ninteractions, ranging from beneficial associations (syntrophy\nand mutualism) to deleterious relationships (parasitism).\nSyntrophic associations, for example, have long\nbeen recognized as a fundamental component of organic\ncarbon mineralization in anaerobic environments (Schink,\n2002). Parasitic interactions between microorganisms,\nhowever, are far less frequently described and perhaps\nmore difficult to define. In most cases, symbiotic microbial\nassociations involve close physical coupling between\npartners, and through these intimate interspecies interactions,\ncan lead to metabolic innovation and niche expansion.\nRegardless of the nature of the symbiosis, it is\nbecoming clear that these intimate microbial associations\nare likely prevalent in nature, and await the proper tools\nfor discovery.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23702, title ="Identification of Diazotrophic Microorganisms in Marine Sediment via Fluorescence In Situ Hybridization Coupled to Nanoscale Secondary Ion Mass Spectrometry (FISH-NanoSIMS)", author = "Dekas, Anne E. and Orphan, Victoria J.", number = "486", pages = "281-305", month = "January", year = "2011", doi = "10.1016/B978-0-12-381294-0.00012-2 ", isbn = "9780123812940", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110518-083725900", note = "© 2011 Elsevier Inc. \n\nAvailable online 23 December 2010. \n\nWe thank Christopher House, Yunbin Guan, John Eiler, Kendra Turk, Annelie Pernthaler, Sameer Walavalkar, Kevin McKeegan, Raj Singh, and Mark Ellisman for helping to develop and optimize the method. We thank James Howard, Sameer Walavalkar, and Eric Matson for sample and data contributions to this chapter. We also thank two anonymous reviewers for helpful suggestions. This research was supported by grants from the Department of Energy (Career award, #DE-SC0003940) and Gordon and Betty Moore Foundation (to VJO) and the National Science Foundation (graduate fellowship to AED).", revision_no = "15", abstract = "Growing appreciation for the biogeochemical significance of uncultured microorganisms is changing the focus of environmental microbiology. Techniques designed to investigate microbial metabolism in situ are increasingly popular, from mRNA-targeted fluorescence in situ hybridization (FISH) to the \"-omics\" revolution, including metagenomics, transcriptomics, and proteomics. Recently, the coupling of FISH with nanometer-scale secondary ion mass spectrometry (NanoSIMS) has taken this movement in a new direction, allowing single-cell metabolic analysis of uncultured microbial phylogenic groups. The main advantage of FISH-NanoSIMS over previous noncultivation-based techniques to probe metabolism is its ability to directly link 16S rRNA phylogenetic identity to metabolic function. In the following chapter, we describe the procedures necessary to identify nitrogen-fixing microbes within marine sediment via FISH-NanoSIMS, using our work on nitrogen fixation by uncultured deep-sea methane-consuming archaea as a case study.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/18649, title ="Distributions of putative aerobic methanotrophs in diverse pelagic marine environments", author = "Tavormina, Patricia L. and Ussler, William, III", journal = "ISME Journal", volume = "4", number = "5", pages = "700-710", month = "May", year = "2010", doi = "10.1038/ismej.2009.155", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100611-084926449", note = "© 2010 International Society for Microbial Ecology. \n\nReceived 3 August 2009; Revised 17 December 2009; Accepted 17 December 2009; Published online 11 February 2010. \n\nWe thank Mary Lidstrom, Dan Arp and Martin Klotz\nfor reference strains, Steve Giovannoni's group for\nBATS DNA, Bob Vrijenhoek for the Fiji/Lau expedition\nand Shana Goffredi, Olivia Mason, and 3 anonymous\nreviewers for assistance, critical comments, and improving\nthe quality of this study. Support was provided by NASA\n(NNG06GB34G to VJO), NSF (MCB-0348492 to VJO and\nOCE-0085549 to SBJ, the Gordon and Betty Moore\nFoundation (to VJO) and the David and Lucile Packard\nFoundation (to WU). \n\nWe thank Mary Lidstrom, Dan Arp and Martin Klotz for reference strains. Steve Giovannoni’s group for BATS DNA, Bob Vrijenhoek for the Fiji/Lau expedition and Shana Goffredi, Olivia Mason, and 3 anonymous reviewers for assistance, critical comments, and improving the quality of this study. Support was provided by NASA (NNG06GB34G to VJO), NSF (MCB-0348492 to VJO and OCE-0085549 to SBJ), the Gordon and Betty Moore Foundation (to VJO) and the David and Lucile Packard Foundation (to WU).", revision_no = "40", abstract = "Aerobic methane oxidization in the pelagic ocean serves an important role in limiting methane release to the atmosphere, yet little is known about the identity and distribution of bacteria that mediate this process. The distribution of putative methane-oxidizing marine groups, OPU1, OPU3 and Group X, was assessed in different ocean provinces using a newly developed fingerprinting method (monooxygenase intergenic spacer analysis (MISA)) in combination with pmoA clone library analysis and quantitative PCR (qPCR). The distribution of these three distinct monooxygenase groups, previously reported from pelagic marine environments, was examined in 39 samples including active methane seeps in the Gulf of Mexico and Santa Monica Bay, submarine canyon heads along the California continental margin, an oligotrophic subtropical gyre and areas proximal to a hydrothermal vent in the North Fiji back-arc basin. OPU1 and OPU3 were widely and similarly distributed within the meso-and bathypelagic zone (110 to similar to 2000 m water depth) and showed a >50-fold greater abundance near methane seeps relative to non-seep sites. In contrast, Group X was predominantly recovered from samples along the California margin, at both seep and non-seep sites. All three phylotypes were below detection in the epipelagic zone to depths of 100 m. Several additional deeply branching monooxygenase sequences were also identified in this study, indicating the presence of uncharacterized groups of microorganisms potentially involved in the cycling of methane or ammonium.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17668, title ="Bacterial community shifts in taxa and diversity in response to localized organic loading in the deep sea", author = "Goffredi, Shana K. and Orphan, Victoria J.", journal = "Environmental Microbiology", volume = "12", number = "2", pages = "344-363", month = "February", year = "2010", doi = "10.1111/j.1462-2920.2009.02072.x ", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100304-142423277", note = "© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 8 June, 2009; accepted 29 July, 2009. \n\nThis work was supported in part by the Gordon and Betty\nMoore Foundation (to V.J.O.) and the US National Science\nFoundation (MCB-0454860 to S.K.G.). The authors thank: the\nR.O.V. Tiburon pilots and R.V.Western Flyer crew, the R.O.V.\nVentana pilots and R.V. Pt. Lobos crew, chief scientist R.C.\nVrijenhoek for allowing our participation in research cruises, S. Johnson and W.J. Jones for shipboard support, R. Wilpiszeski for laboratory assistance at Caltech, G. Martin for laboratory space at Occidental College, D. Gruber for conducting protease assays with funding via the Occidental College summer research program, R. Lee for TOC and isotope analyses, B. Ussler for help with methane measurements, N. Dalleska for IC-PMS assistance, O. Mason and B. Harrison for help with ARB, W. Ziebis for hydrogen analysis and V. Rich for invaluable scientific and editorial advice. Images in Fig. 1 were provided by the Monterey Bay Aquarium Research Institute (MBARI). The authors thank Bob Vrijenhoek (MBARI) and members of his lab as well as the captain and crew of the R.V. Western Flyer and pilots of the R.O.V. Tiburon who played an instrumental role in this study. [Correction added on 1 November 2009, after first online publication: the preceding sentence was added after first online publication.]", revision_no = "14", abstract = "The deep sea is a unique and extreme environment characterized by low concentrations of highly recalcitrant carbon. As a consequence, large organic inputs have potential to cause significant perturbation. To assess the impact of organic enrichment on deep sea microbial communities, we investigated bacterial diversity in sediments underlying two whale falls at 1820 and 2893 m depth in Monterey Canyon, as compared with surrounding reference sediment 10–20 m away. Bacteroidetes, Epsilonproteobacteria and Firmicutes were recovered primarily from whale fall-associated sediments, while Gammaproteobacteria and Planctomycetes were found primarily within reference sediments. Abundant Deltaproteobacteria were recovered from both sediment types, but the Desulfobacteraceae and Desulfobulbaceae families were observed primarily beneath the whale falls. UniFrac analysis revealed that bacterial communities from the two whale falls (~30 km apart) clustered to the exclusion of corresponding reference sediment communities, suggesting that deposition of whale fall biomass is more influential on deep sea microbial communities than specific seafloor location. The bacterial population at whale-1820 at 7 months post deposition was less diverse than reference sediments, with Delta- and Epsilonproteobacteria and Bacteroidetes making up 89% of the community. At 70 months, bacterial diversity in reference sediments near whale-2893 had decreased as well. Over this time, there was a convergence of each community's membership at the phyla level, although lower-taxonomic-level composition remained distinct. Long-term impact of organic carbon loading from the whale falls was also evident by elevated total organic carbon and enhanced proteolytic activity for at least 17–70 months. The response of the sedimentary microbial community to large pulses of organic carbon is complex, likely affected by increased animal bioturbation, and may be sustained over time periods that span years to perhaps even decades.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17627, title ="Pseudofossils in relict methane seep carbonates resemble endemic microbial consortia", author = "Bailey, Jake V. and Raub, Timothy D.", journal = "Palaeogeography, Palaeoclimatology, Palaeoecology", volume = "285", number = "1-2", pages = "131-142", month = "January", year = "2010", doi = "10.1016/j.palaeo.2009.11.002", issn = "0031-0182", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100302-113604574", note = "© 2009 Elsevier B.V. \n\nReceived 26 April 2009; revised 1 October 2009; accepted 3 November 2009. Available online 10 November 2009. \n\nWe thank George Rossman and Liz Boyd for technical guidance and support during Raman spectrum collection, Ray Lee for carbon isotope analyses, Frank Corsetti for helpful discussions and two anonymous reviewers for comments that greatly improved this manuscript. We also thank Joe Kirschvink and Jess Adkins for supporting major instrumentation used in our analyses. Funding for this work was provided by the NASA Astrobiology Institute (Grant award # 3903-CIT-NASA.A76A) as part of the Penn State Astrobiology Research Center (PSARC) and grants from the Gordon and Betty Moore Foundation (to VJO), a research scholarship from the German Research Foundation (to ANM), and the Agouron Institute Geobiology Postdoctoral Fellowship (to JVB). Magnetic microscopy was supported by a Human Frontiers Science Program grant (RGP0028/2007-C) and rock-magnetic analysis was supported by NASA Exobiology grant (NNX07AK12G) to J.L. Kirschvink. Collection of samples in the Eel River Basin was supported by the NOAA-National Underseas Research Program (UAF 05-0132).", revision_no = "14", abstract = "Pleistocene-age methane seep carbonates from the Eel River Basin, California contain aggregate-like structures composed of tightly-packed hollow spheres that morphologically resemble syntrophic archaeal–bacterial consortia known to catalyze the anaerobic oxidation of methane (AOM). Tetragonal microstructures also present in the carbonates resemble seep-endemic Methanosarcinales cell clusters. Despite morphological similarities to the seep-endemic microbes that likely mediated the authigenesis of Eel River Basin carbonates and sulfides, detailed petrographic, SEM, and magnetic microscopic imaging, remanence rock magnetism, laser Raman, and energy dispersive X-ray spectroscopy, suggest that these microstructures are not microfossils, but rather mineral structures that result from the diagenetic alteration of euhedral Fe-sulfide framboids. Electron microscopy shows that during diagenesis, reaction rims composed of Fe oxide form around framboid microcrystalites. Subsequent dissolution of greigite or pyrite crystals leaves behind hollow cell-like casings (external molds) — a transformation that occurs on timescales of ~100 kyr or less. Despite their superficial resemblance to morphologically-distinctive extant microbes in local sediments, the presence of acellular precursor grains, as well as of partially-altered transitional forms, complicate the interpretation of these and other framboidal microstructures that have been reported from the rock record.