This thesis is centered around the role that sulfur plays in the cycling of carbon and in microbial energetics. In the oceans, sulfate is the most important electron acceptor for the remineralization of organic matter after oxygen has been depleted, and sulfate reduction is particularly relevant in coastal environments and in marine and freshwater sediments. The opposite process, reduced sulfur oxidation, allows autotrophic microorganisms to fix carbon in environments where oxygen is scarce. Organic sulfur is also a relevant component of the sulfur cycle, since sulfur is the sixth most abundant element in biomass, it can protect organic matter from degradation, and it is composed of hundreds of molecules that are produced mainly by microorganisms, with potentially relevant ecological roles.
\r\n\r\nThis work has been divided into two parts. In the first one, we attempt to expand our understanding on different aspects of the sulfur cycle. In Chapter 2, published in Limnology and Oceanography, we focus on dimethylsulfoniopropionate (DMSP), the most abundant organic sulfur compound in the oceans with roles of UV, cryo, and osmoprotection, and involved in the formation of sulfate aerosols. We propose a framework to differentiate between the microbial degradation pathways of DMSP based on the sulfur isotope fractionations imprinted by each one of them. In Chapter 3, we perform a survey of sulfate, sulfide, and reduced sulfur intermediates, as well as redox-sensitive elements, in porewaters of a ~40 cm core from the San Clemente Basin (California) and three 1.2-2 m cores near Cocos Ridge (Costa Rica). We correlate these concentrations with the sediment microbial community composition to unveil the specifics of organic matter and sulfur cycling at these localities. In Chapter 4, we explore the utility of sulfur isotope fractionations to characterize different pathways involved in microbial sulfur oxidation (MSO), and examine the role of nutrient limitation and growth rates on the magnitude of the fractionation.
\r\n\r\nIn the second part of this thesis, we aim at understanding biomineralization by consortia between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which comprise more than 90% of the microbial biomass in deep sea sediments around hydrocarbon seeps. In Chapter 5 (in review at Proceedings of the National Academy of Sciences) we establish that modern ANME-SRB aggregates precipitate amorphous silica in undersaturated solutions in sediments and carbonates, often in the form of rims, which pinpoints to a potentially new microbial biomineralization mechanism. In Chapter 6, we posit the use of this proxy, together with distinctive spectral and isotopic signals, to find potential microfossils of ANME-SRB aggregates in the rock record of the Earth and other planetary bodies where methane seepage has occurred throughout geologic time. This suite of tools is used in conjunction to identify ANME-SRB aggregates in the Tepee Buttes (Colorado, 75 Mya) seep carbonates.
", "doi": "10.7907/9bn5-z794", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16110", "collection": "thesis", "collection_id": "16110", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06122023-184806431", "primary_object_url": { "basename": "SWilbert_Thesis.pdf", "content": "final", "filesize": 26984088, "license": "other", "mime_type": "application/pdf", "url": "/16110/1/SWilbert_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Role of Context-Dependent Metabolic Interactions in Organizing Microbial Communities", "author": [ { "family_name": "Wilbert", "given_name": "Steven Alexander", "orcid": "0009-0008-4409-8974", "clpid": "Wilbert-Steven-Alexander" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "We can image the strikingly beautiful compositions of natural microbial communities, but we still lack an understanding of the factors that shape their organization. Understanding the drivers of these structures at the microscale may allow us to better predict and control large-scale community functions in dynamic environments. In this thesis, I developed quantitative image analysis pipelines for uncovering the spatiotemporal growth of aggregate biofilms within a developing oxygen gradient by expanding upon the Agar Block Biofilm Assay (ABBA). I then developed the Agar Disk Biofilm Assay (ADBA) for improved imaging resolution. These tools push the bounders of laboratory experiments to better capture the complexity of natural environments. Next, I built a synthetic microbial community reflecting a metabolic pathway often partitioned between members found in nature: Pseudomonas aeruginosa (PA) strains with a denitrification pathway genetically split at the nitric oxide (NO) node. I characterized the growth of a strict consumer and a strict producer of NO and found that PA metabolizes NO in a manner that supports growth, a previously underappreciated energy conservation strategy. Local oxygen flips this interaction from beneficial to detrimental by increasing toxicity. I found these principles drive context-dependent cellular organization. This work underscores the contributions of partitioned metabolic pathways, redox-active metabolites, and dynamic micro-niches to the organization of microbial communities. Finally, combining my efforts towards method development and an appreciation for how redox-active metabolites drive context-dependent microbial interactions, I show how phenazines promote a previously unrecognized form of slow growth under nutrient limited environments. Taken together, this thesis highlights the importance of understanding dynamic micron-scale microbial interactions and presents several methodological improvements to capture it.", "doi": "10.7907/7sv2-gj10", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15090", "collection": "thesis", "collection_id": "15090", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01202023-073514292", "primary_object_url": { "basename": "LTsypin_Thesis.pdf", "content": "final", "filesize": 12781413, "license": "other", "mime_type": "application/pdf", "url": "/15090/1/LTsypin_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "The Discovery and Biological Mechanisms of a Widespread Phenazine's Oxidation", "author": [ { "family_name": "Tsypin", "given_name": "Lev Maximovich", "orcid": "0000-0002-0642-8468", "clpid": "Tsypin-Lev-Maximovich" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "During the 2017 Microbial Diversity course at the Marine Biological Laboratory in Woods Hole, MA, Scott Saunders and Yinon Bar-On started enrichment cultures in hopes of dis-covering biological oxidation of phenazine-1-carboxylic acid (PCA). I took these enrich-ment cultures and described their PCA oxidation activity. From one of the mixed cultures, I isolated a bacterial strain that recapitulated the behavior of the enrichment. I identified it as a strain of Citrobacter portucalensis via a whole-genome analysis and called the strain \"MBL\" in reference to the Marine Biological Laboratory. Using a combination of analytical chemistry, quantitative fluorescence measurements, and genetic engineering, I showed that C. portucalensis MBL couples PCA oxidation to each mode of anaerobic respiration it employs with nitrate, fumarate, dimethyl sulfoxide (DMSO), and trimethylamine-N-oxide (TMAO) as terminal electron acceptors (TEAs). I further found that most of the PCA oxidation activi-ty depends on electron flux through the quinone/quinol pool but can be driven by certain terminal reductase complexes when no quinones are available, particularly in the case of ni-trate reductases. Every bacterial strain I tested catalyzed PCA oxidation when provided the appropriate TEA. My described mechanism for bacterial PCA oxidation is generalizable and implies that this previously undocumented phenomenon should occur wherever PCA is produced in rhizosphere environments.
", "doi": "10.7907/rmsf-e465", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15009", "collection": "thesis", "collection_id": "15009", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08272022-063655097", "type": "thesis", "title": "Bioorthogonal Noncanonical Amino Acid Tagging for Understanding Bacterial Persistence", "author": [ { "family_name": "Liu", "given_name": "Xinyan", "orcid": "0000-0003-3258-5720", "clpid": "Liu-Xinyan" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of \u201cpersister\u201d cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. On the other hand, bacteria often have multiple molecular mechanisms to adapt to fluctuating environments. Understanding these mechanisms, and their redundancy, requires examinations in depth at the molecular level. This thesis describes a time- and cell state-selective proteome-labeling approach that enables researchers to investigate heterogeneous systems and molecular redundancy.
\r\n\r\nIn Chapter 1, we review the concept of bacterial persistence. The definition of bacterial persistence is introduced. Both the differences and connections between bacterial persistence and resistance are covered. In particular, we discuss research related to Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen found in many cystic fibrosis patients. State-of-the-art technologies to investigate bacterial persistence are discussed, and we conclude that advanced tools are needed to advance research on bacterial persistence further.
\r\n\r\nIn Chapter 2, we highlight the concept of bioorthogonal noncanonical amino acid tagging (BONCAT). BONCAT is a powerful tool developed in the Tirrell and Schuman laboratories allowing the incorporation of noncanonical amino acids (ncAA) into newly-synthesized proteins. We review established strategies for proteomics, especially cell-selective proteomics. We introduce the concept and mechanism of BONCAT and address the advantages of BONCAT in the investigation of phenotypic heterogeneity and bacterial persistence.
\r\n\r\nIn Chapter 3, we describe our work using BONCAT for understanding bacterial persistence. In particular, we investigated the process of persister resuscitation, as it is closely related to the reoccurrence of P. aeruginosa infections. The characteristics of the heterogeneity of persister cells during persister awakening were examined by survival assays and by ScanLag, an automated colony-based system allowing high-throughput acquisition of time-lapse images, quantification, and analysis of growth of bacterial colonies. Two BONCAT methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (Aha), which avoids extensive usage of antibiotic markers and allows direct integration with PA14 transposon insertion library, or with L-azidonorleucine (Anl), which has the advantage of specificity, as well as direct application in nutrition-rich medium. Through BONCAT enrichment experiments, we found proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were up-regulated in the regrowth phase. We further explored whether the up-regulation was a result of the modulation of HigB-HigA toxin-antitoxin system.
\r\n\r\nIn Chapter 4, we describe our work for understanding molecular redundancy. The chapter follows up on our observation of up-regulation of pyochelin-related proteins during persister regrowth. We discuss the hypothesis that pyochelin confers a growth advantage in persister cells subject to carbon-limited conditions. In addition, we discuss the potential role of Fur, a ferric uptake regulator, in bacterial persistence.
