Bacterial genomes are natural mosaics subject to whole genome engineering by horizontal gene transfer in the wild. Existing approaches to whole-genome engineering have concentrated either on the de novo synthesis of single-source genomes from synthetic DNA, or on small-scale editing at the level of nucleotides to kilobases. The intermediate domain, combining large, megabase-scale segments of DNA from different genomes within the same chromosome for the deliberate construction of chimeric bacterial genomes, has been underexplored. This thesis covers this research gap by presenting methods for constructing megabase-scale bacterial genome chimeras.
\r\n\r\nChapter 2 presents methods for addition chimera construction using the Additive Conjugative-CAST Engineering (ACE) technology to expand recipient genomes with up to 2-Mb of a donor sequence in a single step. Transfers were demonstrated over large phylogenetic distance, namely 1-Mb cross-order and 100-kb cross-class transfers from Escherichia coli to Vibrio natriegens and to Agrobacterium tumefaciens, respectively. 522-kb cross-order transfers from Pseudomonas protegens to E. coli provide a window to rebooting foreign genome segments in a different genetic background.
\r\n\r\nChapter 3 extends these methods to substitution chimeras using the technique Replacements by ACE (ReplACE) to rewrite native recipient genome segments with corresponding donor genome segments. We show the recoding of a 1-Mb segment of the E. coli MDS42 genome with E. coli Syn61, and the two-step hybridization of the MDS42 genome with Shigella flexneri to create a Shigoli chimera strain with 50% of its genomic material from both parent strains. We investigate the expression changes from this chimerization event before proceeding to rewrite the genome of MDS42 systematically with that of gut commensal E. coli EcAZ1 to explore genetic determinants of colonization.
\r\n\r\nChapter 4 articulates the natural barriers of horizontal gene transfer removed with the invention of these two techniques and summarizes the benefits and drawbacks of each method and the biological questions they can address as well as the engineering possibilities they provide. Experimental limitations are considered and future experiments are proposed.
", "doi": "10.7907/074j-1k29", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18690", "collection": "thesis", "collection_id": "18690", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292026-171248382", "type": "thesis", "title": "A Physicochemical Approach to Determining the Functions of Microbial Phenazine Metabolites", "author": [ { "family_name": "Thalhammer", "given_name": "Korbinian O.", "orcid": "0000-0001-6882-8611", "clpid": "Thalhammer-Korbinian-O" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Phenazines are redox-active microbial metabolites produced in diverse ecologic niches from agricultural soils to chronic infections. Hundreds of phenazine analogs are known to exist in nature, but their precise roles in context and the reasons for their chemical diversity remain elusive. While much energy has been devoted to investigating phenazine biology, laboratory experiments designed around phenotypes often neglect important aspects of the environmental conditions in which phenazines function, namely pH and EH. In this thesis, I propose that an alternative route to determining the true evolved functions of phenazines and other secondary metabolites is to first interrogate their physicochemical properties in relevant context and to then allow the results to guide biological questions. As a case study, I made detailed abiotic lipophilicity measurements of the phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa. The measurements revealed an elegant redox-mediated mechanism by which lipophilicity is tuned in vivo, sometimes by several orders of magnitude. The increase in biologic retention implied by this finding was born out experimentally by the discovery that the P. aeruginosa membrane harbors millimolar concentrations of reduced pyocyanin in low oxygen conditions, a finding that upends the prevailing concept of that metabolite as an extracellular toxin actively secreted by producer cells. This finding in turn raised questions about the integrity of the pyocyanin-saturated membrane and the function of respiratory enzymes therein. The first question inspired a preliminary lipidomics study that points to significant lipid remodeling in the presence of phenazines. The second may be addressed in the future by respirometry methods developed here for interrogating phenazine interactions with the P. aeruginosa electron transport chain. In the final chapter, I describe attempts to address whether phenazine-mediated reduction of Fe(III)-bearing clays in the environment is a viable mechanism of anaerobic survival for environmental phenazine producers. The experiments described throughout this work represent a fundamentally chemical approach to biological questions, and the results speak to the value of that perspective.", "doi": "10.7907/dqdz-nn56", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18534", "collection": "thesis", "collection_id": "18534", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05012026-185528615", "type": "thesis", "title": "Smart Bandages for Chronic Wound Sampling, Monitoring, and Management", "author": [ { "family_name": "Wang", "given_name": "Canran", "orcid": "0000-0003-3297-9041", "clpid": "Wang-Canran" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Demirer", "given_name": "Gozde S.", "orcid": "0000-0002-3007-1489", "clpid": "Demirer-G\u00f6zde-S" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Zhang", "given_name": "Anqi", "orcid": "0000-0001-6121-8095", "clpid": "Zhang-Anqi" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Chronic wounds are a major global health issue, incurring staggering economic costs and severely impacting patient well-being. Effective exudate management is crucial, yet current methods fail to balance moisture levels. Real-time analysis of biomarkers like reactive oxygen and nitrogen species could guide treatment, but existing systems lack the capacity required for continuous monitoring. Although wearable electronics have the potential to advance wound care, efficient management and analysis of wound exudate in real time remains challenging owing to its low secretion rate and complex composition. To address these issues, we introduce iCares, a wearable device for wound exudate management and continuous in situ analysis of crucial wound biomarkers. iCares contains a flexible nanoengineered sensor array that measures key reactive species such as NO, H\u2082O\u2082, and O\u2082, along with pH and temperature, providing multiparameter data to inform wound status. The device features a pump-free triad microfluidic modules with a superhydrophobic\u2013superhydrophilic Janus membrane, bioinspired wedge channels, and 3D graded micropillars for efficient unidirectional exudate collection, transport, and refreshing. The sensors demonstrate consistent response and analyte selectivity, validated in wound exudate. Rapidly manufacturable through advanced printing and laser-patterning techniques, iCares seamlessly integrates Bluetooth connectivity and enables scalable, wireless, long-term continuous reactive species monitoring without impeding daily activities. The iCares system was validated through in vivo testing in murine models of infection and fasting, where real-time monitoring was performed. In addition, clinical evaluation was conducted in 20 patients with chronic wounds, as well as in patients monitored before and after surgery, demonstrating the system\u2019s applicability across diverse wound conditions. iCares offers early infection detection and wound classification and outcome prediction using machine learning-enhanced data analysis.", "doi": "10.7907/cq9y-x940", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18682", "collection": "thesis", "collection_id": "18682", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292026-023647406", "type": "thesis", "title": "Metabolic Rewiring Promotes Bacterial Survival Under Oxidative and Reductive Stress", "author": [ { "family_name": "Horak", "given_name": "Richard Davis", "orcid": "0000-0003-0630-5481", "clpid": "Horak-Richard-Davis" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Ruby", "given_name": "Edward G.", "orcid": "0000-0002-4112-4830", "clpid": "Ruby-Edward" }, { "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": "Across the tree of life, all cells must follow unifying metabolic rules. Namely, organisms must balance electron flow to couple energy conservation with energy expenditure. Historically, studies in bacterial metabolism focused on exponential growth where cells are awash in nutrients and electron acceptors, exhibiting high bioenergetic levels. Therefore, from the perspective of both human biology and these fast-growing microbes, loss of redox balance is purely detrimental, leading to suppressed energetic states, growth arrest, and even death. Yet bacteria are commonly found under such conditions