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17062, title ="Chemotrophic Microbial Mats and Their Potential for Preservation in the Rock Record", author = "Bailey, Jake V. and Orphan, Victoria J.", journal = "Astrobiology", volume = "9", number = "9", pages = "843-859", month = "November", year = "2009", doi = "10.1089/ast.2008.0314", issn = "1557-8070", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100105-130209242", note = "© 2009 Mary Ann Liebert, Inc. \n\nWe thank Bo Barker Jørgensen, Heide Schulz-Vogt, Tina\nTreude, Jens Greinert, Chris House, Sarah Greene, Beth Orcutt, Katrina Edwards, and Ian R. MacDonald for contributing or for helping to acquire images used in this\nmanuscript. We also thank Heide Schulz-Vogt, Tomaso Bontognali, and four anonymous reviewers for helpful\ncomments that greatly improved this manuscript. J.V.B. is\nsupported by the Agouron Institute Geobiology Postdoctoral\nFellowship Program (Grant # AI-F-GB12.09.2). The Gulf of\nMexico, Eel River Basin, and Costa Rica Margin images were\nobtained during projects funded by the U.S. National Science\nFoundation (OCE-0085549 to S.B.J.; MCB-0348492 to V.J.O.;\nand OCE-0825791 to V.J.O.). Images from the Loihi Seamount\nwere taken during cruise MGLN10MV, Jason Dive J2-242 (Woods Hole Oceanographic Institute) as part of the Iron Microbiology Observatory Project.", revision_no = "13", abstract = "Putative microbialites are commonly regarded to have formed in association with photosynthetic microorganisms, such as cyanobacteria. However, many modern microbial mat ecosystems are dominated by chemotrophic bacteria and archaea. Like phototrophs, filamentous sulfur-oxidizing bacteria form large mats at the sediment/water interface that can act to stabilize sediments, and their metabolic activities may mediate the formation of marine phosphorites. Similarly, bacteria and archaea associated with the anaerobic oxidation of methane (AOM) catalyze the precipitation of seafloor authigenic carbonates. When preserved, lipid biomarkers, isotopic signatures, body fossils, and lithological indicators of the local depositional environment may be used to identify chemotrophic mats in the rock record. The recognition of chemotrophic communities in the rock record has the potential to transform our understanding of ancient microbial ecologies, evolution, and geochemical conditions. Chemotrophic microbes on Earth occupy naturally occurring interfaces between oxidized and reduced chemical species and thus may provide a new set of search criteria to target life-detection efforts on other planets.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16813, title ="Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia", author = "Dekas, Anne E. and Poretsky, Rachel S.", journal = "Science", volume = "326", number = "5951", pages = "422-426", month = "October", year = "2009", doi = "10.1126/science.1178223 ", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20091125-115957263", note = "© 2009 American Association for the Advancement of Science.\n\nWe thank C. House, A. Schmitt, K. McKeegan, Y. Guan,\nJ. Eiler, and L. Remusat for assistance with the ion\nmicroprobe data collection; S. Joye, M. Boyles, M. Walton,\nJ. Howard, N. Dalleska, O. Mason, A. Green,\nP. Tavormina, S. Goffredi, C. Gammon, and the shipboard\nparty and crew of the R/V Atlantis and DSSV Alvin for\nsupport in the field and laboratory; and J. Howard,\nW. Fischer, J. Amend, C. Anderson, D. Fike, D. Sigman,\nV. Rich, J. Bailey, D. Newman, J. Grotzinger, T. Hoehler,\nJ. Delacruz, and three anonymous reviewers for helpful\nsuggestions regarding this manuscript. Funding was\nprovided by NSF (grant MCB-0348492), the Gordon and\nBetty Moore Foundation, and an NSF Graduate Research\nFellowship (A.E.D.). The Caltech Center for Microanalysis\nand nanoSIMS 50L are funded by the Gordon and Betty\nMoore Foundation, and the University of California Los\nAngeles ion microprobe is partially supported by the NSF\nInstrumentation and Facilities Program.", revision_no = "15", abstract = "Nitrogen-fixing (diazotrophic) microorganisms regulate productivity in diverse ecosystems; however, the identities of diazotrophs are unknown in many oceanic environments. Using single-cell–resolution nanometer secondary ion mass spectrometry images of ^(15)N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N_2, as well as structurally similar CN^–, and share the products with sulfate-reducing bacterial symbionts. These archaeal/bacterial consortia are already recognized as the major sink of methane in benthic ecosystems, and we now identify them as a source of bioavailable nitrogen as well. The archaea maintain their methane oxidation rates while fixing N_2 but reduce their growth, probably in compensation for the energetic burden of diazotrophy. This finding extends the demonstrated lower limits of respiratory energy capable of fueling N_2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17327, title ="The effect of sulfate concentration on (sub)millimeter-scale sulfide δ^(34)S in hypersaline cyanobacterial mats over the diurnal cycle", author = "Fike, David A. and Finke, Niko", journal = "Geochimica et Cosmochimica Acta", volume = "73", number = "20", pages = "6187-6204", month = "October", year = "2009", doi = "10.1016/j.gca.2009.07.006 ", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100127-153113402", note = "© 2009 Elsevier Ltd. \n\nReceived 9 January 2009; accepted 6 July 2009. Associate editor: Juske Horita. Available online 12 July 2009. \n\nWe would like to acknowledge the Gordon and Betty Moore\nFoundation and the Caltech Center for Geochemical and Cosmochemical Microanalysis for funding (to V.J.O.) as well as support from National Aeronautics and Space Administration grant NAI02-003-0001 issued through the Astrobiology Program and NASA grant 07-EXOB07-0093 issued through the Exobiology Program. D.A.F. was additionally supported by the Caltech O.K. Earl Postdoctoral Fellowship. N.F. was supported by an EU-Marie Curie Postdoctoral Fellowship Contract MOIF-CT-2005-22154. We would like to thank Y. Guan and J. Eiler for analytical assistance and invaluable discussions, and T. Lyons for conventional d34S analysis of aqueous sulfate. We are also indebted to the NASA Ames group (K. Turk, M. Kubo, L. Jahnke and D. Des Marais) for support and assistance with sample collections.", revision_no = "13", abstract = "Substantial isotopic fractionations are associated with many microbial sulfur metabolisms and measurements of the bulk δ^(34)S isotopic composition of sulfur species (predominantly sulfates and/or sulfides) have been a key component in developing our understanding of both modern and ancient biogeochemical cycling. However, the interpretations of bulk δ^(34)S measurements are often non-unique, making reconstructions of paleoenvironmental conditions or microbial ecology challenging. In particular, the link between the μm-scale microbial activity that generates isotopic signatures and their eventual preservation as a bulk rock value in the geologic record has remained elusive, in large part because of the difficulty of extracting sufficient material at small scales. Here we investigate the potential for small-scale (~100 μm–1 cm) δ^(34)S variability to provide additional constraints for environmental and/or ecological reconstructions. We have investigated the impact of sulfate concentrations (0.2, 1, and 80 mM SO_4) on the δ^(34)S composition of hydrogen sulfide produced over the diurnal (day/night) cycle in cyanobacterial mats from Guerrero Negro, Baja California Sur, Mexico. Sulfide was captured as silver sulfide on the surface of a 2.5 cm metallic silver disk partially submerged beneath the mat surface. Subsequent analyses were conducted on a Cameca 7f-GEO secondary ion mass spectrometer (SIMS) to record spatial δ^(34)S variability within the mats under different environmental conditions. Isotope measurements were made in a 2-dimensional grid for each incubation, documenting both lateral and vertical isotopic variation within the mats. Typical grids consisted of ~400–800 individual measurements covering a lateral distance of ~1 mm and a vertical depth of ~5–15 mm. There is a large isotopic enrichment (10–20‰) in the uppermost mm of sulfide in those mats where [SO_4] was non-limiting (field and lab incubations at 80 mM). This is attributed to rapid recycling of sulfur (elevated sulfate reduction rates and extensive sulfide oxidation) at and above the chemocline. This isotopic gradient is observed in both day and night enrichments and suggests that, despite the close physical association between cyanobacteria and select sulfate-reducing bacteria, photosynthetic forcing has no substantive impact on δ^(34)S in these cyanobacterial mats. Perhaps equally surprising, large, spatially-coherent δ^(34)S oscillations (~20–30‰ over 1 mm) occurred at depths up to ~1.5 cm below the mat surface. These gradients must arise in situ from differential microbial metabolic activity and fractionation during sulfide production at depth. Sulfate concentrations were the dominant control on the spatial variability of sulfide δ^(34)S. Decreased sulfate concentrations diminished both vertical and lateral δ^(34)S variability, suggesting that small-scale variations of δ^(34)S can be diagnostic for reconstructing past sulfate concentrations, even when original sulfate δ^(34)S is unknown.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16031, title ="Extensive carbon isotopic heterogeneity among methane seep microbiota", author = "House, Christopher H. and Orphan, Victoria J.", journal = "Environmental Microbiology", volume = "11", number = "9", pages = "2207-2215", month = "September", year = "2009", doi = "10.1111/j.1462-2920.2009.01934.x", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090923-143137088", note = "Copyright © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 25 November, 2008; accepted 11 March, 2009. Published Online: 7 June 2009. \n\nWe thank Zhidan Zhang and Tsegereda Embaye for laboratory assistance, and the captain and crew of the R/V Western Flyer and ROV Tiburon for their tireless efforts during the field expedition. We also thank Katherine H. Freeman for the opportunity to measure the δ^(13)C isotopic composition of our Escherichia coli cells by EA-CF-IRMS in the PSU Isotope Biogeochemistry Laboratory. This work was funded by the Penn State Astrobiology Research Center (through the National Astrobiology Institute), NOAA-NURP (UAF 05-0132), the National Science Foundation (MCB-0348492 and OCE-0085549), and the ACS-PRF. The UCLA ion Microprobe is partially supported by a grant from the National Science Foundation Instrumentation and Facilities Program. Also, the ion microprobe work in this paper was supported by a grant from the Moore Foundation. Graduate support (B.T.) for this project was provided by the Penn State Biogeochemical Research Initiative for Education funded by NSF (IGERT) Grant DGE-9972759. \n\nSupporting information: Additional Supporting Information may be found in the online version of this article:\nFig. S1. An drawing of the in situ incubation device (‘porewater peeper’) used to place Si-wafers and glass slides into seep sediment. There are a series of seven 1-inch-diameter glass slides and seven 1 inch Si-wafers placed so that the surfaces were in contact with a 1 ml internal volume of anoxic 20 g l-1 NaCl solution. The slides and wafers are behind either a 0.2 mm polycarbonate filter (Whatman) or a 12 mm polycarbonate filter. There is also a mesh covering protecting the filters. Insert: photograph of peeper after 14 months in seep sediment. The mesh covering can be seen in each of the holes.