", "doi": "10.7907/q6bx-kt39", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14982", "collection": "thesis", "collection_id": "14982", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07202022-060317708", "primary_object_url": { "basename": "sanders_cecilia_2022_thesis_draft_21jul2022.pdf", "content": "final", "filesize": 79720599, "license": "other", "mime_type": "application/pdf", "url": "/14982/9/sanders_cecilia_2022_thesis_draft_21jul2022.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Geological and Geochemical Explorations of the Salitre Formation Phosphorite, Eastern Brazil", "author": [ { "family_name": "Sanders", "given_name": "Cecilia Brooke", "orcid": "0000-0002-2663-164X", "clpid": "Sanders-Cecilia-Brooke" } ], "thesis_advisor": [ { "family_name": "Grotzinger", "given_name": "John P.", "orcid": "0000-0001-9324-1257", "clpid": "Grotzinger-J-P" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Grotzinger", "given_name": "John P.", "orcid": "0000-0001-9324-1257", "clpid": "Grotzinger-J-P" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "This thesis documents my explorations of an ancient seafloor environment through sedimentary geology and stable isotope geochemistry. The geologic record of this seafloor \u2014 its life, environmental conditions, lithification, burial and exposure \u2014 consists of hundreds of meters of sedimentary rock, outcropping across Bahia and Minas Gerais, Brazil. Though it consists primarily of carbonate grains surrounded by carbonate cements, such as might be found forming in any shallow carbonate platform, this record also contains one of Brazil\u2019s most extensive sedimentary phosphate deposits. In these deposits, phosphate is concentrated as carbonate fluorapatite cements (CFA) in digitate stromatolites, distinctively finger-like, branching accretionary structures likely formed by the accumulation of sediment by microbial mats and biofilms. Chapter 1 introduces the broader motivations of this kind of paleoenvironmental and paleoecological research, for understanding the record of life on Earth and other worlds. Chapter 2 presents new sedimentological and stratigraphic data which interpret the depositional setting of a seafloor 600 million years ago on the rending supercontinent of Gondwana. Chapter 3 presents new carbon and oxygen isotopic measurements and clumped isotope measurements of structural carbonate in phosphatic and non-phosphatic textures of the rock, and uses them to constrain the alteration history of the rock and its effect on the record of primary depositional conditions. Chapter 4 presents new data on the sulfur isotope composition of specific minerals in the rock, combining several disparate analytical methods to draw conclusions about the metabolism of the stromatolites\u2019 microbial architects. Chapter 5 describes the distribution of organic material and style of fossilization, and presents preliminary data which suggest a possible mechanism by which microbial activity may have facilitated phosphate concentration and mineralization. As a whole, this thesis demonstrates the value of multidisciplinary analyses in the reconstruction and understanding of sedimentary phosphorite deposits throughout Earth history, improving our understanding of how and to what extent phosphorites may record the history of life and the environment.", "doi": "10.7907/rd4m-7x08", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14592", "collection": "thesis", "collection_id": "14592", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202022-005152490", "primary_object_url": { "basename": "thesis_draft13.pdf", "content": "final", "filesize": 290818840, "license": "other", "mime_type": "application/pdf", "url": "/14592/9/thesis_draft13.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural Basis of Antibody Recognition of Viruses", "author": [ { "family_name": "Esswein", "given_name": "Shannon Rose", "orcid": "0000-0002-5142-0190", "clpid": "Esswein-Shannon-Rose" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_chem" } ], "abstract": "The Zika epidemic in 2015-2016 and COVID-19 pandemic in 2019-2021 are the latest reminders of the enormous impact of viruses on the world. Zika, a flavivirus transmitted by mosquitos, can cause severe neurodevelopmental abnormalities including microcephaly in the newborns of the infected mothers. Vaccine design is complicated by concern that elicited antibodies may also recognize other epidemic-causing flaviviruses that share a similar envelope protein, such as dengue virus, West Nile Virus, and yellow fever virus. This cross-reactivity, if non-neutralizing, may worsen symptoms of a subsequent infection through antibody-dependent enhancement (ADE). To better understand the neutralizing antibody response and risk of ADE, we compared germline and mature antibody binding to Zika and other flaviviruses. We showed that affinity maturation of the light chain variable domain is important for strong binding of VH3-23/VK1-5 neutralizing antibodies to Zika virus envelope domain III (EDIII) and identified interactions that contribute to weak, cross-reactive binding to West Nile Virus EDIII. These findings informed our design of EDIII-conjugated mosaic nanoparticles as a pan-flavivirus vaccine candidate. Sera from immunization trials with nanoparticles displaying EDIIIs of Zika and dengue serotypes 1-4 showed cross-reactive binding to Zika, dengue 1-4, and West Nile Virus, a promising step towards the development of safe and effective flavivirus vaccines.
\r\n\r\nCoronaviruses are another group of viruses responsible for widespread morbidity and mortality, including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) epidemics and current SARS-CoV-2 pandemic. Given concerns regarding new SARS-CoV-2 variants and the possibility for additional zoonotic betacoronaviruses to cause future outbreaks, we investigated how the epitopes on the SARS-CoV-2 receptor binding domain (RBD) targeted by VH3-30-derived antibodies correlate with their neutralization potency and breadth of betacoronavirus recognition. Analyses showed how variations in antibody light chains and CDRH3 lengths facilitate the diverse RBD epitopes, cross-reactivity, and neutralization profiles of VH3-30 Abs, illustrating their importance for vaccine design and therapeutic antibody development.
", "doi": "10.7907/krjp-se81", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14138", "collection": "thesis", "collection_id": "14138", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05082021-185615529", "primary_object_url": { "basename": "McCardellReed2021thesis.pdf", "content": "final", "filesize": 7370942, "license": "other", "mime_type": "application/pdf", "url": "/14138/7/McCardellReed2021thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Genetic Circuits for the Control of Multi-Strain Bacterial Populations", "author": [ { "family_name": "McCardell", "given_name": "Reed Dillard", "orcid": "0000-0002-0955-3133", "clpid": "McCardell-Reed-Dillard" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Microbial species rarely exist alone. Nearly everywhere you could think to look, microorganisms of various species live together in harmony. Microbes together in their communities are incredibly powerful actors wherever they are found; they perform small miracles---the conversion of milk into yogurt---and large ones---production of most of the planet's oxygen and organic carbon.
\r\n\r\nOur burgeoning knowledge of microbial life combined with modern technologies to manipulate it create a critical, exciting opportunity to harness microbial power for the betterment of technology, people, and the planet. This thesis presents a body of work which explores the manipulation of microbial communities using the intersectional bio-engineering approach of synthetic biology. We demonstrate how molecular tools evolved by bacteria can be repurposed to create rationally designed systems for controlling features of bacterial populations.
\r\n\r\nWe begin by examining a genetic circuit that caps the size of a bacterial population by coordinating the deaths of population members -- the population capping or \"pop cap\" circuit. Briefly, E. coli cells in the pop cap circuit are engineered to synthesize a chemical -- a quorum sensing (QS) signal -- that reports the density of the population, sense this chemical, and produce the ccdB toxin to destroy themselves in response. The molecular tools that make up this circuit are drawn from organisms across the spectrum of bacterial diversity. Brought together, they create a feedback control circuit that controls population size by causing member cells to die when a target population size has been reached. To improve the performance of this population controller and reduce the influence of the environment on the circuit, we add the aiiA quorum sensing signal degradase to allow the experimenter control over the degradation rate of the QS density signal. Additionally, we explore RNA and protein mechanisms to sequester the death-causing toxin---inactivating it---allowing us to release a population cap. The resulting \"cap and release\" circuit is a flexible motif that can be scaled to control multi-strain populations, expanding the scope of control beyond the single-strain populations regulated by the base pop cap circuit.
\r\n\r\nUsing the scalable cap and release motif, we design a genetic circuit to regulate a multi-strain community. Two different cell strains expressing symmetric, interconnected cap and release systems form the \"A=B\" circuit, so named for its ability to control the composition of the community to a target ratio of A cells to B cells, or Apopulation = \u03b1Bpopulation. Through dynamical system models of the system, we explore the effects of active QS signal degradation on composition control performance and perform a parameter sensitivity analysis of the system to help determine the best method for building a functioning A=B system in the laboratory. We use a high throughput construction and screening protocol to create variants of the A=B system with identical architectures, but slightly differing component production rates. We crown the most successful variant with a series of experiments to determine if it indeed recapitulates our model's predictions for its performance. Our implementation of the A=B circuit can successfully regulate the composition of a community, with interesting additional effects on total population density.
\r\n\r\nThe cap and release and A=B circuits need parts that can do three things: 1) send a signal between cells to communicate information, 2) compare two signals, 3) regulate cell growth or death. We highlight bacteriocins, bacterial protein exotoxins that are released from a producer cell to kill other cells of similar species, as attractive tools for bacterial community engineering both for their multi-functionality and modular protein structure. By themselves, bacteriocins can perform all the functions needed for population control: they transmit themselves between cells, have unique high-affinity sequestering antitoxin proteins, and are toxins to receiver cells. We begin the process of their characterization and usage as synthetic biological \"parts\" by creating non-native expression systems that match native expression strengths. Using these experimenter-controlled systems we design preliminarily test a bacteriocin-based bacterial community control circuit. Additionally, given the E. coli colicin bacteriocins' unique, nearly plug-and-play modular domain structure, we explore possibilities for engineering colicin proteins themselves for increased functional diversity or uses outside of growth regulation.
", "doi": "10.7907/wgpp-vj97", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14220", "collection": "thesis", "collection_id": "14220", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012021-160326752", "type": "thesis", "title": "Manganese Through Time and Other Stories Concerning Cyanobacteria and the World Around Them", "author": [ { "family_name": "Lingappa", "given_name": "Usha Farey", "orcid": "0000-0001-5691-6788", "clpid": "Lingappa-Usha-Farey" } ], "thesis_advisor": [ { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Grotzinger", "given_name": "John P.", "orcid": "0000-0001-9324-1257", "clpid": "Grotzinger-J-P" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "This thesis is a collection of investigations concerning the interplay between Cyanobacteria and the inorganic/physical world. Chapters II-VI focus on manganese, an element Cyanobacteria have been\r\nintimately entangled with for billions of years. Chapter II is a review/perspective paper on the dynamics of\r\nmanganese in the environment through time and the many ways manganese interfaces with dioxygen.\r\nChapter III deciphers environmental and biological signatures recorded in ancient rocks from the pivotal\r\nmoment in Earth history when oxygenic photosynthesis first evolved. Chapter IV explores the ecology of\r\ndesert varnish, and provides an adaptive physiological mechanism underpinning manganese enrichment.\r\nChapter V examines the ability of modern Cyanobacteria to catalyze manganese oxidation. Chapter VI\r\nexplains as kindly as possible that the field of manganese aquatic chemistry has fundamentally\r\nmisunderstood the chemistry of Mn(III) and highlights how the current methods being used are\r\nproblematic because of this misunderstanding. Chapters VII and VIII are not about manganese and instead\r\nconcern other aspects of the physical world and their interface with Cyanobacteria. Chapter VII is about the\r\nimpact of Hurricane Irma on a cyanobacterial mat ecosystem. Chapter VIII is about the use of ooids as an\r\nenvironmentally friendly replacement for plastic microbeads in facial scrubs, in which Cyanobacteria\r\nmake a cameo as endoliths that facilitate ooid dissolution.
", "doi": "10.7907/9ysw-jt52", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13962", "collection": "thesis", "collection_id": "13962", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09232020-172452048", "type": "thesis", "title": "Spatial and Temporal Dynamics of Microorganisms Living Along Steep Energy Gradients and Implications for Ecology and Geologic Preservation in the Deep Biosphere", "author": [ { "family_name": "Mullin", "given_name": "Sean William Alexander", "orcid": "0000-0002-6225-3279", "clpid": "Mullin-Sean-William-Alexander" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "The deep biosphere represents a massive repository of life with unknown effects on global biogeochemical cycles. Even the fundamental life strategies of the endemic microorganisms that inhabit this biome remain enigmatic; some studies have indicated that subsurface organisms subsist in energetic regimes below the theoretical lower limit for life. A boom-bust life cycle, mediated by tectonic disturbances and subsurface fractures, may help explain these phenomena. This work addresses and expands on this question, first by exploring the response of continental deep biosphere microorganisms to an in situ organic matter amendment, then by analyzing the microbial community dynamics of the sediments and carbonate along a naturally-occurring energy gradient at a methane seep. Our experiments in the continental deep biosphere confirmed that mineralogical heterogeneity can drive differential colonization of the native microorganisms, implying that selection and adaptation to in situ conditions occurs, differentiating individual microbial niches. We also observed the formation of secondary framboidal iron sulfide minerals, a well-known phenomenon in marine sediments but not extensively observed in the deep subsurface, that were correlated to the presence of abundant sulfur-metabolizing microorganisms. Chapters 2 and 3 are instead focused on the microbial ecology of a methane seep on the Pacific margin of Costa Rica. Cold methane seeps themselves represent sharp boundaries between the generally low-energy background seafloor and abundant chemical energy in the form of methane. Chapter 2 describes that the microorganisms living at these seeps occupy a significantly narrower spatial scale than the endemic megafauna. In addition, by correlating community dissimilarity and geographic distance, the functional center of the seep was identified, allowing for insight into the ecological differentiation between clades of anaerobic methanotrophic archaea (ANME). Chapter 3 examines in greater detail the endolithic microbial community, principally composed of ANME-1. By conducting transplantation experiments of carbonates on the seafloor, we tested the response of the in situ endolithic communities and found that carbonates moved distinctly outside the active zone changed less than communities moved to regions of less activity.