across diverse environments from industrial bioreactors to chronic infections to agricultural fields. This thesis was motivated by the remaining mystery behind how and why bacteria exist in such low energy survival states. Specifically, I focus on metabolic shifts during non-growth survival in the opportunistic pathogen Pseudomonas aeruginosa due to redox imbalance, as well as the potential benefits to such transitions. In the first section, I focus on oxidative stress, exploring bacterial survival during oxic nutrient starvation. I find that phenazines and toxoflavin \u2013 endogenous redox-active metabolites produced by P. aeruginosa and Burkholderia species respectively \u2013 lower the bioenergetic state of P. aeruginosa. Such bioenergetic self-poisoning would be traditionally deemed detrimental. Yet I find this phenomenon provides cells with increased tolerance to a variety of clinical antibiotics, suggesting cells might have agency over lowering their energetic state and that there is a benefit to doing so. In the following chapters, I turn my attention to reductive stress, examining the metabolic strategies P. aeruginosa uses to support anaerobic survival in the absence of terminal electron-acceptors. I discover that P. aeruginosa uses a phosphoketolase-mediated alternative glucose catabolic pathway under reductive stress, reminiscent of fermentative growth metabolisms in many obligate anaerobes. Moreover, this phosphoketolase plays a key role in mediating ribonucleotide homeostasis during survival-triggered macromolecule turnover. I find that many bacteria unable to grow in the absence of respiration contain phosphoketolases and show that at least two of these species, Dyella japonica and Paraburkholderia graminis, similarly rely on these enzymes for anaerobic survival. Finally, I speculate a generalizable role for phosphoketolases in supporting ribonucleotide turnover across bacterial taxa. These studies expose the large gaps remaining in our understanding of growth arrest metabolisms, even in well-studied model organisms. I hope this thesis motivates further exploration of these enigmatic yet important bacterial lifestyles.", "doi": "10.7907/240t-cx19", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18616", "collection": "thesis", "collection_id": "18616", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242026-035435474", "primary_object_url": { "basename": "marlin thesis.pdf", "content": "final", "filesize": 36379175, "license": "other", "mime_type": "application/pdf", "url": "/18616/1/marlin thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "From Ocean Floor to Stratosphere: Investigating Astrobiologically Relevant Processes and Molecules on Solar System Bodies", "author": [ { "family_name": "Marlin", "given_name": "Theresa", "orcid": "0009-0003-0670-5474", "clpid": "Marlin-Theresa" } ], "thesis_advisor": [ { "family_name": "de Kleer", "given_name": "Katherine R.", "orcid": "0000-0002-9068-3428", "clpid": "de-Kleer-K-R" }, { "family_name": "Cable", "given_name": "Morgan L.", "orcid": "0000-0002-3680-302X", "clpid": "Cable-Morgan-Leigh" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Knutson", "given_name": "Heather A.", "orcid": "0000-0002-5375-4725", "clpid": "Knutson-H-A" }, { "family_name": "de Kleer", "given_name": "Katherine R.", "orcid": "0000-0002-9068-3428", "clpid": "de-Kleer-K-R" }, { "family_name": "Cable", "given_name": "Morgan L.", "orcid": "0000-0002-3680-302X", "clpid": "Cable-Morgan-Leigh" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "In astrobiology, the quest to understand the origin of life on Earth is often paired with an outward focus on whether life can be found elsewhere in the universe. The most accessible corner of the universe in which to search for extraterrestrial life is our own Solar System \u2014- the planets, their satellites, and the minor bodies present throughout. An examination of the essential elements for life on Earth returns three critical factors: liquid water, complex chemistry, and a source of energy. Searching for these three elements in bodies beyond Earth returns a suite of candidates: the ocean worlds of the outer Solar System. These moons, while too cold to harbor liquid water on their surfaces, are thought to have subsurface liquid water reservoirs which could potentially harbor life. Of particular note are two Saturnian satellites: Enceladus, with predicted hydrothermal activity and water-rock interactions, and\r\nTitan, with a dense atmosphere filled with complex organics with potential prebiotic significance. This thesis broadly addresses astrobiological questions on Earth, Enceladus, and Titan via laboratory chemistry and observational astronomy.
\r\n\r\n(I): Chemical gardens as analogs for hydrothermal vents on ocean worlds. This chapter probes laboratory-synthesized analogs of hydrothermal vents as reaction catalysts with alpha keto acids as reactants under various conditions. NMR spectroscopy results support the formation of prebiotically-relevant molecules including glycine (an amino acid) and maloyl formate (a precursor to alpha keto glutarate (AKG)).
\r\n\r\n(II): Propyne: determination of physical properties and unit cell parameters under Titan-relevant conditions. Many questions remain about the organic species that are photochemically produced in Titan\u2019s atmosphere. Propyne (CH3CCH) has been detected in gas phase and is a candidate for Titan\u2019s mixed-species ice clouds. This work presents a characterization of solid propyne using Raman spectroscopy, infrared spectroscopy, and X-ray diffraction.
\r\n\r\n(III):Zonal winds in Titan\u2019s middle atmosphere from a stellar occultation observed with Keck adaptive optics. Winds in Titan\u2019s atmosphere vary seasonally, and stellar occultations present a unique chance to probe them. This work presents spatially-resolved images of the refracted \u201clightspots\" visible around Titan\u2019s limb during an occultation, and discusses the zonal wind profile which best matches the observed data.
", "doi": "10.7907/dcap-p838", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18519", "collection": "thesis", "collection_id": "18519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282026-200138296", "type": "thesis", "title": "Synthetic Control of the Biological Central Dogma", "author": [ { "family_name": "Gerber", "given_name": "Bryan Michael", "orcid": "0000-0002-3979-1095", "clpid": "Gerber-Bryan-Michael" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The Central Dogma of biology dictates the flow of information within all living organisms. By focusing on the construction of synthetic DNA, the transcription of target RNA, and the translation of select proteins, we can improve the synthetic control of engineered organisms. Prioritizing the mis-connection rate present when attaching two DNA molecules, I propose two conceptual improvements to DNA assembly technologies. The high efficiency of the first technique, called Sidewinder, is demonstrated through the construction of a GFP mutation library from DNA oligos, whose diversity is confirmed by high-fidelity sequencing and functional phenotypic analysis. The second DNA assembly technique, High Temperature (HighT) assembly, is demonstrated by high efficiency plasmid ligation, direct integration into bacterial genomes by two independent recombinase HighT assembly-to-integration systems, and the construction of multiple eukaryotic genes including MAPT, VEGFA, BRCA1 and the Sonic Hedgehog embryonic development gene 10-kB genomic DNA segment from the functionally extinct Northern White Rhino. Applications of synthetic DNA are then explored through the import of orthogonal transcription and translation molecular machinery into cells, where they directly regulate protein production. On the transcription level, variations in inducible split-T7 polymerase systems are used to create an orthogonal signaling pathway for low leak and tunable transcriptional control of target genes. Unnatural amino acid incorporation is used to translationally regulate genomically modified essential genes, where exposure to this molecule is demonstrated to enable translation of select essential genes. Through the lens of the Central Dogma, in this thesis I will explore various frameworks to build and fine tune the cells we may aspire to create.", "doi": "10.7907/1t51-1e91", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18607", "collection": "thesis", "collection_id": "18607", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05222026-005425424", "type": "thesis", "title": "To see a World in a Grain of Cells: Statistical Auguries of Nonequilibrium", "author": [ { "family_name": "Salmon", "given_name": "Gabriel L.", "orcid": "0000-0003-2163-8399", "clpid": "Salmon-Gabriel-L" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Precisely what new mathematical and biological behaviors are unlocked as cells---and their collectives---operate out of equilibrium? In this thesis, we take simple steps towards accounting for the characteristic scales of biological energy expenditures and tracing their functional destinies.