\nTable S1. FISH-SIMS d13C data for microorganisms in PC55 from the Eel River Basin.", revision_no = "25", abstract = "To assess and study the heterogeneity of δ^(13)C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ^(13)C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ^(13)C values (> 50‰) in this microbial community. The δ^(13)C of ANME-1 rods ranged from −24‰ to −87‰. The δ^(13)C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ^(13)C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ^(13)C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ^(13)C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/14795, title ="Manganese- and iron-dependent marine methane oxidation", author = "Beal, Emily J. and House, Christopher H.", journal = "Science", volume = "325", number = "5937", pages = "184-187", month = "July", year = "2009", doi = "10.1126/science.1169984", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090804-142607950", note = "© 2009 American Association for the Advancement of Science. \n\n18 December 2008; accepted 2 June 2009. \n\nWe would like to thank M. Arthur for the use of his mass spectrometer, Z. Zhang and S. Goffredi for laboratory assistance, D. Walizer for technical assistance, and D. Jones for help with phylogenetics. We also thank the shipboard scientists, crew, and pilots of R/V Atlantis and R/V Western Flyer. Funding for this project has come from the National Science Foundation (MCB-0348492), National Aeronautics and Space Administration (NASA) Astrobiology Institute under NASA-Ames Cooperative Agreement NNA04CC06A, and the Penn State Biogeochemical Research Initiative for Education (BRIE) funded by NSF (IGERT) grant DGE-9972759. Sequences were submitted to GenBank and have accession numbers FJ264513 to FJ264602 and FJ264604 to FJ264884.", revision_no = "20", abstract = "Anaerobic methanotrophs help regulate Earth’s climate and may have been an important part of the microbial ecosystem on the early Earth. The anaerobic oxidation of methane (AOM) is often thought of as a sulfate-dependent process, despite the fact that other electron acceptors are more energetically favorable. Here, we show that microorganisms from marine methane-seep sediment in the Eel River Basin in California are capable of using manganese (birnessite) and iron (ferrihydrite) to oxidize methane, revealing that marine AOM is coupled, either directly or indirectly, to a larger variety of oxidants than previously thought. Large amounts of manganese and iron are provided to oceans from rivers, indicating that manganese- and iron-dependent AOM have the potential to be globally important.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/15698, title ="Patterns of ^(15)N assimilation and growth of methanotrophic ANME-2 archaea and sulfate-reducing bacteria within structured syntrophic consortia revealed by FISH-SIMS", author = "Orphan, Victoria J. and Turk, Kendra A.", journal = "Environmental Microbiology", volume = "11", number = "7", pages = "1777-1791", month = "July", year = "2009", doi = "10.1111/j.1462-2920.2009.01903.x", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090909-123033461", note = "Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 10 October, 2008; accepted 6 February, 2009. \n\nWe thank A. Schmitt, K. McKeegan and G. Jarzebinski from the UCLA ion microprobe facility, and C. Gammon, A. Pernthaler and T. Embaye for their technical assistance with this project. We also acknowledge S. Goffredi, D. Fike, K.-U. Hinrichs and two anonymous reviewers for critical reading of this manuscript and thank S. Joye and A. Pearson for fruitful discussions. We are grateful to the captain, pilots, crew and shipboard research parties of the R/V Western Flyer and R/V Atlantis (AT 15-11) for their invaluable support. This work was supported by grants from the National Science Foundation (MCB-0348492), NOAA Undersea Research Program (UAF 05-0132), and the Gordon and Betty Moore Foundation (to V.J.O.). A.M.G. was supported by a NSF Graduate Research Fellowship. \n\nAppendix S1. Heterogeneity in δ13C and 15N assimilation in sediment incubations exhibiting low anaerobic oxidation of methane activity.\n\nFig. S1. Relationship between δ13C (‰) and 15N (atom %) for shell ANME-2/DSS after a 5-day incubation with 15N-labelled ammonium or amino acids from (A) PC-55 (diamonds; n = 6) and PC-76 (squares; n = 10). ‘z’ symbol denotes paired δ13C/15N values for a mixed ANME-2/DSS aggregate. (B) Paired δ13C/15N values for PC-59 cell aggregates after 4-day incubation (open symbols) and 85-day incubation (closed symbols) with ammonium and amino acids. Shell aggregates are represented by a square symbol; mixed aggregates (triangle) and mono-specific ANME-2 clusters are represented by a circle. Plus signs denote ANME/DSS shell aggregates from control incubation without exogenous 15N-labelled nitrogen. In both panels, plotted values include heaviest δ13C (red colour) and lightest δ13C (black) data points for each ANME-2 or ANME/DSS aggregate measured during the FISH-SIMS analysis.\n\nFig. S2. Comparison of the relationship between aggregate size and enrichment in 15Nbiomass (atom %) for shell consortia.\nA. Maximum 15N enrichment after 5-day incubation for shell aggregates in PC-76 and PC-55.\nB. Box plot showing the average 15N value, range, and 95% confidence intervals for shell aggregates with diameters between 2 and 7 µm (mean 15N atom % = 1.8, n = 9) and aggregates ranging between 7 and 20 µm (mean 15N atom % = 1.4, n = 6).\nC. Maximum 15N enrichment after 112-day incubation for shell aggregates in PC-76. Although a general trend of greater 15N assimilation by smaller shell aggregates was present, the statistical significance of aggregate size and 15N enrichment was not observed (P = 0.44, Wilcoxon test).\n\nTable S1. Fluorescence in situ hybridization (FISH) quantification of per cent change in ANME and bacteria over time.\n\nTable S2. Temporal variation in ratio of ANME-2/DSS shell aggregate size in PC-76.", revision_no = "16", abstract = "Methane release from the oceans is controlled in large part by syntrophic interactions between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (DSS), frequently found as organized consortia. An understanding of the specifics of this symbiotic relationship and the metabolic heterogeneity existing between and within individual methane-oxidizing aggregates is currently lacking. Here, we use the microanalytical method FISH-SIMS (fluorescence in situ hybridization-secondary ion mass spectrometry) to describe the physiological traits and anabolic activity of individual methanotrophic consortia, specifically tracking ^(15)N-labelled protein synthesis to examine the effects of organization and size on the metabolic activity of the syntrophic partners. Patterns of 15N distribution within individual aggregates showed enhanced ^(15)N assimilation in ANME-2 cells relative to the co-associated DSS revealing a decoupling in anabolic activity between the partners. Protein synthesis in ANME-2 cells was sustained throughout the core of individual ANME-2/DSS consortia ranging in size range from 4 to 20 μm. This indicates that metabolic activity of the methane-oxidizing archaea is not limited to, or noticeably enhanced at the ANME−2/DSS boundary. Overall, the metabolic activity of both syntrophic partners within consortia was greater than activity measured in representatives of the ANME-2 and DSS observed alone, with smaller ANME-2/DSS aggregates displaying a tendency for greater ^(15)N uptake and doubling times ranging from 3 to 5 months. The combination of ^(15)N-labelling and FISH-SIMS provides an important perspective on the extent of heterogeneity within methanotrophic aggregates and may aid in constraining predictive models of activity and growth by these syntrophic consortia.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/15699, title ="Geobiological investigations using secondary ion mass spectrometry: microanalysis of extant and paleo-microbial processes", author = "Orphan, V. J. and House, C. H.", journal = "Geobiology", volume = "7", number = "3", pages = "360-372", month = "June", year = "2009", doi = "10.1111/j.1472-4669.2009.00201.x", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090909-130537989", note = "© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd. \n\nReceived 15 February 2009; accepted 13 April 2009. \n\nWe thank A. Dekas, D. Fike, A. Green and Z. Zhang for their data contributions, Y. Guan and L. Remusat for technical support with the Caltech nanoSIMS, K. Freeman for conventional analysis of δ^(13)C in E. coli, and A. Schmidt, K. McKeegan and G. Jarzebinski at UCLA for collaborating on the gallium ion source installation. We also thank S. Goffredi, K. McKeegan, J. Eiler, J. Valley, and two anonymous reviewers for insightful comments and critical reading that improved this work. Funding for this project was provided by the Gordon and Betty Moore Foundation, the National Science Foundation (MCB-0348492), the Penn State and Ames Astrobiology Research Centers (through the NASA Astrobiology Institute), Exobiology grants (07-EXOB07-0093 and NNG05GN50G) and NOAA-NURP (UAF 05-0132). The UCLA ion Microprobe is partially supported by a grant from the National Science Foundation Instrumentation and Facilities Program.", revision_no = "15", abstract = "The application of secondary ion mass spectrometry (SIMS) has tremendous value for the field of geobiology, representing a powerful tool for identifying the specific role of micro-organisms in biogeochemical cycles. In this review, we highlight a number of diverse applications for SIMS and nanoSIMS in geobiological research. SIMS performs isotope and elemental analysis at microscale enabling the investigation of the physiology of individual microbes within complex communities. Additionally, through the study of isotopic or chemical characteristics that are common in both living and ancient microbial communities, SIMS allows for direct comparisons of potential biosignatures derived from extant microbial cells and their fossil equivalents.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47448, title ="High resolution SIMS-based sulfide δ^(34)S: A new tool for characterizing microbial activity in a variety of depositional environments", author = "Fike, David A. and Orphan, Victoria J.", journal = "Geochimica et Cosmochimica Acta", volume = "73", number = "13, S", pages = "A375", month = "June", year = "2009", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327430", note = "© 2009 Elsevier.", revision_no = "12", abstract = "The sulfur isotopic compositions of sedimentary sulfates\nand sulfides are useful for understanding modern microbial\necology and for reconstructing paleoenvironmental conditions\nassociated with the deposition of ancient sediments. In many\nmodern microbially-dominated sedimentary environments,\nsuch as microbial mats, or methane seeps, the redox gradients\ncan be steep with the transition from oxic to sulfidic condition\nover the space of mm to cm. In these environments, it is\nfrequently difficult (either for logistical reasons or sample\nvolume requirements) to sample at a sufficiently high\nresolution to capture the geochemical and microbiological\ndetails associated with these redox transitions. We build upon\nearlier work [1] to demonstrate the ability to capture aqueous\nsulfide as silver sulfide, which can then be analyzed using a\nCameca NanoSIMS 50L or 7F/Geo for its isotopic\ncomposition at a spatial resolution down to ~ 1 - 50 um. This\nallows for the construction of 2D isotopic datasets that\ndocument vertical isotope gradients as well as lateral\nheterogeneity [2]. Here we present the application of this\nsulfide capture technique to three different modern\nenvironments: (1) microbial mats from Guerrero Negro, Baja\nCalifornia Sur, Mexico; (2) the chemocline of meromictic\nLake Mahoney, British Columbia, Canada; and (3) methane\nseep-associated marine sediments offshore Costa Rica.