", "doi": "10.7907/f3k8-ck13", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13854", "collection": "thesis", "collection_id": "13854", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08122020-110818529", "primary_object_url": { "basename": "Metcalfe_PhD.pdf", "content": "final", "filesize": 248598908, "license": "other", "mime_type": "application/pdf", "url": "/13854/37/Metcalfe_PhD.pdf", "version": "v17.0.0" }, "type": "thesis", "title": "Symbiotic Diversity and Mineral-Associated Microbial Ecology in Marine Microbiomes", "author": [ { "family_name": "Metcalfe", "given_name": "Kyle Shuhert", "orcid": "0000-0002-2963-765X", "clpid": "Metcalfe-Kyle-Shuhert" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Rossman", "given_name": "George Robert", "orcid": "0000-0002-4571-6884", "clpid": "Rossman-G-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "This thesis investigates ecological interactions in the seafloor between microbial taxa (Chapters 1 and 2) and between these microorganisms and their mineral hosts (Chapters 2 through 4). In seafloor sediments, electron acceptors are often limited, forcing microorganisms inhabiting these sediments to acquire symbiotic partners and/or perform extracellular electron transfer to insoluble electron acceptors. Seafloor methane seeps present an endmember case wherein extremely reducing fluids charged with methane advect through sediment. In these benthic ecosystems, anaerobic methanotrophic archaea (ANME) form symbiotic partnerships with sulfate-reducing bacteria (SRB), but it remained unclear if certain ANME exhibit a preference for certain SRB partners. In Chapter 1, I present results documenting such a pattern of partnership specificity in ANME-SRB consortia. In Chapter 2, I further examine these patterns in rare ANME taxa through development and application of a density-separation protocol refined from published work. This protocol exploits the co-association of microbial taxa on mineral surfaces to aid in the detection of novel symbioses, and further is useful to detect microbial interactions with certain minerals. In Chapter 3, I focus on the interaction between ANME-SRB consortia and authigenic silicates that have been observed on consortium exteriors, finding evidence to support that the precipitation of these silicates is actively mediated by ANME-SRB. In Chapter 4, I perform geochemical modeling benchmarked by synchrotron X-ray analysis to examine the imprint of extracellular electron transport by metal-reducing microorganisms on Precambrian manganese-rich sedimentary rocks.", "doi": "10.7907/ve4r-k526", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13842", "collection": "thesis", "collection_id": "13842", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182020-211405749", "primary_object_url": { "basename": "Chadwick_Thesis_v3.pdf", "content": "final", "filesize": 58503253, "license": "other", "mime_type": "application/pdf", "url": "/13842/37/Chadwick_Thesis_v3.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "How to Beat Diffusion: Explorations of Energetics and Spatial Relationships in Microbial Ecosystems", "author": [ { "family_name": "Chadwick", "given_name": "Grayson Lee", "orcid": "0000-0003-0700-9350", "clpid": "Chadwick-Grayson-Lee" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "This thesis investigates four microbial systems, with a particular focus for how spatial considerations shape the behavior and evolution of microorganisms. After a general introduction in Chapter 1, Chapter 2 presents the results of experiments demonstrating how cellular activity varies through space within an anode-reducing biofilm. Chapter 3 presents a comprehensive comparative genomic analysis of all known marine anaerobic methanotrophic archaea, supporting the notion that these organisms share many energetic similarities with the anode reducing organisms in Chapter 2. These are interpreted as specific adaptations to life in highly structured microbial communities. Chapter 4 describes the enrichment and characterization of a new member of the purple sulfur bacteria, and the adaptations that may improve substrate acquisition beyond the normal limitations of diffusion. Chapter 5 describes the convergent evolution of novel Complex I gene clusters that have incorporated new proton pumping subunits, and the modifications made to the protein structure to facilitate the incorporation of these new subunits into the quaternary structure of the complex.
", "doi": "10.7907/svrp-rb07", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13801", "collection": "thesis", "collection_id": "13801", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082020-172255300", "type": "thesis", "title": "3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis", "author": [ { "family_name": "Zhu", "given_name": "Yanzhe", "orcid": "0000-0002-2260-1830", "clpid": "Zhu-Yanzhe" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Venkateswaran", "given_name": "Kasthuri Jhetty", "clpid": "Venkateswaran-K-J" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows:
\r\n\r\n1) COMSOL Multiphysics\u00ae was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated.
\r\n\r\n2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 \u00b5m channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 \u03bcm to 334 \u03bcm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 \u03bcm, each standard 20 \u03bcL reaction could produce ~10\u2074 droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3\u00d710\u00b3 target copies or cells per \u03bcL, and the detection limit is 0.1 copies or cells per \u03bcL.
\r\n\r\n3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations.
\r\n\r\n4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link.
\r\n\r\nIn summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.
", "doi": "10.7907/vk3d-7212", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13664", "collection": "thesis", "collection_id": "13664", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03262020-092455420", "type": "thesis", "title": "A Quantitative and High-Throughput Approach to Gene Regulation in Escherichia coli", "author": [ { "family_name": "Ireland", "given_name": "William Thornton", "orcid": "0000-0003-0971-2904", "clpid": "Ireland-William-Thornton" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Measurements in biology have reached a level of precision that demands quantitative modeling. This is particularly true in the field of gene regulation, where concepts from physics such as thermodynamics have allowed for accurate models to be made.
\r\n \r\nMany issues remain. DNA sequencing is routine enough to sequence new genomes in days and cheap enough to use deep sequencing to perform precision measurements, but our ability to interpret the wealth of genomic data is lagging behind, especially in the realm of gene regulation. The primary reason is that we lack any information what so ever as to the basic regulatory details of approximately 65 percent of operons even in E. coli, the best understood organism in biology. As a result we cannot use our hard won modeling efforts to understand any of these operons.
\r\n \r\nThis work takes steps to address these issues. First we use 30 LacI mutants as a test case to prove that we can make quantitatively accurate models of gene expression and sequence-dependent binding energies of transcription factors and RNA polymerase.
\r\n\r\nNext we note that much of the quantitative insight available on transcriptional regulation relies on work on only a few model regulatory systems such as LacI as was considered above. We develop an approach, through a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling that can be used to dissect bacterial promoters in a systematic and scalable way. We demonstrate that we can uncover a qualitative list of transcription factor binding sites as well as their associated quantitative details from both well-studied and previously uncharacterized promoters in E. coli.
\r\n\r\nFinally we extend the above method to over 100 E. coli promoters using over 12 growth conditions. We show the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulation. In many cases, we identify which transcription factors mediate their regulation. The method introduced clears a path for fully characterizing the regulatory genome of E. coli and advances towards the goal of using this method on a wide variety of other organisms including other prokaryotes and eukaryotes such as Drosophila melanogaster.
", "doi": "10.7907/0sk3-hd69", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11693", "collection": "thesis", "collection_id": "11693", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-181520170", "primary_object_url": { "basename": "PhD_Thesis_Ramesh_Pradeep_Final.pdf", "content": "final", "filesize": 4837595, "license": "other", "mime_type": "application/pdf", "url": "/11693/1/PhD_Thesis_Ramesh_Pradeep_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Imaging and Control of Engineered Cells using Magnetic Fields", "author": [ { "family_name": "Ramesh", "given_name": "Pradeep", "orcid": "0000-0001-6243-8145", "clpid": "Ramesh-Pradeep" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in 'ultraparamagnetic' macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this 'ultraparamagnetic' concept.
\r\n\r\nIn addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest.
\r\n \r\nFinally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.
\r\n", "doi": "10.7907/KY00-7Y74", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10859", "collection": "thesis", "collection_id": "10859", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-122340793", "primary_object_url": { "basename": "GPD thesis 2018.pdf", "content": "final", "filesize": 22341118, "license": "other", "mime_type": "application/pdf", "url": "/10859/1/GPD thesis 2018.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Colonization of the Intestinal Surface by Indigenous Microbiota", "author": [ { "family_name": "Donaldson", "given_name": "Gregory Paul", "orcid": "0000-0002-8551-374X", "clpid": "Donaldson-Gregory-Paul" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "3MT Competition (Caltech)" }, { "literal": "div_bbe" } ], "abstract": "The mammalian gut evolved to foster the development and maintenance of a community of specific bacterial symbionts that persist for years. Bacteroides fragilis is one of a number of species that are able to colonize the mucus of the large intestine in mice and humans. This thesis explores the mechanisms and functions of mucosal colonization, most notably by using reductionist approaches with gnotobiotic mice. Harnessing genetics on both the host and microbial side allowed the dissection of a pathway by which immunoglobulin A enhances mucosal colonization by B. fragilis. Novel colonization assays were developed to explore the importance of mucosal colonization to bacterial fitness. Finally, an enrichment method for host-associated bacterial transcriptomics was used to define the behavior of this symbiont within the mucus layer.
", "doi": "10.7907/6EZ0-3007", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10666", "collection": "thesis", "collection_id": "10666", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312018-211454203", "type": "thesis", "title": "Clumped and Intramolecular Isotopic Perspectives on the Behavior of Organic and Inorganic Carbon in the Shallow Crust and Deep Biosphere", "author": [ { "family_name": "Lloyd", "given_name": "Max Kaufmann", "orcid": "0000-0001-9367-2698", "clpid": "Lloyd-Max-Kaufmann" } ], "thesis_advisor": [ { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Rossman", "given_name": "George Robert", "clpid": "Rossman-G-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Although the upper crust is the most accessible swath of the subsurface, the geochemical processes therein are challenging to observe. Here, I use site-specific and \u2018clumped\u2019 isotope analyses (where multiple rare, heavy isotopes are bound in single molecules) of carbonates and organic solids to explore petrology, brittle tectonics, diagenesis, and biodegradation in the shallow crust. In carbonates, I employ contact aureoles, regional metamorphic terranes, thermal and geochemical models, and high-pressure experiments, to extract nuanced thermal and chemical histories. In the organic realm, I develop new methods to measure the clumped 13C \u2013 2H composition of methoxyl groups (R \u2013 O \u2013 CH3) from kerogen, lignin, and other complex organic substrates. This work expands the field of clumped isotope geochemistry into the realm of solid compounds, and enables new, site-specific isotopic perspectives on the shallow crustal transformations of organic carbon substrates and the microbial assemblages that live on them.