\r\n\r\nAfter embarking on an initial Census of lively dissipation (Chapter 1) to set the stage, Part I explores cells on the bioenergetic brink of survival and death. In Chapter 2 we explore the sweep of the smallest measured cellular metabolic rates; reflect on their empirical and philosophical underpinnings; and endeavor an initial order of magnitude accounting of the biophysical processes that could most plausibly dominate the energy budgets of starving cells. Followed by this tour, Chapter 3 hones in on fresh, provocative experiments probing anaerobic maintenance metabolism of Pseudomonas, proposing simple math to predict how microbes die in balance with their energetic resources and interpreting measured reactions to salt.
\r\n\r\nPart II travels to intracellular gene regulation. Among other regulatory settings, in Chapter 4 we investigate the most common regulated architecture of gene regulation in prokaryotes and ask how its behaviors categorically change under different investments in biochemical drive. Chapter 5 narrates how graph theory gives powerful tools for humanly thinking about even large regulatory state graphs.
\r\n\r\nIn Part III, we work closely with inspiring new experimental collaborations and data on patterns of microtubules made by molecular motors. Chapter 6 reports on measuring biochemical energy profiles, and their rates of change, over time and space. Using this phenomenology, in Chapter 7 we develop theory to ask about the fundamental costs required to build or maintain biochemical gradients.
\r\n\r\nLast, in Chapter 8 (Part IV) we ask how a common mathematics might unify a large class of biological dynamics with disperse initial conditions but coherent final conditions, a setting we refer to as exploratory dynamics.
\r\n\r\nCollectively, we hope these case studies give quantitative glimpses of precisely how energy so exquisitely animates biology.
", "doi": "10.7907/ehyd-3072", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17274", "collection": "thesis", "collection_id": "17274", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272025-172840679", "primary_object_url": { "basename": "Narayanan_AK_Thesis.pdf", "content": "final", "filesize": 22695777, "license": "other", "mime_type": "application/pdf", "url": "/17274/3/Narayanan_AK_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Diversity, Activity, and Adaptations of Phage Communities in Anoxic Hydrocarbon-Rich Marine Sediments", "author": [ { "family_name": "Narayanan", "given_name": "Aditi Kalpagam", "orcid": "0000-0003-0627-1859", "clpid": "Narayanan-Aditi-Kalpagam" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The viruses of the global ocean, especially those infecting prokaryotic taxa, are known to play an important role in maintaining the genetic and taxonomic diversity of their host communities and in the cycling of atmospheric carbon and key nutrients like nitrogen and iron. However, the vast majority of these conclusions are drawn from the surface ocean and upper water column, while the sediments, which constitute one of the largest biomes on earth, are understudied in comparison. Of special interest are areas on the ocean floor where methane and other hydrocarbons are produced and released by geological activity and oxidized by a consortium of archaea and bacteria. Using direct genomic sequencing of the viruses from a variety of simplified sediment-free enrichments of hydrocarbon oxidizers, I compare viral communities sampled from different locations and incubated under a range of temperatures to understand the role these parameters might play in shaping distribution and community structure. I then present the most comprehensive picture thus far of viral diversity and distribution from a methane cold seep and discuss whether the viral assemblages are influenced by the steep geochemical gradients that characterize seep sediments. From these datasets, I propose that viral communities in methane-oxidizing sediments are tailored specifically to the physical constraints of the sediment matrix rather than to the dominant members of the cellular community or to other physicochemical parameters such as temperature, sampling location, or depth below the seafloor. I then outline the development of two methods, stable-isotope probing coupled to nanoSIMS and biorthogonal non-canonical amino acid tagging, to work in heterogenous sediment virus samples rather than the liquid pure cultures on which they had previously relied. Implementation of these methods, which allow us to temporally constrain viral production and virus-influenced nutrient flow, resulted in the hypothesis that viral production likely responds to shifts in the major metabolic processes within an ecosystem and may influence cellular community composition.", "doi": "10.7907/v5x8-7447", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17284", "collection": "thesis", "collection_id": "17284", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282025-171526879", "type": "thesis", "title": "Construction of Long, Complex, and Diverse DNA Sequences", "author": [ { "family_name": "Robinson", "given_name": "Noah Evan", "orcid": "0009-0000-2481-9596", "clpid": "Robinson-Noah-Evan" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The DNA molecule encodes the information required for biological systems to carry out a broad range of functions. The understanding of this relationship has sparked inquiries across vast fields of biology and biological engineering as we read, edit, and write the genetic information of organisms. Great advancements have been made toward these pursuits, from revolutions in DNA reading with long read sequencing and the ability to generate terabytes of data from a single run to the breakthroughs in DNA editing with the major advancements in CRISPR/Cas technologies over the last decade. However, writing DNA, as the ability to construct DNA of any length, complexity, or diversity, lags significantly behind our capacity for reading and editing.
\r\n\r\nDNA oligo synthesis can only reach short lengths of a few hundred nucleotides of single stranded DNA. The field of DNA assembly develops the methods for stitching together DNA oligos and DNA fragments into larger constructs. The current field applies a broad range of approaches that each occupy their own niche due to their unique set of advantages and disadvantages. No existing technique is able to assemble a large number of DNA fragments simultaneously with high accuracy and without placing restrictions on the sequences being assembled. This is because all existing DNA assembly technologies rely on the information contained within the complementary sequences of the DNA molecules being constructed to direct the assembly.
\r\n\r\nTo meet the demand for robust DNA assembly, we have developed a new DNA assembly technique named Sidewinder which separates the information that guides assembly from the final assembled sequence using DNA 3-Way junctions. We demonstrate the transformative nature of the Sidewinder technique with highly robust and accurate assembly of complex DNA sequences of both high GC and high repeats, a 40-piece multi-fragment assembly, a parallel construction of multiple distinct genes in the same reaction, and construction of a combinatorial library with a large number of diversified positions across the entire length of the gene for high coverage of a library of 442,368 variants.
\r\n\r\nWhere Sidewinder excels at the assembly of oligos to the kilobase scale, we have made a series of advancements to an existing 2-Way junction assembly technique, USER cloning, for the accurate and efficient assembly of PCR-based DNA inputs. We characterize these improvements with a series of assemblies where we achieve an average accuracy over 95%, gain 3 orders of magnitude increase in yield of transformants, and conduct large multi-fragment assemblies beyond what was previously possible with the technique. We then interface these two state-of-the-art capacities for the rapid and efficient construction of a complex 10 kilobase sequence de novo and entirely cell-free.