\nCoherent variations up to 20 permil in δ34S are observed over\nranges as small as 1 mm at all depths examined. These data\nhighlight the additional ecological information that can be\nextracted from high resolution isotopic data, which may\nimprove our understanding of the activity of the microbial\necosystems driving biogeochemical cycling in these systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/15701, title ="Methods for unveiling cryptic microbial partnerships in nature", author = "Orphan, Victoria J.", journal = "Current Opinion in Microbiology", volume = "12", number = "3", pages = "231-237", month = "June", year = "2009", doi = "10.1016/j.mib.2009.04.003", issn = "1369-5274", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090909-131707316", note = "Copyright © 2009 Elsevier. \n\nAvailable online 15 May 2009. \n\nI thank A Dekas and A Pernthaler for contributing the Magneto-FISH and nanoSIMS images used in this review. I am also grateful to S Goffredi and D Newman for constructive comments. Research by VJO included in this review was supported by grants from the Gordon and Betty Moore Foundation, Davidow Research Fund, and National Science Foundation (MCB-0348492).", revision_no = "12", abstract = "Syntrophy and mutualism play a central role in carbon and nutrient cycling by microorganisms. Yet our ability to recognize these partnerships in nature or to effectively study their behavior in culture has been hindered by the inherent interdependence of syntrophic associations, their dynamic behavior, and their frequent existence at thermodynamic limits. Now solutions to these challenges are emerging in new methodologies. These include: comparative metagenomics and transcriptomics; discovery-based methods such as Magneto-FISH; and metabolic substrate tracking using stable isotopes coupled either with density gradient separation (SIP) or with FISH-SIMS. These novel approaches are redefining the way we study microbial mutualism and are making intimate microbial associations accessible to both identification and characterization in their native habitats.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/14500, title ="Variations in archaeal and bacterial diversity associated with the sulfate-methane transition zone in continental margin sediments (Santa Barbara Basin, California)", author = "Harrison, Benjamin K. and Zhang, Husen", journal = "Applied and Environmental Microbiology", volume = "75", number = "6", pages = "1487-1499", month = "March", year = "2009", doi = "10.1128/AEM.01812-08", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090706-143447493", note = "© 2009 American Society for Microbiology. \n\nReceived 5 August 2008. Accepted 5 January 2009.", revision_no = "17", abstract = "The sulfate-methane transition zone (SMTZ) is a widespread feature of continental margins, representing a diffusion-controlled interface where there is enhanced microbial activity. SMTZ microbial activity is commonly associated with the anaerobic oxidation of methane (AOM), which is carried out by syntrophic associations between sulfate-reducing bacteria and methane-oxidizing archaea. While our understanding of the microorganisms catalyzing AOM has advanced, the diversity and ecological role of the greater microbial assemblage associated with the SMTZ have not been well characterized. In this study, the microbial diversity above, within, and beneath the Santa Barbara Basin SMTZ was described. ANME-1-related archaeal phylotypes appear to be the primary methane oxidizers in the Santa Barbara Basin SMTZ, which was independently supported by exclusive recovery of related methyl coenzyme M reductase genes (mcrA). Sulfate-reducing Deltaproteobacteria phylotypes affiliated with the Desulfobacterales and Desulfosarcina-Desulfococcus clades were also enriched in the SMTZ, as confirmed by analysis of dissimilatory sulfite reductase (dsr) gene diversity. Statistical methods demonstrated that there was a close relationship between the microbial assemblages recovered from the two horizons associated with the geochemically defined SMTZ, which could be distinguished from microbial diversity recovered from the sulfate-replete overlying horizons and methane-rich sediment beneath the transition zone. Comparison of the Santa Barbara Basin SMTZ microbial assemblage to microbial assemblages of methane seeps and other organic matter-rich sedimentary environments suggests that bacterial groups not typically associated with AOM, such as Planctomycetes and candidate division JS1, are additionally enriched within the SMTZ and may represent a common bacterial signature of many SMTZ environments worldwide. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/14087, title ="Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California", author = "Orphan, V. J. and Jahnke, L. L.", journal = "Geobiology", volume = "6", number = "4", pages = "376-393", month = "September", year = "2008", doi = "10.1111/j.1472-4669.2008.00166.x", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090427-131232717", note = "© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd. \n\nReceived 28 December 2007; accepted 6 May 2008. \n\nThe authors are indebted to Brad Bebout for use of his laboratory facilities and support during this project. We also thank Bob Vjrenhoek and Ed DeLong for generous use of\nthe MBARI sequencing facilities and Steve Carpenter, Mike Kubo, Mary Hogan, Beth Orcutt and Tori Hoehler for assistance with various aspects of this work. We acknowledge\ntwo anonymous reviewers for comments that greatly improved\nthis manuscript. This research was made possible by grants\nfrom the NASA Exobiology program (DJD) and NRA-01-01-EXB-035 (LLJ), grant no. NNG05GN62G (RES), a Moore Foundation Young Investigator award (VJO), and a National Research Council fellowship awarded to VJO and the logistical support of ESSA Exportadora del Sal, S.A., de C.V., Guerrero Negro, Baja California Sur, Mexico.", revision_no = "15", abstract = "Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C_(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/13272, title ="Lipid biomarker and phylogenetic analyses to reveal archaeal biodiversity and distribution in hypersaline microbial mat and underlying sediment", author = "Jahnke, L. L. and Orphan, V. J.", journal = "Geobiology", volume = "6", number = "4", pages = "394-410", month = "September", year = "2008", doi = "10.1111/j.1472-4669.2008.00165.x", issn = "1472-4677", url = "https://resolver.caltech.edu/CaltechAUTHORS:JAHgbi08", note = "© 2008 Blackwell Publishing Ltd. \n\nReceived 28 December 2007; accepted 9 May 2008; published Online 28 June 2008; in print September 2008. \n\nWe thank the Exportadora de Sal, S.A., for permission to work in their facility and for technical assistance. We thank the Mexican government for permission to carry out research at Guererro Negro, Baja California Sur. We thank three anonymous reviewers for their efforts and valuable comments. We thank Yanek Hebting for suggesting improvements to the ether cleavage methodology. We thank Robert Vrijenhoek (MBARI) and Ed DeLong for generous use of the sequencing facilities. Victoria Orphan was supported by an NRC postdoctoral fellowship and by a grant from the Moore Foundation. Linda Jahnke and David Des Marais were supported by the NASA Exobiology Program and the NASA Astrobiology Institute. Roger Summons was supported by the NSF and the NASA Exobiology Program.", revision_no = "19", abstract = "This study has utilized the tools of lipid biomarker chemistry and molecular phylogenetic analyses to assess the archaeal contribution to diversity and abundance within a microbial mat and underlying sediment from a hypersaline lagoon in Baja California. Based on abundance of ether-linked isoprenoids, archaea made up from 1 to 4% of the cell numbers throughout the upper 100 mm of mat and sediment core. Below this depth archaeal lipid was two times more abundant than bacterial. Archaeol was the primary archaeal lipid in all layers. Relatively small amounts of caldarchaeol (dibiphytanyl glyceroltetraether) were present at most depths with phytanyl to biphytanyl molar ratios lowest (~10 : 1) in the 4–17 mm and 100–130 mm horizons, and highest (132 : 1) in the surface 0–2 mm. Lipids with cyclic biphytanyl cores were only detected below 100 mm. A novel polar lipid containing a C30 isoprenoid (squalane) moiety was isolated from the upper anoxic portion of the core and partially characterized. Hydrocarbon biomarker lipids included pentamethylicosane (2–10 mm) and crocetane (primarily below 10 mm). Archaeal molecular diversity varied somewhat with depth. With the exception of samples at 0–2 mm and 35–65 mm, Thermoplasmatales of marine benthic group D dominated clone libraries. A significant number of phylotypes representing the Crenarchaeota from marine benthic group B were generally present below 17 mm and dominated the 35–65 mm sample. Halobacteriaceae family made up 80% of the clone library of the surface 2 mm, and consisted primarily of sequences affiliated with the haloalkaliphilic Natronomonas pharaonis.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37731, title ="Micron-scale mapping of sulfur cycling across the oxycline of a cyanobacterial mat", author = "Fike, David A. and Gammon, Crystal L.", journal = "Geochimica et Cosmochimica Acta", volume = "72", number = "12", pages = "A268", month = "July", year = "2008", doi = "10.1016/j.gca.2008.05.009", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130402-154755386", note = "© 2008 Published by Elsevier Ltd.", revision_no = "14", abstract = "We present a parallel microgeochemical and\nmicrobiological study of μm-scale sulfur cycling within\nhypersaline microbial mats from Guerrero Negro, Baja\nCalifornia Sur, Mexico. Diel variations (day/night) in sulfur cycling were investigated in field incubations as well as in mats grown under controlled conditions in the laboratory at NASA Ames Research Center. Sulfur cycling in the laboratory mats was examined under a variety of different sulfate concentrations to evaluate the role this had on sulfide concentration and isotopic composition. Sulfate levels in the overlying water column were: 80 mM SO_4 (natural level at\nGuerrero Negro); 1 mM SO_4; and 200 uM SO_4. Dissolved\nsulfide within the mat was captured on silver discs and\nanalyzed for its abundance and δ^(34)S isotopic composition\nusing high resolution secondary ion mass spectrometry\n(SIMS) on a Cameca 7F Geo.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38423, title ="Micron-scale mapping of sulfur cycling across the oxycline of a cyanobacterial mat: a paired nanoSIMS and CARD-FISH approach", author = "Fike, David Andrew and Gammon, Crystal Lynn", journal = "ISME Journal", volume = "2", number = "7", pages = "749-759", month = "July", year = "2008", doi = "10.1038/ismej.2008.39", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130510-110748396", note = "© 2008 International Society for Microbial Ecology. \n\nReceived 10 January 2008; revised 17 March 2008; accepted 18\nMarch 2008; published online 5 June 2008. \n\nWe would like to acknowledge the Gordon and Betty Moore Foundation and the Caltech Center for Geochemical and Cosmochemical Microanalysis for funding (to VJO) as well as support from National Aeronautics and Space Administration Grant NAI02-003-0001 issued through the Astrobiology Program. DAF was supported by the Caltech OK Earl Postdoctoral Fellowship. CLG was supported by a National Science Foundation Graduate Research Fellowship. We would like to thank Yunbin Guan, John Eiler and Tina Treude for analytical assistance and invaluable discussions, Bill Ussler for discussions and assistance with methodological development, Frank Stadermann for discussions and Dirk de Beer for a critical reading of this manuscript. We are also indebted to the NASA Ames group (Tori Hoehler, Niko Finke, Kendra Turk, Mike Kubo, Linda Jahnke and David DesMarais) for support and assistance with sample collections.", revision_no = "13", abstract = "The metabolic activities of microbial mats have likely regulated biogeochemical cycling over most of Earth's history. However, the relationship between metabolic activity and the establishment of isotopic geochemical gradients in these mats remains poorly constrained. Here we present a parallel microgeochemical and microbiological study of micron-scale sulfur cycling within hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico. Dissolved sulfide within the mats was captured on silver discs and analyzed for its abundance and δ^(34)S isotopic composition using high-resolution secondary ion mass spectrometry (nanoSIMS). These results were compared to sulfide and oxygen microelectrode profiles. Two-dimensional microgeochemical mapping revealed well-defined laminations in sulfide concentration (on scales from 1 to 200\u2009μm), trending toward increased sulfide concentrations at depth. Sulfide δ^(34)S decreased from ~+10‰ to −20‰ in the uppermost 3\u2009mm and oscillated repeatedly between −10‰ and −30‰ down to a depth of 8\u2009mm. These variations are attributed to spatially variable bacterial sulfate reduction within the mat. A parallel examination of the spatial distribution of known sulfate-reducing bacteria within