", "doi": "10.7907/Z96T0JV5", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10812", "collection": "thesis", "collection_id": "10812", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04172018-174233725", "primary_object_url": { "basename": "GitaMahmoudabadiThesis-postProofFinal.pdf", "content": "final", "filesize": 35079371, "license": "other", "mime_type": "application/pdf", "url": "/10812/1/GitaMahmoudabadiThesis-postProofFinal.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Virology By The Numbers: A Quantitative Exploration of Viral Energetics, Genomics, and Ecology", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita", "orcid": "0000-0002-8812-7246", "clpid": "Mahmoudabadi-Gita" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Gelbart", "given_name": "William", "clpid": "Gelbart-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Over the past couple of decades, technological advancements in sequencing and imaging have unequivocally proven that the world of viruses is far bigger and more consequential than previously imagined. There are 1031 viruses estimated to inhabit our planet, outnumbering even bacteria. Despite their astronomical numbers and staggering sequence diversity, environmental viruses are poorly characterized. In this thesis we will demonstrate our three-pronged exploration of viruses through the lenses of energetics (Chapters 2 and 3), genomics (Chapter 4) and ecology (Chapter 5). We will first focus on one of the defining features of viruses, namely their reliance on their host for energy, and demonstrate the energetic cost of building a virus and mounting an infection. In our second study, we present one of the largest surveys of complete viral genomes, providing a comprehensive and quantitative snapshot of viral genomic trends for thousands of viruses. In our third study, we shift our focus towards ecological questions surrounding the large number of commensal phages inhabiting the human body. We discovered that phage community composition could serve as a fingerprint, or a \"phageprint\" \u2013 highly personal and stable over time. To our knowledge, this study is one of the largest studies of human phages and the first to demonstrate the feasibility of human identification based on phage sequences.
", "doi": "10.7907/Z9Q81B91", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10246", "collection": "thesis", "collection_id": "10246", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012017-152345273", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 68205818, "license": "other", "mime_type": "application/pdf", "url": "/10246/114/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Microbial Evolution and the Rise of Oxygen: The Roles of Contingency and Context in Shaping the Biosphere through Time", "author": [ { "family_name": "Ward", "given_name": "Lewis Michael", "orcid": "0000-0002-9290-2567", "clpid": "Ward-Lewis-Michael" } ], "thesis_advisor": [ { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" } ], "thesis_committee": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "McGlynn", "given_name": "Shawn E.", "clpid": "McGlynn-S-E" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "We are shaped by our environment, but we then shape it in turn. This interplay between life and the Earth, and how these interactions have shaped both parties through time, is the heart of the discipline of geobiology. My research is fundamentally motivated by a desire to understand how life and the Earth have changed together through time to reach the state that they\u2019re at today, and to understand from this history how the coevolution of planet and life may be different on other worlds. The focus of my work has been on how the structure and productivity of the biosphere across time and space has been shaped by the metabolic opportunities provided by the environment\u2014as a result of both biotic and abiotic factors\u2014and the metabolic pathways that are available to life, as a result of evolutionary contingency in the evolution of pathways and their inheritance and horizontal transfer.
\r\n\r\nThe biosphere on Earth today is incredibly productive due to the coupled dominant metabolisms of oxygenic photosynthesis and aerobic respiration, yet these can\u2019t always be assumed to have been present\u2014considering life more broadly, for instance in the context of the early Earth and other planets, we have to grapple with how evolutionary contingency and planetary environments interact to constrain the metabolic opportunities and rates of productivity available to the biosphere. In this dissertation, I broadly consider how the size and structure of Earth\u2019s biosphere has changed through time as surface environments evolve and metabolic innovations accumulate. These investigations make use of information gleaned from the rock record of the early Earth, as well as the biological record of the history of life as preserved in the genomes, biochemistry, and ecology of extant organisms. These coupled records provide opportunities for constraining estimates of the opportunities for life throughout Earth history and elsewhere in the universe.
\r\n", "doi": "10.7907/Z9BZ642S", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10319", "collection": "thesis", "collection_id": "10319", "cite_using_url": "https://resolver.caltech.edu/CaltechThesis:06082017-134458390", "primary_object_url": { "basename": "Thesis_Hang_Yu.pdf", "content": "final", "filesize": 16027895, "license": "other", "mime_type": "application/pdf", "url": "/10319/18/Thesis_Hang_Yu.pdf", "version": "v11.0.0" }, "type": "thesis", "title": "Understanding the Symbiosis in Anaerobic Oxidation of Methane Through Metabolic, Biosynthetic and Transcriptomic Activities", "author": [ { "family_name": "Yu", "given_name": "Hang Hank", "orcid": "0000-0002-7600-1582", "clpid": "Yu-Hang-Hank" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Microorganisms provide essential ecological services to our planet. Their combined activities control and shape our environment as we know today. In the deep sea, a microbial mediated process known as anaerobic oxidation of methane (AOM) consumes large amounts of methane, a potent greenhouse gas and a valuable energy resource. How this symbiosis works is poorly understood.
\r\n\r\nIn this thesis, I tested current hypotheses on the symbiotic mechanisms in AOM microbial consortia, consisting of a partnership between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Sediments collected from methane seeps offshore Oregon and California and dominated by AOM consortia were used in these investigations. A range of compounds were amended to sediment microcosms, and their effects on the metabolic activities of ANME or SRB were monitored by tracking the rates of methane oxidation or sulfate reduction on timescales varying from hours to months. A lack of stimulation or inhibition on the AOM consortia, combined with long-term community profiles, suggest that diffusible compounds are unlikely to be involved in the symbiosis in AOM. I further examine ANME genomes, focusing the role of sulfur in methane seep ecosystems. Phylogenetic analyses revealed multiple poorly characterized genes in the sulfur pathway, and comparisons with methanogenic archaea related to ANME provided a better understanding of their roles in the cell. Transcriptional responses combined with protein modeling were used to predict the potential substrate of a sulfite reductase related enzyme. These predictions were validated using genetics, and together point to an assimilatory rather than dissimilatory sulfur pathway in methane-utilizing archaea in general. Then, the AOM symbiosis was decoupled for the first time using soluble electron acceptors. ANME remained metabolically and biosynthetically active without their SRB partner, suggesting that the electrons are transferred directly in this partnership. This observation was investigated to a greater depth with transcriptomics. Membrane proteins and multiheme cytochromes critical in extracellular electron transfer in ANME and SRB were expressed. These results together illuminate the path electrons may take to exit or enter the AOM consortia. Overall, multiple activity analyses used here piece together a clearer view on how the symbiosis in AOM works, with potential applications in future energy generation from methane.
\r\n", "doi": "10.7907/Z9XD0ZQQ", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10212", "collection": "thesis", "collection_id": "10212", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262017-143517395", "type": "thesis", "title": "Insights into Pathways of Nitrous Oxide Generation from Novel Isotopologue Measurements", "author": [ { "family_name": "Magyar", "given_name": "Paul Macdonald", "orcid": "0000-0003-0234-247X", "clpid": "Magyar-Paul-Macdonald" } ], "thesis_advisor": [ { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Rossman", "given_name": "George Robert", "orcid": "0000-0002-4571-6884", "clpid": "Rossman-G-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "The accumulation of nitrous oxide (N2O) in the atmosphere is a significant manifestation of human perturbations of the nitrogen cycle. This thesis reports the development and first applications of a novel isotopic technique for characterizing nitrous oxide sources. Chapter 1 describes the development of methods to use the newly available technology of high- resolution dual-inlet multi-collector mass spectrometry to measure six isotopic parameters in nitrous oxide. It reports the standardization and initial biological application of these methods. Chapter 2 presents a model for the generation of isotope effects in an important N2O generating enzyme, the bacterial nitric oxide reductase; this model and published isotopic constraints are used to provide insights into the mechanism of that enzyme. Chapter 3 describes the six-dimensional isotopic characterization of nitrous oxide from bacterial denitrifiers, while Chapter 4 describes nitrous oxide generated by ammonia oxidizing bacteria.
", "doi": "10.7907/Z93776RJ", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9064", "collection": "thesis", "collection_id": "9064", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07212015-103405937", "type": "thesis", "title": "Physical, Metabolic, and Energetic Investigations of Methane-Metabolizing Microbial Communities", "author": [ { "family_name": "Marlow", "given_name": "Jeffrey James", "clpid": "Marlow-Jeffrey-James" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoehler", "given_name": "Tori M.", "clpid": "Hoehler-T-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Understanding the roles of microorganisms in environmental settings by linking phylogenetic identity to metabolic function is a key challenge in delineating their broad-scale impact and functional diversity throughout the biosphere. This work addresses and extends such questions in the context of marine methane seeps, which represent globally relevant conduits for an important greenhouse gas. Through the application and development of a range of culture-independent tools, novel habitats for methanotrophic microbial communities were identified, established settings were characterized in new ways, and potential past conditions amenable to methane-based metabolism were proposed. Biomass abundance and metabolic activity measures \u2013 both catabolic and anabolic \u2013 demonstrated that authigenic carbonates associated with seep environments retain methanotrophic activity, not only within high-flow seep settings but also in adjacent locations exhibiting no visual evidence of chemosynthetic communities. Across this newly extended habitat, microbial diversity surveys revealed archaeal assemblages that were shaped primarily by seepage activity level and bacterial assemblages influenced more substantially by physical substrate type. In order to reliably measure methane consumption rates in these and other methanotrophic settings, a novel method was developed that traces deuterium atoms from the methane substrate into aqueous medium and uses empirically established scaling factors linked to radiotracer rate techniques to arrive at absolute methane consumption values. Stable isotope probing metaproteomic investigations exposed an array of functional diversity both within and beyond methane oxidation- and sulfate reduction-linked metabolisms, identifying components of each proposed enzyme in both pathways. A core set of commonly occurring unannotated protein products was identified as promising targets for future biochemical investigation. Physicochemical and energetic principles governing anaerobic methane oxidation were incorporated into a reaction transport model that was applied to putative settings on ancient Mars. Many conditions enabled exergonic model reactions, marking the metabolism and its attendant biomarkers as potentially promising targets for future astrobiological investigations. This set of inter-related investigations targeting methane metabolism extends the known and potential habitat of methanotrophic microbial communities and provides a more detailed understanding of their activity and functional diversity.", "doi": "10.7907/Z9W66HPS", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9768", "collection": "thesis", "collection_id": "9768", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262016-132232540", "primary_object_url": { "basename": "ETR_Thesis_Final.pdf", "content": "final", "filesize": 120182434, "license": "other", "mime_type": "application/pdf", "url": "/9768/43/ETR_Thesis_Final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Molecular and Geochemical Insights into Microbial Life Centimeters to Kilometers Below the Seafloor", "author": [ { "family_name": "Trembath-Reichert", "given_name": "Elizabeth", "orcid": "0000-0002-3979-8676", "clpid": "Trembath-Reichert-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Fischer", "given_name": "Woodward W.", "clpid": "Fischer-W-W" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "At the broadest scale, this thesis is an investigation of how life modulates the movement of essential elements (carbon, sulfur, nitrogen, and silicon) on modern and geologic timescales. Chapters 1 and 2 explore carbon and sulfur cycling microbial communities found centimeters below the seafloor in hydrocarbon-rich methane seep ecosystems. At the Hydrate Ridge methane seep, we investigated how microbial partnerships direct the flow of methane and sulfide in these benthic oases by using identity-based physical separation methods developed in our lab (Magneto-FISH) in conjunction with community profiling and metagenomic sequencing. This method explores the middle ground between single cell and bulk sediment analysis by separating target microbes and their physically associated community for downstream sequencing applications. Magneto-FISH captures were done at a range of microbial taxonomic group specificities and sequenced with both clone library and next-gen iTag 16S rRNA gene methods. Chapter 1 provides a demonstration of how FISH probe taxonomic specificity correlates to resultant Archaeal taxonomic diversity in Magneto-FISHed seep sediments, with specific attention to preparation of Archaea-enriched samples for downstream metagenomic sequencing. In Chapter 2, a Bacteria-focused parallel environmental isolation and sequencing effort was subjected to co-occurrence analyses which suggested there may be far more microbial associations in methane seep systems than are currently appreciated, including partnerships that do not involve the canonical anaerobic methane oxidizing archaea and sulfate reducing bacteria. With samples from IODP Expedition 337 Shimokita coalbed biosphere, Chapter 3 provides evidence for an active microbial assemblage kilometers below the sea floor in the deepest samples ever collected by marine scientific ocean drilling. Using in situ temperature Stable Isotope Probing (SIP) incubations and NanoSIMS, we investigated whole community activity (with the passive tracer D2O) and substrate specific activity with C1-carbon compounds methylamine and methanol. We found deuterium-based turnover times to be faster (years) than previous deep biosphere estimates (hundreds to thousands of years), but methylotrophy rates to be slower than previous carbon metabolic rates.