", "doi": "10.7907/qtq1-dv04", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17294", "collection": "thesis", "collection_id": "17294", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292025-055458332", "primary_object_url": { "basename": "silverman_shaelyn_2025_thesis.pdf", "content": "final", "filesize": 35182409, "license": "other", "mime_type": "application/pdf", "url": "/17294/3/silverman_shaelyn_2025_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "From Pure Cultures to Particles: Tracing Microbial Metabolism Through Amino Acid \u00b2H/\u00b9H Ratios", "author": [ { "family_name": "Silverman", "given_name": "Shaelyn Nicole", "orcid": "0000-0001-9201-6904", "clpid": "Silverman-Shaelyn-Nicole" } ], "thesis_advisor": [ { "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" } ], "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": "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" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Microbial metabolisms exert profound impact on our planet\u2019s atmosphere and surface geochemistry. Most available tools to study microbial metabolism in the environment provide only snapshots of activity at the time of sampling. However, holistic understanding of microbial function requires the ability to quantitatively reconstruct their activities prior to sampling, for which tools are currently limited. The overarching research presented in this thesis addresses this challenge through development of a new isotopic tool, amino acid hydrogen isotope (\u03b42HAA) analysis, into a useful tracer of microbial metabolism in the environment. We begin by solving a major analytical challenge: correcting for contributions of exchangeable amine-bound hydrogen in derivatized amino acids, which unlocks the ability to accurately measure \u03b42HAA values in organisms via gas chromatography-pyrolysis-isotope ratio mass spectrometry. We demonstrate in aerobic heterotrophic bacteria and phytoplankton that \u03b42HAA values are controlled by metabolism (specifically, carbon flow in cells), and we apply this isotopic tool to natural samples of marine particulate organic matter (POM), demonstrating substantial potential turnover of photoautotrophic proteins into heterotrophic proteins (up to 57 \u00b1 18%) in POM with depth at different ocean sites. We further explore the microscale dynamics of marine bacteria on diatom aggregates to contextualize our understanding of controls on marine POM degradation. In particular, we find that both intra- and interspecies interactions profoundly shape microbial colonization dynamics, which in turn likely affect bulk particle degradation rates. Together, this body of work demonstrates the profound utility of \u03b42HAA analysis as a tracer of microbial metabolism\u2014a timely development given the need to trace and quantify the metabolic responses of microbial communities to ongoing environmental perturbations.", "doi": "10.7907/5v12-1149", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17329", "collection": "thesis", "collection_id": "17329", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312025-215903214", "primary_object_url": { "basename": "barlow_alexandra_2025.pdf", "content": "final", "filesize": 39706810, "license": "other", "mime_type": "application/pdf", "url": "/17329/1/barlow_alexandra_2025.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Biochemical and Structural Basis of Get3d\u2019s Role in Photosynthesis", "author": [ { "family_name": "Barlow", "given_name": "Alexandra Nichole", "orcid": "0000-0001-6515-2396", "clpid": "Barlow-Alexandra-Nichole" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Tail-anchored (TA) membrane proteins, defined by a single C-terminal transmembrane domain, are inserted into the endoplasmic reticulum (ER) membrane via the guided entry of tail-anchored proteins pathway. The central targeting factor of this pathway is Get3, an ATPase that receives TA clients from upstream chaperones and mediates their delivery to the ER. Here, we identify and characterize a unique Get3 homolog, termed Get3d, distinguished by a C-terminal \u03b1-crystallin domain (\u03b1CD). We show that Get3d is conserved across plants and photosynthetic bacteria and demonstrate that it localizes to the chloroplast in plants. We present the X-ray crystal structure of Get3d, revealing unique features including the \u03b1CD and a client-binding chamber in the closed state. Biochemical analyses confirm that Get3d is an active ATPase capable of binding TA proteins in vitro. To investigate its physiological role, we identified the plant-like Get3d homolog in Synechocystis sp. PCC 6803 and generated deletion and complementation strains. Loss of Get3d impairs cell growth and pigment production, and proteomic analyses reveal widespread dysregulation, including up-regulation of transcriptional regulators and down-regulation of redox-associated proteins\u2014suggesting a role in redox homeostasis. Complementation studies show that ATPase activity is necessary for restoring the expression of key photosynthesis-related proteins, while the \u03b1CD is critical for maintaining Get3d protein stability in vivo. Finally, co-immunoprecipitation coupled to mass spectrometry identifies putative Get3d interaction partners enriched in membrane-associated and photosynthetic proteins. Together, these findings establish Get3d as a biochemically distinct and functionally essential member of the Get3 family, with a potential role in redox regulation and photosynthetic homeostasis in diverse photosynthetic organisms.", "doi": "10.7907/wht9-1974", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17345", "collection": "thesis", "collection_id": "17345", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-210705210", "type": "thesis", "title": "Domestication of Environmental Bacteria for Biosensing Applications", "author": [ { "family_name": "Larsson", "given_name": "Elin Maria", "orcid": "0000-0003-1341-5937", "clpid": "Larsson-Elin-Maria" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Cao", "given_name": "Mengyi", "orcid": "0000-0002-3117-3401", "clpid": "Cao-Mengyi" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The field of synthetic biology has made impressive progress in the past 25 years, but is still lacking when it comes to our capability to predictably engineer organisms outside of a small group of lab model organisms. In this thesis, I present the efforts to domesticate two soil bacteria important in agriculture for biosensing. The first, Pseudomonas synxantha, a wheat-colonizing bacterium that helps fight off fungal disease, was engineered into a bioreporter for phosphorus limitation. We also made cell-free extract from this organism, to enable rapid characterization of genetic elements. For the second, Xenorhabdus griffiniae, we asked the question of whether this bacterium can sense the presence of its entomopathogenic nematode host Steinernema hermaphroditum. We learned that X. griffiniae is able to sense its host and we were able to build an early variant of a nematode reporter by first characterizing genetic elements in X. griffiniae.", "doi": "10.7907/m077-7633", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17375", "collection": "thesis", "collection_id": "17375", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-214449948", "type": "thesis", "title": "Exploring Versatility of Energy Metabolism and Dynamics of Anabolism and Growth in Anaerobic Methanotrophic Consortia", "author": [ { "family_name": "Guo", "given_name": "Yongzhao", "orcid": "0009-0005-3983-8382", "clpid": "Guo-Yongzhao" } ], "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": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "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" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Two main questions are asked in this thesis, how environmental microorganisms respond and persist in an energy-limiting condition, and how we can investigate and disentangle the dynamics of these microbes\u2019 activity and growth at a high spatiotemporal resolution. To the end, this thesis focuses on a symbiosis of methane oxidizing archaea (ANME) and sulfate reducing bacteria (SRB), who mediate the anaerobic oxidation of methane (AOM), an important process in the global methane cycle. For the former question, Chapter 2 first found that carbon monoxide (CO) was able to serve as an alternative electron donor for ANME-SRB, and notably CO can even reverse the direction of AOM in ANME