the family Desulfobacteraceae was conducted using catalyzed reporter deposition fluorescence in situ hybridization. Significant concentrations of Desulfobacteraceae were observed in both oxic and anoxic zones of the mat and occurred in several distinct layers, in large aggregates and heterogeneously dispersed as single cells throughout. The spatial distribution of these microorganisms is consistent with the variation in sulfide concentration and isotopic composition we observed. The parallel application of the methodologies developed here can shed light on micron-scale sulfur cycling within microbially dominated sedimentary environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/10569, title ="Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin", author = "Tavormina, Patricia L. and Ussler, William, III", journal = "Applied and Environmental Microbiology", volume = "74", number = "13", pages = "3985-3995", month = "July", year = "2008", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:TAVaem08", note = "Copyright © 2008, American Society for Microbiology. \n\nReceived 9 January 2008/ Accepted 8 May 2008. AEM Accepts, published online ahead of print on 16 May 2008. \n\nWe are grateful to M. Lidstrom and M. Kalyuzhnaya (University of Washington) and D. Arp and N. Hommes (Oregon State University) for providing reference strains and S. Goffredi, S. Ussler, and A. Bures for technical assistance with various aspects of this work. We also thank S. Goffredi, D. Fike, C. Gammon, and A. Dekas for critical reading of the manuscript and constructive insight. We are indebted to the pilots of the ROV Tiburon and the crew and shipboard research party of the research vessel Western Flyer on a cruise sponsored by NOAA-NURP in 2005. We also thank three anonymous reviewers for their comments. \n\nThis work was made possible by grants from NASA (NNG06GB34G), NOAA (UAF 05-0132), the National Science Foundation (MCB-0348492), and the Gordon and Betty Moore Foundation (V.J.O.) and by the support provided to MBARI by the David and Lucile Packard Foundation (W.U.).", revision_no = "17", abstract = "Methane vents are of significant geochemical and ecological importance. Notable progress has been made towards understanding anaerobic methane oxidation in marine sediments, however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs, and distantly related to established methanotrophic clades. Water-column associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/11123, title ="Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics", author = "Pernthaler, Annelie and Dekas, Anne E.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "105", number = "19", pages = "7052-7057", month = "May", year = "2008", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:PERpnas08", note = "© 2008 by the National Academy of Sciences. \n\nEdited by David M. Karl, University of Hawaii, Honolulu, HI, and approved March 18, 2008 (received for review November 29, 2007). Published ahead of print May 8, 2008. This article is a PNAS Direct Submission. \n\nWe thank C. House for assistance with the ion microprobe measurements; D. Newman, J. Grotzinger, M. Joye, C. Gammon, D. Fike, I. Head, and two anonymous reviewers for invaluable comments that improved this manuscript; and S. Johnson, F. Rohwer, R. Edwards, B. Orcutt, and the 2006 science party of cruise AT15–11 and pilots of the D.S.R.V. Alvin for their assistance with various aspects of this work. This work was supported by National Science Foundation Award Grant MCB-0348492, the Gordon and Betty Moore Foundation, a Davidow grant to Caltech’s GPS Division (to V.J.O.); National Institutes of Health Grant P50HG004071 (to C.T.B.), in part by a Caltech GPS Texaco fellowship (to A.P.), and a National Science Foundation graduate fellowship (to A.D.). \n\nAuthor contributions: A.P. and V.J.O. designed research; A.P., A.E.D., S.K.G., T.E., and V.J.O. performed research; A.P. and C.T.B. contributed new reagents/analytic tools; A.P., A.E.D., C.T.B., S.K.G., and V.J.O. analyzed data; and A.P., C.T.B., and V.J.O. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nData deposition: The sequence reported in this paper has been deposited in the GenBank database [accession nos. EU622281–EU622312 (16S rRNA) and EU647340–EU647354 (nifH)]. \n\nThis article contains supporting information online at www.pnas.org/cgi/content/full/0711303105/DCSupplemental.", revision_no = "27", abstract = "Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of 15N2 incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37639, title ="Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a deep-sea whale-fall in Monterey Canyon, California", author = "Goffredi, Shana K. and Wilpiszeski, Regina", journal = "ISME Journal", volume = "2", number = "2", pages = "204-220", month = "February", year = "2008", doi = "10.1038/ismej.2007.103", issn = "1751-7362", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130327-080517687", note = "© 2008 International Society for Microbial Ecology. \n\nReceived 13 August 2007; Revised 18 October 2007; Accepted 19 October 2007; Published online 24 January 2008. \n\nThis study was supported by a Davidow grant to Caltech’s Geological and Planetary Sciences division, US National Science Foundation (MCB-0454860 to SKG), and the Gordon and Betty Moore Foundation (to VJO). We thank the Tiburon pilots and Western Flyer crew and chief scientist R Vrijenhoek for allowing our participation in research cruises; S Johnson and WJ Jones for shipboard support; A Pernthaler for advice regarding FISH analyses; B Ussler for help with methane measurements; Patty Tavormina for QPCR data; Tsegereda Embaye for CARD FISH preparations and B Harrison for ARB assistance.", revision_no = "11", abstract = "Whale-falls represent localized areas of extreme organic enrichment in an otherwise oligotrophic deep-sea environment. Anaerobic remineralization within these habitats is typically portrayed as sulfidogenic; however, we demonstrate that these systems are also favorable for diverse methane-producing archaeal assemblages, representing up to 40% of total cell counts. Chemical analyses revealed elevated methane and depleted sulfate concentrations in sediments under the whale-fall, as compared to surrounding sediments. Carbon was enriched (up to 3.5%) in whale-fall sediments, as well as the surrounding sea floor to at least 10\u2009m, forming a ‘bulls eye’ of elevated carbon. The diversity of sedimentary archaea associated with the 2893\u2009m whale-fall in Monterey Canyon (California) varied both spatially and temporally. 16S rRNA diversity, determined by both sequencing and terminal restriction fragment length polymorphism analysis, as well as quantitative PCR of the methyl-coenzyme M reductase gene, revealed that methanogens, including members of the Methanomicrobiales and Methanosarcinales, were the dominant archaea (up to 98%) in sediments immediately beneath the whale-fall. Temporal changes in this archaeal community included the early establishment of methylotrophic methanogens followed by development of methanogens thought to be hydrogenotrophic, as well as members related to the newly described methanotrophic lineage, ANME-3. In comparison, archaeal assemblages in ‘reference’ sediments collected 10\u2009m from the whale-fall primarily consisted of Crenarchaeota affiliated with marine group I and marine benthic group B. Overall, these results indicate that whale-falls can favor the establishment of metabolically and phylogenetically diverse methanogen assemblages, resulting in an active near-seafloor methane cycle in the deep sea.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37640, title ="Methyl sulfides as intermediates in the anaerobic oxidation of methane", author = "Moran, James J. and Beal, Emily J.", journal = "Environmental Microbiology", volume = "10", number = "1", pages = "162-173", month = "January", year = "2008", doi = "10.1111/j.1462-2920.2007.01441.x", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130327-081544064", note = "© 2007 The Authors. Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 15 April, 2007; accepted 11 August, 2007. Article first published online: 30 Sep. 2007. \n\nWe thank Dr Michael Arthur for the use of his mass spectrometer, Denny Walizer for valuable technical assistance, Dr Shana Goffredi for assistance with the T-RFLP analysis and Dr Lisa Levin for providing samples from Hydrate Ridge. Graduate support for this project was provided by the Penn State Biogeochemical Research Initiative for Education (BRIE) funded by NSF (IGERT) Grant DGE-9972759. This work was also funded by the Penn State Astrobiology Research Center (through the National Astrobiology Institute), NOAA-NURP (UAF 05-0132) and the National Science Foundation (MCB-0348492).", revision_no = "12", abstract = "While it is clear that microbial consortia containing Archaea and sulfate-reducing bacteria (SRB) can mediate the anaerobic oxidation of methane (AOM), the interplay between these microorganisms remains unknown. The leading explanation of the AOM metabolism is ‘reverse methanogenesis’ by which a methanogenesis substrate is produced and transferred between species. Conceptually, the reversal of methanogenesis requires low H_2 concentrations for energetic favourability. We used ^(13)C-labelled CH_4 as a tracer to test the effects of elevated H_2 pressures on incubations of active AOM sediments from both the Eel River basin and Hydrate Ridge. In the presence of H_2, we observed a minimal reduction in the rate of CH_4 oxidation, and conclude H_2 does not play an interspecies role in AOM. Based on these results, as well as previous work, we propose a new model for substrate transfer in AOM. In this model, methyl sulfides produced by the Archaea from both CH_4 oxidation and CO_2 reduction are transferred to the SRB. Metabolically, CH_4 oxidation provides electrons for the energy-yielding reduction of CO_2 to a methyl group (‘methylogenesis’). Methylogenesis is a dominantly reductive pathway utilizing most methanogenesis enzymes in their forward direction. Incubations of seep sediments demonstrate, as would be expected from this model, that methanethiol inhibits AOM and that CO can be substituted for CH_4 as the electron donor for methylogenesis.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37736, title ="Archaea, Methane, and Oases of the Deep", author = "Orphan, Victoria J. and House, Christopher H.", journal = "Nova Acta Leopoldina", volume = "96", number = "356", pages = "19-23", month = "January", year = "2008", issn = "0369-5034", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130403-085441691", note = "© 2008 Deutsche Akademie der Naturforscher Leopoldina. \n\nSupport for this work was provided by the Gordon and Betty Moore Foundation, NOAA NURP (UAF-05-0132), and the National Science Foundation (MCB-0348492). The authors wish to thank the numerous students and research scientists who were instrumental in this research including K. Turk, J. Vrentas, J. Metz, S. Johnson, J. Jones, and C. Braby. We also wish to express special thanks to B. Vjrenhoek of MBARl, and the captain, crew and pilots of the R/V Western Flyer and ROY Tiburon.", revision_no = "21", abstract = "The deep sea, fed by a slow trickling input of photosynthetically derived carbon, has historically been considered\na low energy, oligotrophic environment. In localized areas, however, oases of elevated microbial biomass and activity\nwithin the deep sea do exist. Perhaps the most famous are hydrothermal vents, emerging along spreading\ncenters and subduction zones, fueled by hot reduced fluids re-circulated within the Earth's crust. Equally rich,\nalthough less well known, areas of stimulated biomass production and activity also occur in the psychrophilic\ndepths of the seafloor, fueled by large organic accumulations (i.e. food falls) and subsurface reservoirs of methane.\nThe microbial ecology within these locally active deep-sea habitats is unique, supporting novel microbial\nassociations and diverse pathways for carbon remineralization.