", "doi": "10.7907/Z96Q1V6Q", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9776", "collection": "thesis", "collection_id": "9776", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262016-170051580", "primary_object_url": { "basename": "Case_David_2016_Thesis_Full_Revised.pdf", "content": "final", "filesize": 23212031, "license": "other", "mime_type": "application/pdf", "url": "/9776/54/Case_David_2016_Thesis_Full_Revised.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Carbonate-Associated Microbial Ecology at Methane Seeps: Assemblage Composition, Response to Changing Environmental Conditions, and Implications for Biomarker Longevity", "author": [ { "family_name": "Case", "given_name": "David Hamilton", "orcid": "0000-0002-1023-0040", "clpid": "Case-David-Hamilton" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Fischer", "given_name": "Woodward W.", "clpid": "Fischer-W-W" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Levin", "given_name": "Lisa A.", "clpid": "Levin-L-A" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Methane seeps are globally distributed geologic features in which reduced fluid from below the seafloor is advected upward and meets the oxidized bottom waters of Earth\u2019s oceans. This redox gradient fuels chemosynthetic communities anchored by the microbially-mediated anaerobic oxidation of methane (AOM). Both today and in Earth\u2019s past, methane seeps have supported diverse biological communities extending from microorgansisms to macrofauna and adding to the diversity of life on Earth. Simultaneously, the carbon cycling associated with methane seeps may have played a significant role in modulating ancient Earth\u2019s climate, particularly by acting as a control on methane emissions.
\r\n\r\nThe AOM metabolism generates alkalinity and dissolved inorganic carbon (DIC) and at a 2:1 ratio, promoting the abiogenic, or authigenic, precipitation of carbonate minerals. Over time, these precipitates can grow into pavements covering hundreds of square meters on the seafloor and dominating the volumetric habitat space available in seep ecosystems. Importantly, carbonates are incorporated into the geologic record and therefore preserve an inorganic (i.e., d13C) and organic (i.e., lipid biomarker) history of methane seepage. However, the extent to which preserved biomarkers represent a snapshot of microorganisms present at the time of primary precipitation, a time-integrated history of microbial assemblages across the life cycle of a methane seep, or a view of the final microorganisms inhabiting a carbonate prior to incorporation in the sedimentary record is unresolved.
\r\n\r\nThis thesis addresses the ecology of carbonate-associated seep microorganisms. Chapters One and Two contextualize the extant microbial diversity on seep carbonates versus within seep sediments, as determined through 16S rRNA gene biomarkers. Small, protolithic carbonate \u201cnodules\u201d recovered from within seep sediments are observed to be capable of capturing surrounding sediment-hosted microbial diversity, but in some cases also diverge from sediments. Meanwhile, lithified carbonate blocks recovered from the seafloor host microbial assemblages demonstrably distinct from seep sediments (and seep nodules). Microbial 16S rRNA gene diversity within carbonate samples is well-differentiated by the extent of contemporary seepage. In situ seafloor transplantation experiments further demonstrated the microbial assemblages associated with seep carbonates to be sensitive to seep quiescence and activation on short (13-month) timescales. This was particularly true for organisms whose 16S rRNA genes imply physiologies dependent on methane or sulfur oxidation. With an improved understanding of the modern ecology of carbonate-associated microorganisms, Chapter Three applies intact polar lipid (IPL) and core lipid analyses to begin describing whether, and to what extent, geologically relevant biomarkers mimic short-term dynamics observed in 16S rRNA gene profiles versus archive a record of historic microbial diversity. Biomarker longevity is determined to increase from 16S rRNA genes to IPLs to core lipids, with IPLs preserving microbial diversity history on timescales more similar to 16S rRNA genes than core lipids. Ultimately, individual IPL biomarkers are identified which may be robust proxies for determining whether the biomarker profile recorded in a seep carbonate represents vestiges of active seepage processes, or the profile of a microbial community persisting after seep quiescence.
", "doi": "10.7907/Z9TD9V84", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8637", "collection": "thesis", "collection_id": "8637", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182014-160835979", "primary_object_url": { "basename": "final_thesis_electronic_format.pdf", "content": "final", "filesize": 28484306, "license": "other", "mime_type": "application/pdf", "url": "/8637/1/final_thesis_electronic_format.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "From Lakes to Lungs: Assessing Microbial Activity in Diverse Environments ", "author": [ { "family_name": "Kopf", "given_name": "Sebastian Hermann", "clpid": "Kopf-Sebastian-Hermann" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "All major geochemical cycles on the Earth\u2019s surface are mediated by microorganisms. Our understanding of how these microbes have interacted with their environments (and vice versa) throughout Earth's history, and how they will respond to changes in the future, is primarily based on studying their activity in different environments today. The overarching questions that motivate the research presented in the two parts of this thesis -- how do microorganisms shape their environment (and vice versa)? and how can we best study microbial activity in situ? -- have arisen from the ultimate goal of being able to predict microbial activity in response to changes within their environments both past and future.
\r\n\r\nPart one focuses on work related to microbial processes in iron-rich Lake Matano and, more broadly, microbial interactions with the biogeochemical cycling of iron. Primarily, we find that the chelation of ferrous iron by organic ligands can affect the role of iron in anoxic environmental systems, enabling photomixotrophic growth of anoxygenic microorganisms with ferrous iron, as well as catalyzing the oxidation of ferrous iron by denitrification intermediates. These results imply that the ability to grow photomixotrophically on ferrous iron might be more widespread than previously assumed, and that the co-occurrence of chemical and biological processes involved in the coupled biogeochemical cycling of iron and nitrogen likely dominate organic-rich environmental systems.
\r\n\r\nPart two switches focus to in situ measurements of growth activity and comprises work related to microbial processes in the Cystic Fibrosis lung, and more broadly, the physiology of slow growth. We introduce stable isotope labeling of microbial membrane fatty acids and whole cells with heavy water as a new technique to measure microbial activity in a wide range of environments, demonstrate its application in continuous culture in the laboratory at the population and single cell level, and apply the tool to measure the in situ activity of the opportunistic pathogen Staphylococcus aureus within the environment of expectorated mucus from cystic fibrosis patients. We find that the average in situ growth rates of S. aureus fall into a range of generation times between ~12 hours and ~4 days, with substantial heterogeneity at the single-cell level. These data illustrate the use of heavy water as a universal environmental tracer for microbial activity, and highlight the crucial importance of studying the physiology of slow growth in representative laboratory systems in order to understand the role of these microorganisms in their native environments.
", "doi": "10.7907/Z9HQ3WV6", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8864", "collection": "thesis", "collection_id": "8864", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05182015-162708844", "primary_object_url": { "basename": "JohnsonJE-2015_PhdThesis_final.pdf", "content": "final", "filesize": 357112948, "license": "other", "mime_type": "application/pdf", "url": "/8864/84/JohnsonJE-2015_PhdThesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Manganese: Minerals, Microbes, and the Evolution of Oxygenic Photosynthesis", "author": [ { "family_name": "Johnson", "given_name": "Jena Elaine", "clpid": "Johnson-Jena-Elaine" } ], "thesis_advisor": [ { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Webb", "given_name": "Samuel M.", "clpid": "Webb-S-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Oxygenic photosynthesis fundamentally transformed our planet by releasing molecular oxygen and altering major biogeochemical cycles, and this exceptional metabolism relies on a redox-active cubane cluster of four manganese atoms. Not only is manganese essential for producing oxygen, but manganese is also only oxidized by oxygen and oxygen-derived species. Thus the history of manganese oxidation provides a valuable perspective on our planet\u2019s environmental past, the ancient availability of oxygen, and the evolution of oxygenic photosynthesis. Broadly, the general trends of the geologic record of manganese deposition is a chronicle of ancient manganese oxidation: manganese is introduced into the fluid Earth as Mn(II) and it will remain only a trace component in sedimentary rocks until it is oxidized, forming Mn(III,IV) insoluble precipitates that are concentrated in the rock record. Because these manganese oxides are highly favorable electron acceptors, they often undergo reduction in sediments through anaerobic respiration and abiotic reaction pathways.
\r\n\r\nThe following dissertation presents five chapters investigating manganese cycling both by examining ancient examples of manganese enrichments in the geologic record and exploring the mineralogical products of various pathways of manganese oxide reduction that may occur in sediments. The first chapter explores the mineralogical record of manganese and reports abundant manganese reduction recorded in six representative manganese-enriched sedimentary sequences. This is followed by a second chapter that further analyzes the earliest significant manganese deposit 2.4 billon years ago, and determines that it predated the origin of oxygenic photosynthesis and thus is supporting evidence for manganese-oxidizing photosynthesis as an evolutionary precursor prior to oxygenic photosynthesis. The lack of oxygen during this early manganese deposition was partially established using oxygen-sensitive detrital grains, and so a third chapter delves into what these grains mean for oxygen constraints using a mathematical model. The fourth chapter returns to processes affecting manganese post-deposition, and explores the relationships between manganese mineral products and (bio)geochemical reduction processes to understand how various manganese minerals can reveal ancient environmental conditions and biological metabolisms. Finally, a fifth chapter considers whether manganese can be mobilized and enriched in sedimentary rocks and determines that manganese was concentrated secondarily in a 2.5 billion-year-old example from South Africa. Overall, this thesis demonstrates how microbial processes, namely photosynthesis and metal oxide-reducing metabolisms, are linked to and recorded in the rich complexity of the manganese mineralogical record.