archaea to produce methane by the reduction of CO\u2082. Chapter 3 then explored and verified from the other side the potential role of pyrite (nano)particles in supporting AOM via a predicted reaction at the pyrite-water interface to generate sulfate and iron oxides as electron sinks. For the latter question, Chapter 4 took advantage of stable isotopic probing combined with the high sensitivity and spatial resolution of nanometer-scale secondary ion mass spectrometry (nanoSIMS) approach, and proposed a pipeline of multi-isotope imaging to record and in situ read out the single cell activity in the past. Chapter 5 as an in progress work attempts to disentangle the native division process in the yet uncultured ANME archaea by means of serial block face electron microscopy (SBEM) and deep learning imaging analysis. Taken together, this work provides more evidence of the versatile energy metabolism for ANME-SRB symbiosis and at the same time offers solutions to capturing the dynamics of activity and growth in natural microorganisms for the field of environmental microbiology.", "doi": "10.7907/fz82-xt77", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16529", "collection": "thesis", "collection_id": "16529", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06292024-034255793", "primary_object_url": { "basename": "MacKrell_Elliot_2025_thesis.pdf", "content": "final", "filesize": 10855249, "license": "other", "mime_type": "application/pdf", "url": "/16529/13/MacKrell_Elliot_2025_thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Global Analysis of Protein Synthesis and Degradation in Escherichia coli", "author": [ { "family_name": "MacKrell", "given_name": "Elliot James", "orcid": "0009-0006-5619-8548", "clpid": "MacKrell-Elliot-James" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein synthesis and degradation shape the cellular proteome to drive homeostasis and physiological adaptation. Many fundamental aspects of protein regulation have been elucidated through investigation of the Gram-negative bacterium Escherichia coli, which remains a fruitful model organism for uncovering conserved regulatory mechanisms relevant to cell biology, biotechnology, and medicine. Here, we used bioorthogonal noncanonical amino acid tagging (BONCAT) for the time-resolved analysis of protein synthesis and degradation in this organism in several contexts. We profiled protein degradation on a proteome-wide scale in growing and growth-arrested cells, identifying instability in a diverse panel of regulators. Our identifications served as training data in the validation and deployment of a machine learning classifier of in vivo protein stability, which highlighted the role of active degradation in motility and surface adhesion. We then utilized an efficient system of active degradation in this organism to engineer the instability of the mutant methionyl-tRNA synthetase NLL-MetRS for the analysis of protein synthesis in transient physiological states. Destabilized NLL-MetRS variants exhibited half-lives on the order of hours, which improved the fidelity of metabolic labeling in growth-arrested cells. Additionally, we leveraged the sensitivity of BONCAT to investigate protein synthesis in growth-arrested cells expressing a well-studied but controversial member of the widespread toxin-antitoxin family, MazF. Our proteomic profiling suggests this toxin activates several endogenous stress response systems, most notably the cold shock response system. Taken together, these investigations highlight the advantage of time-resolved proteomics in characterizing proteome dynamics.", "doi": "10.7907/n97w-ch36", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16499", "collection": "thesis", "collection_id": "16499", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042024-022255958", "type": "thesis", "title": "Leveraging the \u03a6X174 Protein Antibiotic to Study MraY Structure, Function, and Regulation", "author": [ { "family_name": "Orta", "given_name": "Anna Karen", "orcid": "0000-0002-8526-0383", "clpid": "Orta-Anna-Karen" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The overuse of antibiotics has escalated the prevalence of bacterial resistance to existing treatments, posing a significant threat to global health. This rise in antimicrobial resistance (AMR) has spurred research into innovative therapeutic approaches. Among the most promising strategies is the use of viruses of bacteria for 'phage therapy'. This thesis delves into the interplay between antibacterial resistance and peptidoglycan biosynthesis, highlighting the pivotal role of the membrane protein MraY. We present the first structure of MraY from a pathogenic species, revealing its inhibition by the lysis protein from the bacteriophage \u03a6X174, protein E. Additionally, we analyze lipidic interactions with MraY, proposing a previously unexplored allosteric feedback mechanism for regulating its enzymatic activity. Building on these insights, we expand the application of protein E to non-native hosts, offering new avenues for the development of targeted antibiotic interventions. This work not only advances our understanding of the structural and functional dynamics of MraY but also paves the way for novel antibacterial strategies.", "doi": "10.7907/ggr0-0n72", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16204", "collection": "thesis", "collection_id": "16204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10102023-024622119", "primary_object_url": { "basename": "Wang_Renee_Z_2023_THESIS.pdf", "content": "final", "filesize": 15451019, "license": "other", "mime_type": "application/pdf", "url": "/16204/2/Wang_Renee_Z_2023_THESIS.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "From Photosynthesis to Detoxification: Microbial Metabolisms Shape Earth\u2019s Surface Chemistry", "author": [ { "family_name": "Wang", "given_name": "Ren\u00e9e Zurui", "orcid": "0000-0003-3994-3244", "clpid": "Wang-Ren\u00e9e-Zurui" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" } ], "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": "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" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Earth\u2019s chemistry, through geologic time and in the present, is inextricably linked with biologically mediated reactions. All major elemental cycles on Earth\u2019s surface have arisen from two competing processes \u2013 life shaping its chemical environment through the evolution of key biochemical pathways, and the environment constraining metabolism by dictating which reactions will occur. Understanding this complicated interplay motivates the research presented in this thesis, which studies this phenomenon over two major elemental cycles \u2013 the modern Nitrogen (N) and ancient Carbon (C) cycle.
\r\n\r\nChapters One and Two focus on the evolution of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), the enzyme that catalyzes the key carbon fixation step in modern oxygenic photosynthesis. This reaction also imparts a large kinetic isotope effect (KIE) that causes the fixed carbon to be relatively depleted in natural abundance \u00b9\u00b3C compared to its substrate; this isotopic fingerprint can be seen in both the modern C cycle and in rock records recording the ancient C cycle. Therefore, this KIE has been used both in vitro (outside the cell) by biochemical models to rationalize rubisco\u2019s reaction mechanism, and in vivo (in the cell) as a proxy for environmental CO\u2082 concentrations in the past and present. However, both the in vitro and in vivo measurements are calibrated using modern organisms even though rubisco and oxygenic photosynthesis have undergone profound evolution over geologic time. Therefore, we measured the KIE in vitro and in vivo of a reconstructed ancestral Form IB rubisco dating to >> 1 Ga, and the KIE in vitro of a recently discovered Form I\u2019 rubisco that presents a modern analogue to ancestral Form I rubiscos prior to the evolution of the small subunit. Overall, we find that the KIEs of both rubiscos are smaller than their modern counterparts, which is surprising given that the rock record indicates overall carbon isotope fractionations in vivo are larger in the past. In addition, we find that models strictly based on modern organisms may not apply to the past, questioning the basic assumption that uniformitarianism can be readily applied to biological processes. However, these models can be rescued by accounting for other aspects of cell physiology.