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/36854, title ="Micron-scale resolution of sulfur cycling in a microbial mat", author = "Fike, David and Ussler, William", journal = "Geochimica et Cosmochimica Acta", volume = "71", number = "15", pages = "A278", month = "August", year = "2007", doi = "10.1016/j.gca.2007.06.015", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130211-144010988", note = "© 2007 Published by Elsevier Ltd.", revision_no = "14", abstract = "Microbial mats consist of finely laminated layers of\ndiverse microbial communities. Mat organization is thought\nto result from strong spatial gradients in light intensity and\nredox in the uppermost few millimeters Optical examination\nreveals microbial laminations on scales between 5mm and\n5um throughout the thickness of the microbial mat. However,\nsuch fine laminations at depth have usually been regarded as a\n‘relict architecture’ inherited from an older mat surface. To\nfurther our understanding of microbial processes within this\nlaminated architecture, we have investigated sulfur cycling (as\nrecorded by sulfide production) within a benthic microbial\nmat.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47447, title ="The ties that bind: Dynamics of syntrophic associations in marine methane seeps", author = "Orphan, Victoria J. and Pernthaler, Annelie", journal = "Geochimica et Cosmochimica Acta", volume = "71", number = "15, S", pages = "A743", month = "August", year = "2007", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327312", note = "Copyright © 2007 Elsevier.", revision_no = "13", abstract = "The deep-sea methane seep environment supports active\nand diverse microbial assemblages supported by the anaerobic\noxidation of methane (AOM). Unknown to science less than a\ndecade ago, the microorganisms and the molecular\nmechanisms underlying this enigmatic and globally important\nbiogeochemical process have been the subject of intensive\nstudy worldwide. The identification, activity, distribution, and\npartial metabolic pathway reconstruction of methanotrophic\narchaea and co-associated sulfate reducing bacteria has been\ncharacterized. However fundamental questions still remain\nregarding the necessity of a physically coupled syntrophic\nassociation between sulfate reducing bacteria and methane\noxidizing archaea, the underlying biochemistry enabling\nsulfate-coupled methane oxidation, and the extent of the\ndiversity of microbial assemblages involved in AOM. Using\nmicroanalytical stable isotope analyses of whole cells in\ntandem with genomics enabled molecular methods, we\nexamined the variation in metabolic activity between\nindividual aggregations of microorganisms recovered from\nmethane seep sediments. Significant differences in activity\nwere observed between archaeal-bacterial associations and\nmono-specific aggregations of putative methanotrophic\narchaea and sulfate-reducing populations, supporting\nenhanced metabolism in multi-species aggregates.\nApplication of a new SSU rRNA targeted method for\ncapturing and concentrating specific uncultured microbial\npopulations from methane seep sediments has uncovered\nnovel partnerships and additional insights into the metabolic\npotential of the methanotrophic archaea and co-associated\nbacteria.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38165, title ="Authigenic carbonate formation at hydrocarbon seeps in continental margin sediments: A comparative study", author = "Naehr, Thomas H. and Eichhubl, Peter", journal = "Deep-Sea Research. Part II, Topical Studies in Oceanography", volume = "54", number = "11-13", pages = "1268-1291", month = "June", year = "2007", doi = "10.1016/j.dsr2.2007.04.010", issn = "0967-0645", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130429-140926092", note = "© 2007 Elsevier Ltd. \n\nAccepted 16 April 2007; Available online 20 July 2007. \n\nWe thank the captain and crew of R/V Point Lobos and the pilots of ROV Ventana for their dedicated efforts to obtain many of the samples used in this study. We also thank G. Bohrmann, J. Greinert, D. Orange, D. Stakes, J. Martin, and\nmany other colleagues for years of stimulating discussions. We are grateful for the comments provided by J. Peckmann and an anonymous reviewer, which greatly improved the paper. We also thank M. Dalthorp for her critical remarks and C. Glenn and G. Filippelli for the editorial handling\nof the paper. Financial support for this study was provided by the David and Lucile Packard Foundation, the Monterey Bay Aquarium Research Institute, through a TAMUCC Faculty Summer Research Grant, and NSF-EAR award 0421410.", revision_no = "12", abstract = "Authigenic carbonates from five continental margin locations, the Eel River Basin, Monterey Bay, Santa Barbara Basin, the Sea of Okhotsk, and the North Sea, exhibit a wide range of mineralogical and stable isotopic compositions. These precipitates include aragonite, low- and high-Mg calcite, and dolomite. The carbon isotopic composition of carbonates varies widely, ranging from −60‰ to +26‰, indicating complex carbon sources that include ^(13)C-depleted microbial and thermogenic methane and residual, 13C-enriched, bicarbonate. A similarly large variability of δ^(18)O values (−5.5‰ to +8.9‰) demonstrates the geochemical complexity of these sites, with some samples pointing toward an ^(18)O-enriched oxygen source possibly related to advection of ^(18)O-enriched formation water or to the decomposition of gas hydrate. Samples depleted in ^(18)O are consistent with formation deeper in the sediment or mixing of pore fluids with meteoric water during carbonate precipitation.\n\nA wide range of isotopic and mineralogical variation in authigenic carbonate composition within individual study areas but common trends across multiple geographic areas suggest that these parameters alone are not indicative for certain tectonic or geochemical settings. Rather, the observed variations probably reflect local controls on the flux of carbon and other reduced ions, such as faults, fluid conduits, the presence or absence of gas hydrate in the sediment, and the temporal evolution of the local carbon reservoir.\n\nAreas with seafloor carbonates that indicate formation at greater depth below the sediment–water interface must have undergone uplift and erosion in the past or are still being uplifted. Consequently, the occurrence of carbonate slabs on the seafloor in areas of active hydrocarbon seepage is commonly an indicator of exhumation following carbonate precipitation in the shallow subsurface. Therefore, careful petrographic and geochemical analyses are critical components necessary for the correct interpretation of processes related to hydrocarbon seepage in continental margin environments and elsewhere.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/8287, title ="Consumption of Methane and CO_2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea", author = "Treude, Tina and Orphan, Victoria J.", journal = "Applied and Environmental Microbiology", volume = "73", number = "7", pages = "2271-2283", month = "April", year = "2007", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:TREaem07", note = "Copyright © 2007, American Society for Microbiology. \n\nReceived 16 November 2006/ Accepted 20 January 2007. Published ahead of print on 2 February 2007. \n\nWe acknowledge F. Widdel for his advice concerning the interpretation of the methane formation experiments. We thank K. Nauhaus and N. Finke for fruitful discussions about methane formation in the mat. We thank the officers, crew, and shipboard scientific party of R/V Professor Logachev and the JAGO Team for excellent support during the Black Sea cruise in summer 2001. Special thanks are due to W. Michaelis. We acknowledge B. Ratunde, T. Wilkop, and I. Mueller for providing technical assistance. We thank the GHOSTDABS project and the JAGO Team for providing the reef pictures. We thank Kevin McKeegan for assistance with the SIMS configuration. Two anonymous reviewers are acknowledged for their comments. \n\nThe UCLA ion microprobe is supported by the W.M. Keck Foundation and by a grant from the National Science Foundation Instrumentation and Facilities Program (grant EAR 01-13563). Support for V. Orphan and C. House, as well as for the SIMS analyses, was provided by the NSF Microbial Interactions and Processes Program (grant 03488492). This is a publication of the MUMM (03G0554A) and GHOSTDABS (03G0559A) programs supported by the German Ministry of Education and Research (BMBF) and the German Research Foundation (DFG). Additional support was provided by the Max-Planck-Gesellschaft (Germany) and by the NASA Astrobiology Institute. \n\nPublication no. GEOTECH-258 of the R&D program GEOTECHNOLOGIEN.", revision_no = "10", abstract = "The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH_4 and CO_2 assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO_2 reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ^(13)C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ^(14)C from radiolabeled CH_4 or CO_2 revealed one hot spot for methanotrophy and CO2 fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ^(14)CH_4 or ^(14)CO_2 revealed that there was interconversion of CH_4 and CO_2. The level of CO_2 reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47446, title ="Linking molecular taxonomy with environmental geochemsitry in environments relevant to astrobiology: The anaerobic oxidation of methane in cold seeps & deeply buried marine sediments", author = "House, Christopher H. and Biddle, Jennifer F.", journal = "Origins of life and evolution of the biosphere", volume = "36", number = "3", pages = "314-315", month = "June", year = "2006", issn = "0169-6149", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140723-162327093", note = "© 2006 Springer.", revision_no = "9", abstract = "The linking of molecular taxonomy (including 16s rRNA) to environmental geochemistry\nis a powerful way to work out the interactions, metabolic activities, and food webs of microorganisms in their natural setting, whether it is sediment, soil,\nor a water column. To this end, we developed a method for coupling an extant microorganism’s\ngenetic information with geochemical data derived from the direct\nanalysis of its cell. FISH–SIMS combines fluorescent in-situ hybridization (FISH)\nwith secondary ion mass spectrometry (SIMS). FISH is a culture-independent technique\nused to visually identify naturally occurring microorganisms by staining their\nribosomal RNA. Secondary ion mass spectrometry (SIMS) is a method by which\ngeochemical information can be obtained from microsamples. Using FISH-SIMS, a\nresearcher can measure a target cell’s isotopic or elemental composition in a mixed\nenvironment.\nThe identification and study of methane-consuming microorganisms is an important\nstep toward understanding the methane cycle and microbial response to\nmethane release. The recent identification of two distinct Archaea capable of anaerobic\nmethane oxidationwas in part accomplished using FISH-SIMS. Because natural\nmethane is highly depleted in 13C, FISH-SIMS is particularly powerful at determining\nif a particular cell, collected from the environment, and consumed methane\nas a substrate for its cell carbon. This research demonstrated that both the ANME-1\nand ANME–2 Archaea from the Eel River Methane Seep are highly depleted in\n13C due to growth on methane.\nThe deep marine biosphere is thought to contain abundant microbial inhabitants,\nestimated to be a tenth of the Earth’s total biomass. Sediments from this\nenvironment were recovered during Ocean Drilling Program (ODP) Leg 201, and\nwere analyzed by both molecular biological and organic geochemical techniques.\nOf particular interest in these sediments were four sulfate/methane transition zones\nseen at ODP Sites 1227, 1229 and 1230, two of which coincided with strongly elevated\ncell counts. Archaeal cells in these zones were analyzed for abundance and\nd13C composition by whole cell analysis (FISH-SIMS) and intact membrane lipids\n(HPLC-ESI-MSn). Cell counts showed greater archaeal abundance than bacterial,\nwhich was reflected by intact membrane lipid abundance. Isotopic compositions\nby both techniques (often around −20‰) suggest that methane is not an important\ncarbon source for these cells. Autotrophic carbon fixation appears to be an unlikely\nmetabolism given the relationship between the isotopic composition of DIC and\narchaeal biomass. The isotopic evidence suggests that the bulk archaeal community\nis heterotrophic, possibly mediating the oxidation of methane without consuming\nit as a carbon source.\nThe importance of these techniques is that the cells targeted for study can be\nenvironmental species that cannot currently be grown in the laboratory. These\ntechniques promises to become critical for working out the interactions, metabolic\nactivities, and food webs of microorganisms in their natural setting, whether it is\nsediment, soil, or a water column.