", "doi": "10.7907/Z9RF5S0X", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8017", "collection": "thesis", "collection_id": "8017", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10262013-152651094", "primary_object_url": { "basename": "Lucey_KS_2014Thesis.pdf", "content": "final", "filesize": 9643634, "license": "other", "mime_type": "application/pdf", "url": "/8017/1/Lucey_KS_2014Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Catechol 2,3-Dioxygenase-Assisted Cleavage of Aromatics by \"Anaerobic\" Termite Gut Spirochetes and Genomic Evidence of a Complete Meta-Pathway", "author": [ { "family_name": "Lucey", "given_name": "Kaitlyn Shae", "clpid": "Lucey-Kaitlyn-Shae" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The termite hindgut microbial ecosystem functions like a miniature lignocellulose-metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting-edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS-1 and ZAS-2 as well as T. azotonutricium str. ZAS-9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system\u2019s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O2-stress and to generate acetate, the \u201cbiofuel\u201d of the termite system, are proposed. In particular, catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes are described in the \u201canaerobic\u201d termite hindgut spirochetes T. primitia str. ZAS-1 and ZAS-2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O2-dependent, yet nonrespiratory, metabolisms of plant-derived aromatics should be re-evaluated in termite hindgut communities. Potential future work is also illustrated.", "doi": "10.7907/KQB3-7010", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7347", "collection": "thesis", "collection_id": "7347", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12192012-141624638", "primary_object_url": { "basename": "Dekas_Anne_2013_thesis.pdf", "content": "final", "filesize": 52021793, "license": "other", "mime_type": "application/pdf", "url": "/7347/1/Dekas_Anne_2013_thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Diazotrophy in the Deep: An Analysis of the Distribution, Magnitude, Geochemical Controls, and Biological Mediators of Deep-Sea Benthic Nitrogen Fixation", "author": [ { "family_name": "Dekas", "given_name": "Anne Elizabeth", "orcid": "0000-0001-9548-8413", "clpid": "Dekas-Anne-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Howard", "given_name": "James B.", "clpid": "Howard-J-B" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Biological nitrogen fixation (the conversion of N2 to NH3) is a critical process in the oceans, counteracting the production of N2 gas by dissimilatory bacterial metabolisms and providing a source of bioavailable nitrogen to many nitrogen-limited ecosystems. One currently poorly studied and potentially underappreciated habitat for diazotrophic organisms is the sediments of the deep-sea. Although nitrogen fixation was once thought to be negligible in non-photosynthetically driven benthic ecosystems, the present study demonstrates the occurrence and expression of a diversity of nifH genes (those necessary for nitrogen fixation), as well as a widespread ability to fix nitrogen at high rates in these locations. The following research explores the distribution, magnitude, geochemical controls, and biological mediators of nitrogen fixation at several deep-sea sediment habitats, including active methane seeps (Mound 12, Costa Rica; Eel River Basin, CA, USA; Hydrate Ridge, OR, USA; and Monterey Canyon, CA, USA), whale-fall sites (Monterey Canyon, CA), and background deep-sea sediment (off-site Mound 12 Costa Rica, off-site Hydrate Ridge, OR, USA; and Monterey Canyon, CA, USA). The first of the five chapters describes the FISH-NanoSIMS method, which we optimized for the analysis of closely associated microbial symbionts in marine sediments. The second describes an investigation of methane seep sediment from the Eel River Basin, where we recovered nifH sequences from extracted DNA, and used FISH-NanoSIMS to identify methanotrophic archaea (ANME-2) as diazotrophs, when associated with functional sulfate-reducing bacterial symbionts. The third and fourth chapters focus on the distribution and diversity of active diazotrophs (respectively) in methane seep sediment from Mound 12, Costa Rica, using a combination of 15N-labeling experiments, FISH-NanoSIMS, and RNA and DNA analysis. The fifth chapter expands the scope of the investigation by targeting diverse samples from methane seep, whale-fall, and background sediment collected along the Eastern Pacific Margin, and comparing the rates of nitrogen fixation observed to geochemical measurements collected in parallel. Together, these analyses represent the most extensive investigation of deep-sea nitrogen fixation to date, and work towards understanding the contribution of benthic nitrogen fixation to global marine nitrogen cycling.
", "doi": "10.7907/H9F5-T161", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7755", "collection": "thesis", "collection_id": "7755", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282013-062935856", "primary_object_url": { "basename": "SaxenaAbigail2013_thesis.pdf", "content": "final", "filesize": 13911949, "license": "other", "mime_type": "application/pdf", "url": "/7755/31/SaxenaAbigail2013_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches", "author": [ { "family_name": "Saxena", "given_name": "Abigail Green", "orcid": "0000-0002-8502-6589", "clpid": "Saxena-Abigail-Green" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes 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 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. 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. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.
", "doi": "10.7907/Z9125QKD", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7879", "collection": "thesis", "collection_id": "7879", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06102013-045943358", "primary_object_url": { "basename": "KBergmann_Thesis.pdf", "content": "final", "filesize": 281436489, "license": "other", "mime_type": "application/pdf", "url": "/7879/1/KBergmann_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Constraints on the Carbon Cycle and Climate During the Early Evolution of Animals", "author": [ { "family_name": "Bergmann", "given_name": "Kristin Diane", "clpid": "Bergmann-Kristin-Diane" } ], "thesis_advisor": [ { "family_name": "Grotzinger", "given_name": "John P.", "clpid": "Grotzinger-J-P" }, { "family_name": "Fischer", "given_name": "Woodward W.", "clpid": "Fischer-W-W" } ], "thesis_committee": [ { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" }, { "family_name": "Grotzinger", "given_name": "John P.", "clpid": "Grotzinger-J-P" }, { "family_name": "Fischer", "given_name": "Woodward W.", "clpid": "Fischer-W-W" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "One of the greatest challenges in science lies in disentangling causality in complex, coupled systems. This is illustrated no better than in the dynamic interplay between the Earth and life. The early evolution and diversification of animals occurred within a backdrop of global change, yet reconstructing the potential role of the environment in this evolutionary transition is challenging. In the 200 million years from the end-Cryogenian to the Ordovician, enigmatic Ediacaran fauna explored body plans, animals diversified and began to biomineralize, forever changing the ocean's chemical cycles, and the biological community in shallow marine ecosystems transitioned from a microbial one to an animal one.
\r\n\r\nIn the following dissertation, a multi-faceted approach combining macro- and micro-scale analyses is presented that draws on the sedimentology, geochemistry and paleontology of the rocks that span this transition to better constrain the potential environmental changes during this interval.
\r\n\r\nIn Chapter 1, the potential of clumped isotope thermometry in deep time is explored by assessing the importance of burial and diagenesis on the thermometer. Eocene- to Precambrian-aged carbonates from the Sultanate of Oman were analyzed from current burial depths of 350-5850 meters. Two end-member styles of diagenesis independent of burial depth were observed.
\r\n\r\nChapters 2, 3 and 4 explore the fallibility of the Ediacaran carbon isotope record and aspects of the sedimentology and geochemistry of the rocks preserving the largest negative carbon isotope excursion on record---the Shuram Excursion. Chapter 2 documents the importance of temperature, fluid composition and mineralogy on the delta 18-O min record and interrogates the bulk trace metal signal. Chapter 3 explores the spatial variability in delta 13-C recorded in the transgressive Johnnie Oolite and finds a north-to-south trend recording the onset of the excursion. Chapter 4 investigates the nature of seafloor precipitation during this excursion and more broadly. We document the potential importance of microbial respiratory reactions on the carbonate chemistry of the sediment-water interface through time.
\r\n\r\nChapter 5 investigates the latest Precambrian sedimentary record in carbonates from the Sultanate of Oman, including how delta 13-C and delta 34-S CAS vary across depositional and depth gradients. A new model for the correlation of the Buah and Ara formations across Oman is presented. Isotopic results indicate delta 13-C varies with relative eustatic change and delta 34-S CAS may vary in absolute magnitude across Oman.
\r\n\r\nChapter 6 investigates the secular rise in delta 18-Omin in the early Paleozoic by using clumped isotope geochemistry on calcitic and phosphatic fossils from the Cambrian and Ordovician. Results do not indicate extreme delta 18-O seawater depletion and instead suggest warmer equatorial temperatures across the early Paleozoic.
\r\n ", "doi": "10.7907/KFJX-7S28", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7535", "collection": "thesis", "collection_id": "7535", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03202013-162430365", "primary_object_url": { "basename": "Osburn_thesis_final.pdf", "content": "final", "filesize": 35915213, "license": "other", "mime_type": "application/pdf", "url": "/7535/1/Osburn_thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Isotopic Proxies for Microbial and Environmental Change: Insights from Hydrogen Isotopes and the Ediacaran Khufai Formation", "author": [ { "family_name": "Osburn", "given_name": "Magdalena Rose", "clpid": "Osburn-Magdalena-Rose" } ], "thesis_advisor": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Grotzinger", "given_name": "John P.", "clpid": "Grotzinger-J-P" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Grotzinger", "given_name": "John P.", "clpid": "Grotzinger-J-P" }, { "family_name": "Fischer", "given_name": "Woodward W.", "clpid": "Fischer-W-W" }, { "family_name": "Lyons", "given_name": "Timothy W.", "clpid": "Lyons-T-W" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Microbes have profoundly influenced the Earth\u2019s environments through time. Records of these interactions come primarily from the development and implementation of proxies that relate known modern processes to chemical signatures in the sedimentary record. This thesis is presented in two parts, focusing first on novel proxy development in the modern and second on interpretation of past environments using well-established methods. Part 1, presented in two chapters, builds on previous observations that different microbial metabolisms produce vastly different lipid hydrogen isotopic compositions. Chapter 1 evaluates the potential environmental expression of metabolism-based fractionation differences by exploiting the natural microbial community gradients in hydrothermal springs. We find a very large range in isotopic composition that can be demonstrably linked to the microbial source(s) of the fatty acids at each sample site. In Chapter 2, anaerobic culturing techniques are used to evaluate the hydrogen isotopic fractionations produced by anaerobic microbial metabolisms. Although the observed fractionation patterns are similar to those reported for aerobic cultures for some organisms, others show large differences. Part 2 changes focus from the modern to the ancient and uses classical stratigraphic methods combined with isotope stratigraphy to interpret microbial and environmental changes during the latest Precambrian Era. Chapter 3 presents a detailed characterization of the facies, parasequence development, and stratigraphic architecture of the Ediacaran Khufai Formation. Chapter 4 presents measurements of carbon, oxygen, and sulfur isotopic ratios in stratigraphic context. Large oscillations in the isotopic composition of sulfate constrain the size of the marine sulfate reservoir and suggest incorporation of an enriched isotopic source. Because this data was measured in stratigraphic context, we can assert with confidence that these isotopic shifts are not related to stratigraphic surfaces or facies type but instead reflect the evolution of the ocean through time. This data integrates into the chemostratigraphic global record and contributes to the emerging picture of changing marine chemistry during the latest Precambrian Era.", "doi": "10.7907/KBAN-B073", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6392", "collection": "thesis", "collection_id": "6392", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05132011-121157483", "primary_object_url": { "basename": "Farnsworth_thesis.pdf", "content": "final", "filesize": 1599174, "license": "other", "mime_type": "application/pdf", "url": "/6392/13/Farnsworth_thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Processes Controlling the Fate and Transport of Trace Metals in the Subsurface During Changing Redox Potential", "author": [ { "family_name": "Farnsworth", "given_name": "Claire Elizabeth", "clpid": "Farnsworth-Claire-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Hering", "given_name": "Janet G.", "clpid": "Hering-J-G" } ], "thesis_committee": [ { "family_name": "Hering", "given_name": "Janet G.", "clpid": "Hering-J-G" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "This study investigated the cycling of Mn and Fe as oxic surface water entered shallow sediments and was reduced, and as reduced groundwater became exposed to oxygen and was oxidized. A Mn-oxide doped gel probe sampler was developed to study in situ rates of reductive dissolution and was validated with laboratory studies with ascorbic acid and Shewanella oneidensis MR-1 as model reductants. The sampler was deployed in the bank sediments of Lake Tegel, Berlin, Germany. Modeling of the diffusion-controlled reaction converted the mass loss from the gels in the sampler to a profile of pseudo-first-order rate constants as a function of depth. The rate constants were highest at depths with high dissolved Fe and low operationally defined fractions of reducible oxides of Fe and Mn in the sediments.