\r\n\r\nChapter Three focuses on disentangling the source of key metabolites, like nitrous oxide (N\u2082O) in the modern N cycle. Like Chapters 1 and 2, an isotopic fingerprint that measures the \u2018preference\u2019 of \u00b9\u2075N for the central or outer nitrogen site in N\u2082O (\u201cSite Preference\u201d or \u201cSP\u201d) has primarily been calibrated using dissimilatory, or energy-generating, nitric oxide (NO) reductases (NORs). However, there exists a much larger and phylogenetically widespread class of NO-detoxifying enzymes; in particular, flavohemoglobin proteins (Fhp/Hmp) produce N\u2082O as a strategy to neutralize damaging NO-radicals in anoxic conditions. This enzyme, which generates N\u2082O in non-growing and anoxic conditions, may be more relevant to natural environments where N\u2082O production has been detected. Surprisingly, we found that Fhp imparts a distinct SP on N\u2082O that differs from both bacterial and eukaryotic NORs, and that this value better aligns with existing in situ measurements of N\u2082O from soils. In addition, we find that in strains with both Fhp and NOR, the Fhp signal dominates when cells are first exposed to high concentrations of NO in oxic conditions while growing before being shifted to an anoxic, non-growing state. Therefore, in addition to telling us \u2018Who\u2019s there,\u2019 the SP fingerprint may also be able to tell us something about cell physiology in vivo. We propose a new framework for interpreting the source of N\u2082O based on SP values.
", "doi": "10.7907/kf85-cq89", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16342", "collection": "thesis", "collection_id": "16342", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03292024-213352165", "primary_object_url": { "basename": "Wang_thesis_final.pdf", "content": "final", "filesize": 12860605, "license": "other", "mime_type": "application/pdf", "url": "/16342/3/Wang_thesis_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Chemo-Selective Proteomics for Discovery of Polymicrobial Interactions", "author": [ { "family_name": "Wang", "given_name": "Grace Zimu", "orcid": "0000-0002-0938-304X", "clpid": "Wang-Grace-Zimu" } ], "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": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The future of microbiome research lies in our ability to engineer polymicrobial interactions toward improved host health outcomes, which requires a fundamental molecular understanding of how microbial species sense and respond to ecological competition. Chronic respiratory infection by polymicrobial communities is the leading cause of mortality and morbidity in people living with cystic fibrosis (CF). My thesis work adapts chemo-selective proteomics to dissect molecular mechanisms that drive interspecies dynamics between two notorious opportunistic pathogens dominating chronic CF infection, Pseudomonas aeruginosa and Staphylococcus aureus.
\r\n\r\nIn Chapter 1, I introduce bioorthogonal noncanonical amino acid tagging (BONCAT)-based comparative proteomics, focusing on time-resolved, cell-specific, and cellular state-selective proteomic applications in the dissection of complex microbial systems. In Chapter 2, I discuss a new usage of time-resolved BONCAT\r\nto monitor immediate competition-sensing responses in interbacterial warfare. While coinfection by the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. We\r\ndiscovered that P. aeruginosa senses S. aureus secreted cytotoxic peptides from a distance and preempts potential competition through activation of type six secretion system (T6SS). P. aeruginosa enhances such competition-sensing-induced antagonism through concomitant attraction toward S. aureus peptides, effectively reducing cellular distances between neighboring species and providing a competitive advantage. In Chapter 3, I discuss a new usage of cell-selective BONCAT to target protein synthesis analysis of the lowabundance organism, S. aureus, in a coculture environment predominated by P. aeruginosa. P. aeruginosa robustly outcompetes S. aureus, and conventional shotgun proteomics, which is biased toward highly abundant proteins on principle, could only identify and quantify less than 5% of total protein synthesis by S. aureus in coculture. We demonstrate that chemical enrichment affords a more than 12-fold increase in total protein abundances synthesized by S. aureus. About 50% of protein \u201chits\u201d with statistically significant changes in expression were not detected in pre-enrichment lysates, highlighting BONCAT as a powerful strategy that facilitates high-resolution proteomic analysis of low-abundance organisms in polymicrobial communities.
", "doi": "10.7907/1vw0-gt98", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16355", "collection": "thesis", "collection_id": "16355", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152024-205944516", "primary_object_url": { "basename": "JAC_thesis_FINAL.pdf", "content": "final", "filesize": 16699403, "license": "other", "mime_type": "application/pdf", "url": "/16355/1/JAC_thesis_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Bioenergetics of a Low-Power, Phenazine-Dependent Maintenance Metabolism in Pseudomonas aeruginosa", "author": [ { "family_name": "Ciemniecki", "given_name": "John Alan", "orcid": "0000-0003-2789-6700", "clpid": "Ciemniecki-John-Alanlan" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Ruby", "given_name": "Edward G.", "orcid": "0000-0002-4112-4830", "clpid": "Ruby-Edward" }, { "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": "A common feature of all life is the metabolic transformation of energy from the environment to biochemical energy in the organism. While this process is well-characterized in molecular detail for fast-growing or otherwise fast-metabolizing organisms such as humans, many microorganisms subsist in the environment around us with little to no exogenous energy for extended periods, and we have only vague ideas how. Questions about the metabolic mechanisms and rates underpinning these astounding survival capabilities speak to the fundamental question of the lower energetic limits of life. Motivated by this big-picture question in biology, this thesis represents one line of physiological inquiry into a specific anaerobic survival metabolism of Pseudomonas aeruginosa, an opportunistic bacterial pathogen. Pseudomonas is perhaps best known for its characteristic production of colorful, redox-active, secondary metabolites called phenazines that allow a metabolic process called extracellular electron transfer. Phenazine extracellular electron transfer has been previously shown to unlock a slow, anaerobic glucose catabolism that facilitates the survival of energy-limited populations of cells. My thesis work has elucidated the predominant membrane-bound protein complexes involved in phenazine reduction and the predominant subcellular location of reduction for each of the main phenazines produced by Pseudomonas. I show that the survival metabolism powered by these phenazines places them in a true maintenance state where there is no detectable growth in the population at the single-cell level. The metabolic rate of this maintenance was measured and found to be 1,000 times slower than when the cells are growing in aerobic culture, 100 times slower than estimates of maintenance rates made in continuous culture, and 10 times slower than the mean basal metabolic rate estimated across all life on the planet. These results open the door to investigations of metabolic attenuation, a physiological state that underpins microbial survival in nature and disease. In pursuit of these discoveries, various new experimental assays that allow further investigation into the bioenergetics and biochemistry of phenazine metabolism were developed. Finally, intellectual frameworks are presented that, in conjunction with the discoveries made and methods developed, collectively bring us steps closer to understanding the bioenergetic basis of microbial resiliency.", "doi": "10.7907/n992-ey51", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16437", "collection": "thesis", "collection_id": "16437", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292024-221307093", "primary_object_url": { "basename": "BussMarjorie_thesis_v04.pdf", "content": "final", "filesize": 30293546, "license": "other", "mime_type": "application/pdf", "url": "/16437/1/BussMarjorie_thesis_v04.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Tools for Noninvasive Imaging and Control of Engineered Bacteria In Vivo", "author": [ { "family_name": "Buss", "given_name": "Marjorie Theresa", "orcid": "0000-0002-4266-9197", "clpid": "Buss-Marjorie-Theresa" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Genetically engineered bacteria are promising new cell-based diagnostic and therapeutic agents due to their ability to sense and respond to unique signals, access and interface with hard-to-reach areas of the body, and deliver therapeutics directly to these areas. However, currently tools to noninvasively monitor and control their activity in vivo are limited. Optical imaging methods, which are based on fluorescent and luminescent reporter genes, and optogenetics, which are based on light-activated proteins, are widely used in cell culture and rodent studies. However, these optical methods suffer from the poor penetration depth of light in tissue which limits their use in larger animals or humans. On the other hand, nuclear imaging methods such as PET and SPECT have good imaging depth but rely on radioactive tracers whose synthesis can be complex and exposes patients to radiation. Here I present tools for imaging and control of bacteria that based on non-ionizing forms of energy that easily penetrate tissue: sound waves and magnetic fields.