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37710, title ="Evolutionary innovation: a bone-eating marine symbiosis", author = "Goffredi, Shana K. and Orphan, Victoria J.", journal = "Environmental Microbiology", volume = "7", number = "9", pages = "1369-1378", month = "September", year = "2005", doi = "10.1111/j.1462-2920.2005.00824.x", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130402-081618805", note = "© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd. \n\nReceived 28 June, 2004; revised 14 January, 2005; accepted 26 January, 2005. Article first published online: 23 Jun. 2005. \n\nThe authors thank the Tiburon pilots and Western Flyer crew\nfor obtaining whale bone samples; J. Jones, R. Young and S.\nJohnson for laboratory support; N. Dubilier and A. Pernthaler for advice regarding the FISH analyses; L. Howe at the Stanford Biofilm Research Center for assistance with confocal microscopy, H. Schoppe, K. Rogers and Adelaide Microscopy for microscopy support. The David and Lucile Packard Foundation, the US National Science Foundation (OCE0241613 to R.C.V. and DBI0116203 to R.L.) and the South Australian Museum (G.R.) have supported this work.", revision_no = "13", abstract = "Symbiotic associations between microbes and invertebrates have resulted in some of the most unusual physiological and morphological adaptations that have evolved in the animal world. We document a new symbiosis between marine polychaetes of the genus Osedax and members of the bacterial group Oceanospirillales, known for heterotrophic degradation of complex organic compounds. These organisms were discovered living on the carcass of a grey whale at 2891 m depth in Monterey Canyon, off the coast of California. The mouthless and gutless worms are unique in their morphological specializations used to obtain nutrition from decomposing mammalian bones. Adult worms possess elaborate posterior root-like extensions that invade whale bone and contain bacteriocytes that house intracellular symbionts. Stable isotopes and fatty acid analyses suggest that these unusual endosymbionts are likely responsible for the nutrition of this locally abundant and reproductively prolific deep-sea worm.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37643, title ="Geological, geochemical, and microbiological heterogeneity of the seafloor around methane vents in the Eel River Basin, offshore California", author = "Orphan, V. J. and Ussler, W., III", journal = "Chemical Geology", volume = "205", number = "3-4", pages = "265-289", month = "May", year = "2004", doi = "10.1016/j.chemgeo.2003.12.035", issn = "0009-2541", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130327-085250919", note = "© 2004 Elsevier B.V. \n\nReceived 1 January 2003; received in revised form 20 September 2003; accepted 23 December 2003. \n\nSupport for this work was provided by the David and Lucile Packard Foundation. We thank Howard Mendlovitz for the methane and CO_2 δ^(13)C analyses and the pilots of the ROV Ventana and ROV Tiburon for their expert skill in sample collection. We thank Andreas Teske (UNC), Shana Goffredi, Steve Hallam, Peter Girguis, Edward DeLong (MBARI) and Bill Sullivan (UCSC), for their expert assistance with various aspects of this research and Rick Colwell for the helpful comments on this manuscript. VJO is supported by a National Research Council fellowship.", revision_no = "12", abstract = "Marine methane vents and cold seeps are common features along continental margins worldwide, serving as localized sites for methane release and colonization by microbial and chemosynthetic megafaunal communities. The Eel River Basin (ERB), located on the continental slope off Northern California, contains active methane vents and seep-associated chemosynthetic biological communities (CBC) on the crests of anticlines in ∼520-m water depth. Seep-related features on the seafloor have a patchy distribution and include active bubbling vents, chemosynthetic clam beds, and sulfide-oxidizing bacterial mats. Methane sources supplying local seeps are heterogeneous on all spatial scales and support a large and diverse microbial assemblage involved in the anaerobic oxidation of methane (AOM).\n\nTo develop a comprehensive understanding of the complex biological, geochemical and physical processes associated with, and influencing seafloor methane seepage, a multidisciplinary approach is required. Here we present an integrative, multidisciplinary study that illustrates the diverse processes associated with seafloor methane seepage within the Eel River Basin and the complex interactions defining the geochemistry, mineralogy and microbiology within this environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/1596, title ="Novel Forms of Structural Integration between Microbes and a Hydrothermal Vent Gastropod from the Indian Ocean", author = "Goffredi, Shana K. and Warén, Anders", journal = "Applied and Environmental Microbiology", volume = "70", number = "5", pages = "3082-3090", month = "May", year = "2004", doi = "10.1128/AEM.70.5.3082-3090.2004", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:GOFaem04", note = "© 2004, American Society for Microbiology. \n\nReceived 30 September 2003/ Accepted 2 February 2004 \n\nWe thank the scientific party on Knorr leg KN162-13 and the captains and crew of the R.V. Knorr and the R.O.V. Jason. We also thank Todd Walsh for photographic assistance, J. Salerno and S. Bengtson for original transmission and scanning electron micrographs, R. Lee for isotopic measurements, and C. Cavanaugh, A. L. Reysenbach, and R. Popa for helpful discussions. \n\nThis work was supported by the David and Lucile Packard Foundation, the U.S. National Science Foundation (R.C.V. and C.L.V.D.), and the M. Bergvall Foundation (A.W.).", revision_no = "9", abstract = "Here we describe novel forms of structural integration between endo- and episymbiotic microbes and an unusual new species of snail from hydrothermal vents in the Indian Ocean. The snail houses a dense population of {gamma}-proteobacteria within the cells of its greatly enlarged esophageal gland. This tissue setting differs from that of all other vent mollusks, which harbor sulfur-oxidizing endosymbionts in their gills. The significantly reduced digestive tract, the isotopic signatures of the snail tissues, and the presence of internal bacteria suggest a dependence on chemoautotrophy for nutrition. Most notably, this snail is unique in having a dense coat of mineralized scales covering the sides of its foot, a feature seen in no other living metazoan. The scales are coated with iron sulfides (pyrite and greigite) and heavily colonized by {epsilon}- and {delta}-proteobacteria, likely participating in mineralization of the sclerites. This novel metazoan-microbial collaboration illustrates the great potential of organismal adaptation in chemically and physically challenging deep-sea environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/1595, title ="Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor", author = "Girguis, Peter R. and Orphan, Victoria J.", journal = "Applied and Environmental Microbiology", volume = "69", number = "9", pages = "5472-5482", month = "September", year = "2003", doi = "10.1128/AEM.69.9.5472-5482.2003", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:GIRaem03", note = "Copyright © 2003, American Society for Microbiology. \n\nReceived 28 March 2003/ Accepted 25 June 2003. \n\nSpecial thanks to James Childress for generously loaning equipment, Marcelino Suzuki for extensive assistance with quantitative PCR and collard green recipes, and Tori Hoehler for invaluable commentary and critique. Thanks to Christina Preston, Lynne Christianson, and Shana Goffredi for assistance and guidance with sequencing and Jose de la Torre for assistance with phylogenetic analysis. As always, special thanks to the crew and pilots of the RV Point Lobos and the ROV Ventana. \n\nThis work was supported by the Monterey Bay Aquarium Research Institute and the Packard Foundation.", revision_no = "8", abstract = "Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38036, title ="Geochemical Influence on Diversity and Microbial Processes in High Temperature Oil Reservoirs", author = "Orphan, V. J. and Goffredi, S. K.", journal = "Geomicrobiology Journal", volume = "20", number = "4", pages = "295-311", month = "July", year = "2003", doi = "10.1080/01490450303898", issn = "0149-0451", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130419-103230121", note = "© 2003 Taylor & Francis Inc. \n\nReceived 20 February 2002; accepted 7 April 2003. \n\nWe thank Peter Eichhubl and Steve Franks for generously sharing their unpublished work with us. We also are indebted to Prentice Patterson and the crew from Platform Holly (Mobil and Venoco Inc), Trent Taylor, Christa Schleper, and Chris Preston for assistance with sample collection and Jim Childress for technical advice and use of his analytical equipment. V. J. Orphan is currently supported by a National Research Council Fellowship at NASA Ames Research Center.", revision_no = "10", abstract = "The diversity of thermophilic microbial assemblages detected within two neighboring high temperature petroleum formations was shown to closely parallel the different geochemical regimes existing in each. A high percentage of archaeal 16S rRNA gene sequences, related to thermophilic aceticlastic and hydrogenotrophic methanogens, were detected in the natural gas producing Rincon Formation. In contrast, rRNA gene libraries from the highly sulfidogenic Monterey Formation contained primarily sulfur-utilizing and fermentative archaea and bacteria. In addition to the variations in microbial community structure, microbial activities measured in microcosm experiments using high temperature production fluids from oil-bearing formations also demonstrated fundamental differences in the terminal respiratory and redox processes. Provided with the same suite of basic energy substrates, production fluids from the South Elwood Rincon Formation actively generated methane, while thermophilic microflora within the Monterey production fluids were dominated by hydrogen sulfide producing microorganisms. In both cases, molecular hydrogen appeared to play a central role in the stimulation of carbon and sulfur cycling in these systems. In methanogenic production fluids, the addition of sulfur compounds induced a rapid shift in the terminal electron accepting process, stimulating hydrogen sulfide formation and illustrating the metabolic versatility of the subsurface thermophilic assemblage. The high similarity between microbial community structure and activity corresponding with the prevalent geochemical conditions observed in deep subsurface petroleum reservoirs suggests that the resident microflora have adapted to the subsurface physicochemical conditions and may actively influence the geochemical environment in situ.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47249, title ="Direct phylogenetic and isotopic evidence for multiple groups of archaea involved in the anaerobic oxidation of methane", author = "Orphan, V. J. and House, C. H.", journal = "Geochimica et Cosmochimica Acta", volume = "66", number = "S1", pages = "A571", month = "August", year = "2002", issn = "0016-7037", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140715-170201332", note = "Copyright © 2002 Elsevier.", revision_no = "12", abstract = "The biological oxidation of methane by anaerobic\nmicroorganisms is a significant sink for methane in the marine\nenvironment. Although there is convincing biogeochemical\nevidence for anaerobic oxidation of methane (AOM) by\nmethanotrophic archaea and sulfate-reducing bacteria, the\nidentity of these uncultured microorganisms is only now being\ndescribed. In this study, we examined the diversity archaeal\nand bacterial assemblages involved in AOM using directly\ncoupled isotopic and phylogenetic analyses at the level of\nsingle cells. The combined application of fluorescent in situ\nhybridization and secondary ion mass spectrometry (FISHSIMS)\nidentified two phylogenetically distinct groups of\narchaea (ANME-1 and ANME-2) from marine methane seeps\nthat were extremely depleted in carbon-13 (-83‰) and appear\nto be capable of directly oxidizing methane. These archaeal\ngroups were observed to exist as monospecies aggregates or\nsingle cells as well as in physical association with bacteria\nincluding, but not limited to, members of the sulfate-reducing\nDesulfosarcina. The results from this work illustrate the\ncomplexity of the microbial communities and possible\nmechanisms involved in AOM. FISH-SIMS is an effective\napproach for understanding the dynamic microbial interactions\nwithin diverse methane-associated communities and may\nprovide a useful culture-independent tool for deciphering the\nmetabolic function of other environmentally significant\nmicroorganisms in situ.