\r\n\r\nA laboratory column experiment showed that 1.3-m water table fluctuations, as observed in bank filtration sites around Berlin, were able to provide sufficient dissolved oxygen delivery for Pseudomonas putida GB-1, an obligate aerobe, to oxidize Mn(II) in situ. Accumulation of Mn on the quartz sand in the column at the end of the experiment was limited to the top 60 cm, as measured with X-ray fluorescence (XRF), and X-ray absorption spectroscopy (XAS) analysis confirmed that the solid formed was a Mn(IV) oxide characteristic of biogenic origin. After a period of \u201cfilter ripening\u201d in the column, rates of in situ oxidation were still lower than rates in engineered aerated sand filters.
\r\n\r\nAdjacent to a production well with a water table that fluctuates up to 7 m annually at Lake Tegel, however, sediments collected from a borehole did not show any significant accumulation of Mn or Fe with depth, as measured by XRF; analysis of the speciation of Mn and Fe in the solid phase by XAS suggested a slight increase in the proportions of total Mn as Mn(II) and of total Fe as Fe(II) with depth. At this location, vertical zonation of groundwater may preclude the co-occurrence of reduced Mn and Fe with dissolved oxygen entrapped by water table fluctuations. Whether groundwater changes from oxidizing to reducing conditions or vice versa, the behavior of Mn and Fe reflects a complex interaction between sediments, solutes, microbial activity, and hydrology.
", "doi": "10.7907/TB3A-Z569", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6448", "collection": "thesis", "collection_id": "6448", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262011-085126961", "primary_object_url": { "basename": "BKH_Thesis_110527.pdf", "content": "final", "filesize": 3601319, "license": "other", "mime_type": "application/pdf", "url": "/6448/1/BKH_Thesis_110527.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Microbial Colonization of Minerals in Marine Sediments \u2013 Method Development and Ecological Significance", "author": [ { "family_name": "Harrison", "given_name": "Benjamin Kimball", "clpid": "Harrison-Benjamin-Kimball" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" }, { "family_name": "Rossman", "given_name": "George Robert", "clpid": "Rossman-G-R" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Edwards", "given_name": "Katrina J.", "clpid": "Edwards-K-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Interactions between microorganisms and minerals significantly impact microbial diversity and geochemical cycles in diverse settings. However, methodological difficulty has inhibited past study of microbe\u2013mineral interactions in fine-grained subsurface environments. Conventional sampling poorly resolves microbial diversity at the fine scale necessary to perceive overall community differences between mineral substrates that are thoroughly mixed. In particular, the importance of microbial attachment to minerals in unconsolidated marine sediments remains poorly constrained despite extensive geobiological research in these settings. This study presents an approach for characterizing microbial colonization patterns using mineral separation techniques. Differences in density and magnetic susceptibility are used to enrich target minerals from bulk environmental samples, selecting for those minerals which may have importance as substrates for metabolic activity.
\r\n \r\nThe application of this methodology to methane seep sediments of the Eel River Basin (ERB) on the California margin demonstrates that variations in microbial diversity between minerals are comparable to community differences across broad spatial scales and a range of porewater geochemistry. ERB colonization patterns determined by separation are shown to be reproducible and reflect in situ differences in the microbial community. Affinity of putative sulfide-oxidizing bacteria (primarily identified as Gammaproteobacteria) for mineral partitions enriched in authigenic sulfides suggests microbial attachment may reflect a metabolic role in sulfur cycling under reducing conditions. Mineral attachment is also shown to select between key archaeal phylotypes involved in the anaerobic oxidation of methane (AOM), providing insight into physiological differences between these uncultured groups. Preliminary results demonstrate that mineral attachment may be a significant factor in the microbial diversity of the marine subsurface, and that such community differences will be ecologically relevant.
\r\n", "doi": "10.7907/3891-QQ56", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5997", "collection": "thesis", "collection_id": "5997", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182010-104705359", "primary_object_url": { "basename": "Full_Dissertation_(N._Ballor).pdf", "content": "final", "filesize": 13949251, "license": "other", "mime_type": "application/pdf", "url": "/5997/1/Full_Dissertation_(N._Ballor).pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Hydrogenases and Hydrogen Sensors in the Symbiotic Microbial Communities of Wood-Feeding Termites", "author": [ { "family_name": "Ballor", "given_name": "Nicholas R.", "clpid": "Ballor-Nicholas-R" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Mazmanian", "given_name": "Sarkis", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite\u2019s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities. ", "doi": "10.7907/621E-9221", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6447", "collection": "thesis", "collection_id": "6447", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252011-233927917", "primary_object_url": { "basename": "Tadmor-thesis_complete.pdf", "content": "final", "filesize": 19775295, "license": "other", "mime_type": "application/pdf", "url": "/6447/55/Tadmor-thesis_complete.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Phage-Host Interaction in Nature", "author": [ { "family_name": "Tadmor", "given_name": "Arbel David", "clpid": "Tadmor-Arbel-David" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Though viruses may be the most abundant biological entities on the planet, very little is known about phage-host interaction in the wild due to the absence of proper experimental tools. In the present work we report of a method to pair environmental phages with their bacterial hosts at the single-cell level without having to culture either host or virus. The method utilizes microfluidic digital PCR in conjunction with a metagenome data mining tool that was developed to find a viral marker gene in an unknown environment. We implemented this technique on the microbial community residing in the hindgut of termites. Consequently, we discovered genus-wide infection patterns displaying remarkable intra-genus selectivity, with viral alleles displaying limited lateral gene transfer and/or host switching despite host proximity. To try and explain phage-host interactions from a theoretical perspective, we formulated a simple biophysical model describing the interaction of bacteria and viruses in aqueous environments. We predict that the radius r of a bacterium is the most critical parameter determining its fixed point concentration, which scales as r-4. Given the hypothesis that there is no selection pressure on bacterial radii, our model predicts that the size spectrum of marine bacteria follows a power law with slope -1, close to the observed average spectrum. Moreover, given the total concentration of bacteria in the ocean, our model enables us to estimate the total number of bacterial \u201cspecies\u201d per volume of water providing a lower and upper bound on the total number of species in the oceans. To elucidate the concept of a \u201cspecies\u201d, we consider a bacterial-viral co-speciation model, which is consistent with the observed narrow host range of phages. Our model hints that the bacterial-viral \u201carms race\u201d may be a critical component in the process of co-speciation. We suggest further experiments to test both models. Finally, we consider a recent high resolution measurement of the force as a function of time generated by stress fibers within a single fibroblast cell and suggest a stochastic model that is capable of accounting for the observed data.", "doi": "10.7907/68Q5-D532", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5786", "collection": "thesis", "collection_id": "5786", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072010-142709280", "primary_object_url": { "basename": "XZhang_Complete_Thesis.pdf", "content": "final", "filesize": 24930564, "license": "other", "mime_type": "application/pdf", "url": "/5786/12/XZhang_Complete_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "I. Formate Dehydrogenase Gene Diversity in Lignocellulose-Feeding Insect Gut Microbial Communities. II. Metabolic Impacts on the Hydrogen Isotope Content of Bacterial Lipids ", "author": [ { "family_name": "Zhang", "given_name": "Xinning", "orcid": "0000-0003-2763-1526", "clpid": "Zhang-Xinning" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "I.) Symbiotic CO\u2082-reducing acetogens are important bacterial members of lignocellulose-feeding termite and roach gut communities. Acetogens are the major consumers of H\u2082 derived from lignocellulose fermentation and can contribute up to 1/3 of the acetate that serves as fuel for the insect host. Many acetogens in wood-feeding termites belong to a diverse group of relatively unstudied, uncultured spirochetes within the genus Treponema. Here, I use the gene sequence for hydrogenase-linked formate dehydrogenase, an enzyme utilized in sugar fermentation and the acetogenic metabolism of the spirochete isolate Treponema primitia, to investigate the diversity, evolution, and activity of uncultured acetogenic spirochetes in lignocellulose-feeding insect guts. The results suggest that (a) the trace element selenium has shaped the gene content of acetogenic spirochetes in gut communities over evolutionary time scales, (b) acetogenic spirochete populations have undergone extinctions and radiations associated with an evolutionary bottleneck, convergent evolutions, and possibly even invasion during termite evolution, and (c) termite gut acetogenesis is largely mediated by only a few spirochete species, which represent a small portion of total acetogenic spirochete diversity.
\r\n\r\nII.) The hydrogen-stable isotope compositions (D/H) of lipids in the environment vary greatly. All variations have been assumed to result from changes in the D/H of water, a source of lipid hydrogen. However, several studies suggest that water D/H may not be the only influential factor. In this study, I report that lipid D/H values can vary by 500\u2030 in bacterial cultures despite constant water D/H. This indicates variations in lipid/water fractionation need to be considered when interpreting environmental data. More significantly, I demonstrate that lipid D/H values are systematically related to the utilization of different central metabolic pathways in bacteria. The results suggest that different cellular mechanisms for NADPH synthesis result in lipids with characteristic D/H. Implications for the use of lipid D/H as an isotopic marker of energy metabolism are discussed.
", "doi": "10.7907/ETXQ-D671", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5501", "collection": "thesis", "collection_id": "5501", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01072010-132355433", "primary_object_url": { "basename": "Thesis_Wanwan_Yang_Submitted_Version.pdf", "content": "final", "filesize": 2857578, "license": "other", "mime_type": "application/pdf", "url": "/5501/1/Thesis_Wanwan_Yang_Submitted_Version.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Fast Viability Assessment of Clostridium Spores: Survival in Extreme Environments", "author": [ { "family_name": "Yang", "given_name": "Wanwan", "clpid": "Yang-Wanwan" } ], "thesis_advisor": [ { "family_name": "Ponce", "given_name": "Adrian", "clpid": "Ponce-A" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Ponce", "given_name": "Adrian", "clpid": "Ponce-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Bacterial endospores are formed in genera such as Bacillus and Clostridium in response to adverse environmental changes. Endospores have remarkable resistance to various extreme conditions and can remain dormant for extended periods of time. Clostridium spores are of particular interest due to their significant importance in several industries, such as food processing, wastewater treatment, pharmaceuticals, and health care. They are also the ideal candidates to study Panspermia and potential extraterrestrial life. However, to date, most endospore research has been conducted on Bacillus, and study of the anaerobic spore former, Clostridium, is not adequate.