\r\n\r\nThe first two parts of my thesis focuses on imaging bacteria in vivo with ultrasound, which is a widely available imaging modality that does not use ionizing radiation and has tissue penetration depth of several centimeters. Bacteria can be imaged with ultrasound by expressing acoustic reporter genes (ARGs) which result in the production of gas vesicles (GVs), air-filled protein nanostructures that aquatic microbes use to regulate their buoyancy. However, the first-generation acoustic reporter genes expressed too poorly under in vivo conditions to enable ultrasound imaging of bacteria in therapeutically relevant contexts. Here, we present a new and improved ARG construct that produces high levels of robust gas vesicle expression in the probiotic bacterium E. coli Nissle (EcN), enabling ultrasound imaging of these cells with high sensitivity. This second-generation ARG construct, bARGSer, uses genes derived from Serratia sp. ATCC 39006 and was optimized for plasmid-based expression in EcN. We demonstrate that with bARGSer, we can visualize the spatial distribution of engineered EcN after they home to and colonize tumors upon systemic administration. We also demonstrate that the engineered EcN can be imaged with ultrasound when colonizing the gastrointestinal tract of mice after sensing dietary sugars as well as biomarkers of inflammation. By enabling monitoring of the precise spatial location of engineered probiotic bacteria inside the body, this technology could greatly improve the development and eventual clinical use of this emerging class of microbial cell-based theranostics.
\r\n\r\nThe last part of my thesis focuses on control of bacteria in vivo with magnetic fields. Many bacteria have limited ability to selectively colonize specific targeted regions of the GI tract due to a lack of external control over their location and persistence. Magnetic fields are well suited to provide such control due to their ability to freely penetrate biological tissues, but they are difficult to apply with enough strength to directly manipulate magnetically labeled cells within deep tissue or viscous environments such as in the GI tract. Here, we show that ingestible micron-sized magnetic particles, combined with an externally applied magnetic field, act as in vivo magnetic field gradient amplifiers, enabling the trapping and retention of orally administered probiotic E. coli within the mouse GI tract. This technology improves the ability of these probiotic agents to accumulate at specific locations and stably colonize without antibiotic treatment. By enhancing the ability of GI-targeted cellular agents to be at the right place at the right time, cellular localization assisted by magnetic particles (CLAMP) adds external physical control to an important emerging class of biotherapeutics.
", "doi": "10.7907/mvgg-ch02", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16456", "collection": "thesis", "collection_id": "16456", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312024-213823439", "type": "thesis", "title": "Design and Construction of Bacterial Genomes at the Megabase-Scale", "author": [ { "family_name": "Sanfiorenzo", "given_name": "Charles John", "orcid": "0009-0004-4652-3744", "clpid": "Sanfiorenzo-Charles-John" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Building genomic chimeras would enable melding of the diverse functions and properties of life. However, prior arts in genome synthesis are limited to reconstituting functions within singular genomes rather than combining diverse genomic functions across multiple distinct genomes. Existing methods are also prohibitively expensive, laborious, time-consuming, and not scalable for creating large genomes. Addressing these limitations, the author invented Additive Conjugative-CAST Engineering (ACE) combining conjugation with CRISPR-associated transposition (CAST) to deliver and integrate up to half a genome per step from a donor into a precisely defined position in the recipient\u2019s genome. This work demonstrates ACE\u2019s engineering capacities integrating a 2-megabase donor genomic segment in a single step and at least three megabase in two steps. Importantly, ACE\u2019s generality is confirmed through the creation of genomic chimeras across species, genus, order, and class barriers. With ACE, this work further showcases that such chimeric organisms, denoted genome expanded organisms (GEOs), can be forged from at least three starting bacterial strains, and are stably maintained to express all acquired genomic parts. Principles confounding ACE are expanded onto genome editing technologies, such as Prime Editing, and further explored for the megabase-scale transfer of DNA into eukaryotes. ACE and derivative technologies thereof offer a new paradigm of creating artificial lifeforms to combine and potentially create novel functions beyond the constraints of nature, while probing and elucidating genome plasticity, architecture, and expression patterns of GEOs.
", "doi": "10.7907/yapk-c742", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:14989", "collection": "thesis", "collection_id": "14989", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07272022-064642720", "type": "thesis", "title": "Applications of Genetically Engineered Bacillus subtilis in Biocatalysis and Functional Materials", "author": [ { "family_name": "Hui", "given_name": "Yue", "orcid": "0000-0002-0354-0382", "clpid": "Hui-Yue" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David", "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": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Bacillus subtilis is a gram-positive model bacterium that forms endospores as a response to nutrient limitation and other environmental stresses. The B. subtilis spore contains a dehydrated core, where the bacterial genome is safely stored, and multilayer proteinaceous coats, protecting the spore from various physical and chemical insults. Because of the outstanding resilience of the B. subtilis spore, it has attracted increasing interest for application in biotechnology. In this thesis, we demonstrate the utilization of genetically engineered B. subtilis cells and spores for heterologous protein display and functional material synthesis and characterization.
\r\n\r\nIn Chapter 1, we review the fundamentals of sporulation and germination in B. subtilis. We highlight notable biotechnological applications of native and engineered B. subtilis spores in recent years. We also discuss limitations associated with prior studies that inspire us to pursue the work in this thesis.
\r\n\r\nIn Chapter 2, we describe the T7 RNA polymerase (RNAP) enabled high density protein display on B. subtilis spores (TIED) method. The TIED constructs employ a coat protein promoter \u2013 PcotG, PcotV, or PcotZ \u2013 to drive the expression of the T7 RNAP. Target proteins are fused to the C-terminus of a spore crust protein \u2013 CotY or CotZ \u2013 and subjected to amplification by the T7 promoter. We prepare the endogenous constructs in which coat protein promoters directly regulate fusion protein expression for comparison with TIED. In addition, we develop a supplementary procedure to harvest spores before mother cell lysis, further improving the loading density of the target proteins. We verify the performance of the TIED architectures with a fluorescent reporter protein, mWasabi. Together with the early harvest protocol, the TIED method substantially enhances the total expression level and loading density of the crust-mWasabi fusion proteins relative to the endogenous expression system, as evidenced by bulk fluorescence measurements and microscopy.