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/773, title ="Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments", author = "Orphan, Victoria J. and House, Christopher H.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "99", number = "11", pages = "7663-7668", month = "May", year = "2002", doi = "10.1073/pnas.072210299", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:ORPpnas02", note = "© 2002 by the National Academy of Sciences. \n\nFrom the Cover\n\nCommunicated by John M. Hayes, Woods Hole Oceanographic Institution, Woods Hole, MA, April 6, 2002 (received for review January 23, 2002); Published online before print May 14, 2002, 10.1073/pnas.072210299 \n\nWe thank Steve Hallam, Chris Preston and Peter Girguis for advice assistance, and Bill Sullivan for use of the University of California Santa Cruz Confocal Microscope Facility. We also thank Tori Hoehler and an anonymous reviewer for helpful comments and suggestions. This work was supported by grants from the David and Lucile Packard Foundation, the Penn State Astrobiology Research Center, and the University of California, Los Angeles, (UCLA) Center for Astrobiology, National Aeronautics and Space Administration National Astrobiology Institute. The UCLA ion microprobe is partially supported by a grant from the National Science Foundation Instrumentation and Facilities Program.", revision_no = "9", abstract = "No microorganism capable of anaerobic growth on methane as the sole carbon source has yet been cultivated. Consequently, information about these microbes has been inferred from geochemical and microbiological observations of field samples. Stable isotope analysis of lipid biomarkers and rRNA gene surveys have implicated specific microbes in the anaerobic oxidation of methane (AOM). Here we use combined fluorescent in situ hybridization and secondary ion mass spectrometry analyses, to identify anaerobic methanotrophs in marine methane-seep sediments. The results provide direct evidence for the involvement of at least two distinct archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps. Although both archaeal groups often occurred in direct physical association with bacteria, they also were observed as monospecific aggregations and as single cells. The ANME-1 archaeal group more frequently existed in monospecific aggregations or as single filaments, apparently without a bacterial partner. Bacteria associated with both archaeal groups included, but were not limited to, close relatives of Desulfosarcina species. Isotopic analyses suggest that monospecific archaeal cells and cell aggregates were active in anaerobic methanotrophy, as were multispecies consortia. In total, the data indicate that the microbial species and biotic interactions mediating anaerobic methanotrophy are diverse and complex. The data also clearly show that highly structured ANME-2/Desulfosarcina consortia are not the sole entities responsible for AOM at marine methane seeps. Other microbial groups, including ANME-1 archaea, are capable of anaerobic methane consumption either as single cells, in monospecific aggregates, or in multispecies consortia.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37841, title ="Methane-Consuming Archaea Revealed by Directly Coupled Isotopic and Phylogenetic Analysis", author = "Orphan, Victoria J. and House, Christopher H.", journal = "Science", volume = "293", number = "5529", pages = "484-487", month = "July", year = "2001", doi = "10.1126/science.1061338", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130410-080805831", note = "© 2001 American Association for the Advancement of Science. \n\nReceived 4 April 2001; accepted 5 June 2001. \n\nWe thank C. Paull (MBARI) and W. Ussler (MBARI) for graciously supplying the δ^(13)C of CH_4 data used in this study and M. Harrison (UCLA) for support during the development of this new ion microprobe application. We would also like to thank K. Buck (MBARI), S. Goffredi (MBARI), and the crew of the R/V Western Flyer and ROV Tiburon for their invaluable assistance with this research and C. Coath (UCLA), W. Bach (WHOI), K. Freeman (PSU), P. Girguis (MBARI), O. Beja (MBARI), for stimulating discussions and helpful advice during this project. Funding for this project was provided by the David and Lucile Packard Foundation, the Penn State Astrobiology Research Center and the University of California, Los Angeles, Center for Astrobiology, NASA National Astrobiology Institute. The UCLA ion microprobe is partially supported by a grant from the National Science Foundation Instrumentation and Facilities Program.", revision_no = "13", abstract = "Microorganisms living in anoxic marine sediments consume more than 80% of the methane produced in the world's oceans. In addition to single-species aggregates, consortia of metabolically interdependent bacteria and archaea are found in methane-rich sediments. A combination of fluorescence in situ hybridization and secondary ion mass spectrometry shows that cells belonging to one specific archaeal group associated with the Methanosarcinales were all highly depleted in ^(13)C (to values of –96‰). This depletion indicates assimilation of isotopically light methane into specific archaeal cells. Additional microbial species apparently use other carbon sources, as indicated by significantly higher ^(13)C/^(12)C ratios in their cell carbon. Our results demonstrate the feasibility of simultaneous determination of the identity and the metabolic activity of naturally occurring microorganisms.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/1579, title ="Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments", author = "Orphan, V. J. and Hinrichs, K.-U.", journal = "Applied and Environmental Microbiology", volume = "67", number = "4", pages = "1922-1934", month = "April", year = "2001", doi = "10.1128/AEM.67.4.1922-1934.2001", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:ORPaem01", note = "© 2001, American Society for Microbiology. \n\nReceived 10 October 2000/Accepted 2 February 2001. \n\nFunding for this project was provided by the David and Lucile Packard Foundation and a NASA isotopic biogeochemistry grant, NAG5-9422, to J.M.H. K.-U.H. thanks the Hanse Institute of Advanced Study in Delmenhorst, Germany, for a fellowship, during which the manuscript was completed. \n\nWe thank Andreas Teske, Jon Martin, Jim Barry, and Thomas Naehr for graciously supplying data used in this study. We also thank Shana Goffredi for helpful comments on the manuscript; Josh Plant, Christopher Lovera, and the crew of the R.V. Point Lobos for their invaluable assistance in sample collection and processing; and A. Boetius and D. Valentine for sharing their unpublished manuscripts with us.", revision_no = "7", abstract = "The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant 13C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. 13C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of delta -proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong 13C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant 13C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38166, title ="Molecular and isotopic analysis of anaerobic methane-oxidizing communities in marine sediments", author = "Hinrichs, Kai-Uwe and Summons, Roger E.", journal = "Organic Geochemistry", volume = "31", number = "12", pages = "1685-1701", month = "December", year = "2000", doi = "10.1016/S0146-6380(00)00106-6", issn = "0146-6380", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130429-141001775", note = "© 2000 Elsevier Science Ltd. \n\nWe are grateful to Dennis Sprott (National Research Council of Canada, Ottawa) for providing us with an authentic standard of sn-2-hydroxyarchaeol, and to Simon Brassell (Indiana University), Rich Pancost (NIOZ), and Jaap Sinninghe Damsté (NIOZ) for discussion of mass spectra. We also thank the crew of RV Point Lobos and ROV Ventana for support during sampling. This research was funded by a research fellowship to KH by the Deutsche Forschungsgemeinschaft and by support of NASA grant NAG6-6660 for laboratory expenses. Roger Summons publishes with the approval of the CEO of AGSO. This is Woods Hole Oceanographic Institution contribution 10256.", revision_no = "14", abstract = "Convergent lines of molecular, carbon-isotopic, and phylogenetic evidence have previously indicated (Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G., DeLong, E. F., 1999. Methane-consuming archaebacteria in marine sediments. Nature 398, 802–805.) that archaea are involved in the anaerobic oxidation of methane in sediments from the Eel River Basin, offshore northern California. Now, further studies of those same sediments and of sediments from a methane seep in the Santa Barbara Basin have confirmed and extended those results. Mass spectrometric and chromatographic analyses of an authentic standard of sn-2-hydroxyarchaeol (hydroxylated at C-3 in the sn-2 phytanyl moiety) have confirmed our previous, tentative identification of this compound but shown that the previously examined product was the mono-TMS, rather than di-TMS, derivative. Further analyses of ^(13)C-depleted lipids, appreciably more abundant in samples from the Santa Barbara Basin, have shown that the archaeal lipids are accompanied by two sets of products that are only slightly less depleted in ^(13)C. These are additional glycerol ethers and fatty acids. The alkyl substituents in the ethers (mostly monoethers, with some diethers) are non-isoprenoidal. The carbon-number distributions and isotopic compositions of the alkyl substituents and of the fatty acids are similar, suggesting strongly that they are produced by the same organisms. Their structures, n-alkyl and methyl-branched n-alkyl, require a bacterial rather than archaeal source. The non-isoprenoidal glycerol ethers are novel constituents in marine sediments but have been previously reported in thermophilic, sulfate- and nitrate-reducing organisms which lie near the base of the rRNA-based phylogenetic tree. Based on previous observations that the anaerobic oxidation of methane involves a net transfer of electrons from methane to sulfate, it appears likely that the non-archaeal, ^(13)C-depleted lipids are products of one or more previously unknown sulfate-reducing bacteria which grow syntrophically with the methane-utilizing archaea. Their products account for 50% of the fatty acids in the sample from the Santa Barbara Basin. At all methane-seep sites examined, the preservation of aquatic products is apparently enhanced because the methane-oxidizing consortium utilizes much of the sulfate that would otherwise be available for remineralization of materials from the water column.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37641, title ="Culture-Dependent and Culture-Independent Characterization of Microbial Assemblages Associated with High-Temperature Petroleum Reservoirs", author = "Orphan, V. J. and Taylor, L. T.", journal = "Applied and Environmental Microbiology", volume = "66", number = "2", pages = "700-711", month = "February", year = "2000", doi = "10.1128/AEM.66.2.700-711.2000", issn = "0099-2240", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130327-083124041", note = "© 2000 American Society for Microbiology. \n\nReceived 13 September 1999; Accepted 25 November 1999. \n\nFunding for this project was provided by the University of California Energy Institute, NSF grants OCE95-29804 and OPP94-18442, and the David and Lucile Packard Foundation. This work could not have been done without the cooperation of Mobil, Torch, and Chevron Oil companies, in particular Mobil’s Scott Hornafius, Prentice Patterson, and Geoffrey MacDonald, and Torch’s David White and Sabrina Miller. We also thank Anaerobe System’s Mike Cox for providing the anaerobic chamber, as well as Jim Boles, Shana Goffredi, Jim Childress, Chad Mireau, Martin Keller, and Marcelino Suzuki for their assistance during the course of this study.", revision_no = "10", abstract = "Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90°C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H_2, acetate, and CO_2 as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.", }