\r\n\r\nIn this study, we have developed a general protocol to produce and purify Clostridium spores. Spectroscopy and microscopy based Endospore Viability Assay (Spectro EVA and Micro EVA) were developed and validated to assess the viability of Clostridium spores. Germinability was used as an indicator for spore viability. The basic principle of the two EVAs is to measure the release of a unique biomarker, dipicolinic acid (DPA), via germination as a proxy for endospore viability. In particular, a luminescence time-gated microscopy technique (Micro EVA) has been developed to enumerate germinable Clostridium endospores within an hour. Micro EVA is based on energy transfer from DPA to terbium ions doped in a solid matrix upon UV excitation. The distinctive emission and millisecond lifetime enables time-resolved imaging to achieve single endospore sensitivity. Comparing to traditional CFU cultivation, EVA probes the early stage of germination, resulting in a much faster detection rate (within 60 minutes) than CFU measurement (more than 3 days incubation). Micro EVA has also been successfully applied to quantify Clostridium spores in an extreme cold biosphere, Greenland ice core, and a hyper-arid biosphere, Atacama Desert, two Mars analogs on earth.
\r\n\r\nThe development of EVA provides a faster way to assess viability of Clostridium spores, which has significant importance in various industries. It also enables the determination of the limit and longevity of life, and provides insight on the search of extinct or extant life on Mars and other celestial bodies.
\r\n\r\n", "doi": "10.7907/8BBD-7Y02", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:2211", "collection": "thesis", "collection_id": "2211", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282008-140316", "primary_object_url": { "basename": "00_Complete_Thesis.pdf", "content": "final", "filesize": 7764982, "license": "other", "mime_type": "application/pdf", "url": "/2211/1/00_Complete_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Mechanisms and Evolution of Magnetotactic Bacteria", "author": [ { "family_name": "Nash", "given_name": "Cody Zane", "clpid": "Nash-Cody-Zane" } ], "thesis_advisor": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Magnetotactic bacteria (MB) contain intracellular magnetic crystals of iron oxides and/or iron sulfides. These crystals and the membranes which enclose them are together known as magnetosomes. The crystals formed by MB fall into a narrow size range and have species-specific crystal morphologies. Magnetosomes are physically connected to the rest of the cell by actin-like filaments that are thought to allow the MB to take advantage of their passive orientation in the Earth\u2019s magnetic field to navigate more efficiently across chemical gradients. The large excess of crystals in most strains suggests that magnetosomes may also function as an iron reservoir or as a redox battery.
\r\n\r\nThis thesis describes a number of investigations of the MB. First, a set of genes was identified as being conserved uniquely among the MB by comparative genomics. This method was validated by finding many of the genes already known to be involved in magnetotaxis. Many additional genes were identified and some of these genes were found to cluster together. Three of these clusters were genetically interrupted to determine their role in magnetite biomineralization.
\r\n\r\nSecond, a transposon mutagenesis was undertaken to identify genes necessary for the magnetic phenotype of MB. Out of 5809 mutants screened, nineteen were found to be non- or partially magnetic. Fourteen of these have insertion sites in genes known to be involved in magnetotaxis. Five more were found to have insertions in previously unsuspected genes. The mutant phenotypes of the five mutants include the complete absence of magnetosomes, elongate crystals, reduced numbers of crystals and incomplete mineralization. These mutant strains were used to develop ferromagnetic resonance theory of isolated single-domain particles and biogenic particle identification.
\r\n\r\nThird, MB were discovered in hot springs and in hyper-saline, hyper-alkaline Mono Lake, CA. This extends the environmental range of MB to astro- and paleobiologically relevant environments. Magnetotactic Archaea were tentatively identified from Mono Lake, CA and are the first magnetotactic representatives of that domain.
\r\n", "doi": "10.7907/9M6S-MS85", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1588", "collection": "thesis", "collection_id": "1588", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05022008-220144", "primary_object_url": { "basename": "JMendez_Thesis_.pdf", "content": "final", "filesize": 17876944, "license": "other", "mime_type": "application/pdf", "url": "/1588/18/JMendez_Thesis_.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Iron and Manganese in the Ocean: Investigation of Atmospheric Input by Dust and Coastal Ocean Time Series", "author": [ { "family_name": "Mendez", "given_name": "Jeffrey Michael", "clpid": "Mendez-Jeffrey-Michael" } ], "thesis_advisor": [ { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Trace metals such as iron (Fe) and manganese (Mn) are essential micronutrients in the biogeochemistry of the ocean (Turner and Hunter, 2001), and dry deposition is a substantial source of both Fe and Mn to the surface ocean (Duce and Tindale, 1991; Guieu et al., 1994). Kinetic and thermodynamic values for the release of metals from dust are needed for computer models which incorporate dust as part of their ocean system. Here we investigate the thermodynamic and kinetics parameters involved in the dissolution of metals from dust in seawater. We added dust from the Sahara and the Western United States to seawater in a variety of ways to investigate the dissolution patterns of Fe and Mn. Results show different apparent thermodynamic constants for manganese (Mn) and iron (Fe). The final Mn concentrations are proportional to the added dust concentration and light intensity, and independent of initial dissolution rate. Fe concentrations in fresh seawater reach a maximum concentration of less than 2 nM. However, depletion of organic ligands lead to the precipitation of Fe oxide from solution, and the addition of siderophores enhanced both the total Fe capacity of the seawater and the rate of Fe dissolution from dust. The first order rate constant for the dissolution of dust differed by dust source and was dependent on oxalate concentration and intensity of natural UV light. We conclude that final Mn concentrations are limited by available Mn on the dust surface, while Fe concentrations are limited by the ligand concentrations in the seawater, which ultimately are determined by the biological community. Because the coastal ocean plays a significant role in global biogeochemical cycles, (Smith and Hollibaugh, 1993; Tsunogai and Noriki, 1991), we conducted a coastal ocean time series to investigate the basic modes and cycles which characterize the ocean. We found that Mn is highly dependent on seasonal rain events, with surface water concentrations observed as high as 30 nM after rain events. Fe within the coastal ocean is highly variable and can be used as a tool to track water mass movements and mixing patterns.\r\n", "doi": "10.7907/Q7JK-MV77", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:319", "collection": "thesis", "collection_id": "319", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01242007-141030", "primary_object_url": { "basename": "Jiao_Thesis.pdf", "content": "final", "filesize": 4638581, "license": "other", "mime_type": "application/pdf", "url": "/319/1/Jiao_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Physiological and Mechanistic Studies of Phototrophic Fe(II) Oxidation in Purple Non-sulfur Bacteria", "author": [ { "family_name": "Jiao", "given_name": "Yongqin", "orcid": "0000-0002-6798-5823", "clpid": "Jiao-Yongqin" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Asimow", "given_name": "Paul David", "orcid": "0000-0001-6025-8925", "clpid": "Asimow-P-D" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Phototrophic Fe(II)-oxidizing bacteria use electrons from ferrous iron [Fe(II)] and energy from light to drive reductive CO\u2082 fixation. This metabolism is thought to be ancient in origin, and plays an important role in environmental iron cycling. It has been implicated in the deposition of Banded Iron Formations, a class of ancient sedimentary iron deposits. Consistent with this hypothesis, we discovered that hydrogen gas, a thermodynamically favorable electron donor to Fe(II), in an Archean atmosphere would not have inhibited phototrophic Fe(II) oxidation. To understand this physiology and the connection to BIF formation at the molecular level, the mechanisms of phototrophic Fe(II) oxidation were examined in two purple non-sulfur bacteria, Rhodopseudomonas palustris TIE-1 and Rhodobacter sp. SW2.
\r\n\r\nImportant advances were made in elucidating genes critical to phototrophic Fe(II) oxidation. In R. palustris TIE-1, the first genetically tractable phototrophic Fe(II) oxidizer isolated, transposon mutagenesis identified a putative integral membrane protein and a potential cobalamin (vitamin B\u2081\u2082) biosynthesis protein involved in Fe(II) oxidation.
\r\n\r\nIncreased expression of a putative decaheme c-type cytochrome, encoded by pioA, was observed when cells were grown under Fe(II)-oxidizing conditions. Two genes located immediately downstream of pioA in the same operon, pioB and pioC, encode a putative outer membrane beta-barrel protein and a putative high potential iron-sulfur protein, respectively. Deletion studies demonstrated that all three genes are involved in phototrophic Fe(II) oxidation.
\r\n\r\nIn Rhodobacter sp. SW2, a three-gene operon, foxEYZ, was found to be involved in phototrophic Fe(II) oxidation through heterologous expression in a close relative, Rhodobacter capsulatus SB1003. The first gene, foxE, encodes a novel c-type cytochrome located in the periplasm. Expression of foxE alone confers light-dependent Fe(II) oxidation activity to SB1003, but maximal activity is achieved when foxE is co-expressed with foxY and foxZ. FoxY appears to contain the redox cofactor pyrroloquinoline quinone and FoxZ a cytoplasmic membrane transporter. Recombinant PioC was overexpressed and partially purified from Escherichia coli.
\r\n\r\nThis research presents a detailed study of the physiology and genetics of phototrophic Fe(II) oxidation in two purple non-sulfur bacteria, and provides our first insight into the molecular mechanisms of this metabolism.
", "doi": "10.7907/XC8V-K304", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:4931", "collection": "thesis", "collection_id": "4931", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12102004-144939", "primary_object_url": { "basename": "101204.pdf", "content": "final", "filesize": 2856256, "license": "other", "mime_type": "application/pdf", "url": "/4931/1/101204.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Laboratory Models of Microbial Biosignatures in Carbonate Rocks", "author": [ { "family_name": "Bosak", "given_name": "Tanja", "orcid": "0000-0001-5179-5323", "clpid": "Bosak-Tanja" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Ingersoll", "given_name": "Andrew P.", "orcid": "0000-0002-2035-9198", "clpid": "Ingersoll-A-P" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Enigmatically shaped laminated carbonate rocks called stromatolites dominated shallow marine environments for the first 80% of Earth\u2019s history, and are potentially the oldest macrofossils. While these ancient rocky cones and domes occasionally resemble some modern microbial structures, it is unclear whether their formation required biological processes or they could have been produced abiotically. To develop criteria for assessing the biogenicity of ancient stromatolites, we precipitated calcium carbonate in the laboratory in the presence and absence of modern microorganisms under chemical conditions relevant for the early Earth. Using this novel approach, we disproved the paradigm that microbial sulfate reduction, a metabolism important for the formation of modern stromatolites, was responsible for the precipitation of their ancient counterparts. We also produced the first laboratory evidence that sub-micron and micron-sized pores occured in rapidly precipitating carbonate rocks only when microbes were present. Applying a set of experimentally established criteria to modern environmental samples and ancient stromatolites, we found similar biogenic microporosity in some modern fast-precipitating carbonates and in ancient stromatolites. In our abiotic laboratory precipitates, we observed calcite grains that resembled putatively biogenic features from the rock record called peloids. We explained their shape and growth pattern by purely inorganic parameters, underscoring the need for caution when interpreting seemingly biogenic fabrics in the rock record of Earth and other planets. Finally, we showed that active anoxygenic photosynthesis by Rhodopseudomonas palustris could stimulate the precipitation of calcite even in solutions that were well-buffered by a high concentration of dissolved inorganic carbon. Future studies of the relationship between photosynthetic biofilms, the environmental parameters such as light and currents, and the morphology of carbonate precipitates are key to recognizing potential biosignatures produced by similar organisms in the in situ precipitated stromatolites and other microbialites.", "doi": "10.7907/NJ3F-PJ25", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" } ]