\r\n\r\nIn Chapter 3, we implement the TIED architectures described in Chapter 2 for enzyme display on B. subtilis spores. We demonstrate the spore-based biocatalyst platform with three enzymes \u2013 lipase A and lipase B secreted by vegetative B. subtilis, and an engineered peroxidase, APEX2. We manifest that TIED enables massive accumulation of all three enzymes on the spore surface, with loading densities in the range of 106-107 enzymes per spore. Further, TIED-enzymes show comparable catalytic performance to the respective free-form enzymes, enhanced catalytic activity in methanol, and increased temperature stability. We conduct Michaelis-Menten studies to elucidate the kinetic characteristics of TIED-enzymes and their free form counterparts. Finally, we demonstrate that TIED-enzymes are not only recyclable, but also fully renewable after loss of activity through induction of germination and sporulation, demonstrating the potential for perpetual regeneration of the immobilized biocatalysts.
\r\n\r\nIn Chapter 4, we describe a new class of living composite materials (LCMs), in which genetically engineered B. subtilis cells and spores are effectively crosslinked into the surrounding polymeric scaffold. The resulting LCMs can be dried to yield portable materials. When re-immersed in aqueous media, entrapped cells and spores in previously- dried LCMs exhibit metabolic activity, including synthesis and secretion of recombinant proteins. Notably, we show that the scaffold based on photopolymerization of N-(hydroxymethyl) acrylamide (NHMAA) achieves effective cellular confinement, showing no evidence of cellular leakage over a period of 72 hours. We envision that the design principles elucidated in this work can provide a promising route to functional living materials engineered for biomedical and other applications.
", "doi": "10.7907/cdja-ck19", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15225", "collection": "thesis", "collection_id": "15225", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-181810775", "primary_object_url": { "basename": "Marken_Thesis_Final.pdf", "content": "final", "filesize": 4502877, "license": "other", "mime_type": "application/pdf", "url": "/15225/1/Marken_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Experimental and Theoretical Frameworks for Enabling Environmental Synthetic Biology", "author": [ { "family_name": "Marken", "given_name": "John Paul", "orcid": "0000-0001-9696-088X", "clpid": "Marken-John-Paul" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Although the field of synthetic biology has made great advances toward becoming a mature engineering discipline over its first quarter-century, the vast majority of these efforts have focused on improving the design and performance of genetic circuits intended to operate in well-controlled, laboratory settings. The goal of safely deploying engineered microbes to reliably perform their programmed functions in natural, uncontrolled environments begets its own set of foundational challenges that will require new frameworks that shift our existing mindsets about the way we engineer biological systems.
\r\n\r\nThese frameworks, because they focus on enabling system properties that were not priorities for conventional synthetic biology research, can constitute a new field of research which I refer to as environmental synthetic biology. The central priorities of environmental synthetic biology include (1) developing and characterizing effective ways to introduce engineered biological systems into natural environments, (2) ensuring that the performance of these systems can remain robust and predictable in the face of environmental variability, (3) developing and characterizing ways to control and monitor the behavior of an engineered system after deployment in an inaccessible environment, and (4) developing fundamental architectures to enable autonomous system operation and adaptation within environmental contexts.
\r\n\r\nIn this thesis, I present the initial steps towards the development of three frame- works that address these priorities of environmental synthetic biology. The first framework, described in Chapter 2, demonstrates the potential of using DNA as the substrate for addressable and adaptable intercellular communication in engineered populations. This enables the ability to one day create multicellular systems that can autonomously reconfigure their own architecture in the face of changing environmental conditions. The second framework, described in Chapters 3 and 4, presents a new mathematical representation of biomolecular reaction systems that enables geometric bounds on the space of possible behaviors under all possible configurations for a particular system architecture. The third, ongoing framework emphasizes the importance of explicitly incorporating the physiological state of the host cell into the assessment of a genetic circuit\u2019s behavior by exploring the impact of cellular growth arrest on transcriptional response curves. The preliminary results of this work are presented in Chapter 5.
", "doi": "10.7907/h50w-p058", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15071", "collection": "thesis", "collection_id": "15071", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12022022-073109279", "primary_object_url": { "basename": "2022_12_05_JTM_Thesis_v037.pdf", "content": "final", "filesize": 8086854, "license": "other", "mime_type": "application/pdf", "url": "/15071/1/2022_12_05_JTM_Thesis_v037.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineering and Rapid Prototyping for Biology in Extreme Conditions", "author": [ { "family_name": "Meyerowitz", "given_name": "Joseph Toshiro", "orcid": "0000-0002-3426-0885", "clpid": "Meyerowitz-Joseph-Toshiro" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-3426-0885", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In this thesis we show three projects in which biological systems are engineered for increased robustness to environmental stressors such as toxic small molecules. Several lignocellulose-derived growth inhibitors commonly found in industrial feedstocks for fermentation were used to grow a panel of yeast knockouts for several efflux pumps and detoxifying enzymes. Some specific knockout strains showed slowed growth on specific growth inhibitors, while other knockout strains showed the same growth rate as the wild-type. One efflux pump was identified for vanillin, YHK8, and was overexpressed in yeast. The overexpression strain did not show an improved tolerance to vanillin, and grew more slowly than the wild-type. To regulate the expression of the vanillin pump, a sensor for vanillin was created. The starting enzyme was the wild-type qacR transcription factor, and several variants were generated using computational protein design. The designs were synthesized and tested using in vitro transcription-translation (TX-TL) as part of a rapid prototyping process. This rapid prototyping considerably sped up the design-build-test process. Finally, four bacteria, Pseudomonas synxantha 2-79, Pseudomonas chlororaphis PCL1391, Pseudomonas aureofaciens 30-84, and E. coli are tested against the same lignocellulose growth inhibitors. The Pseudomonas spp. show an improved tolerance to the growth inhibitors. We then develop some ability to engineer and prototype in all three species. A panel of promoter parts were integrated into the P. synxantha genome to produce a collection of test strains. These same promoter parts were also used as DNA templates for TX-TL reactions. The in vivo measurements of promoter strength and in vitro measurements show similar relative strengths between the parts, showing the Pseudomonas-based TX-TL systems can be used for design-build-test activities in these non-model organisms. This alternate approach to developing tolerance, starting with a species that already has a working tolerance to the stressor in question, changes the problem to one of building engineering capabilities in the new chassis.", "doi": "10.7907/9gbb-n831", "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:15163", "collection": "thesis", "collection_id": "15163", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112023-130637882", "primary_object_url": { "basename": "Liaw_Eric_2023_Thesis.pdf", "content": "final", "filesize": 10234218, "license": "other", "mime_type": "application/pdf", "url": "/15163/2/Liaw_Eric_2023_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "A Novel, Rapid Phenotypic Assay for a Beta-Lactam Antibiotic Susceptibility and an Analysis of its Theoretical Limits", "author": [ { "family_name": "Liaw", "given_name": "Eric Jer-Jiun", "orcid": "0000-0003-2244-8335", "clpid": "Liaw-Eric-Jer-Jiun" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Current management of bacterial infections is limited by the slow turnaround time of culture-based antibiotic susceptibility testing (AST). Culture-free phenotypic AST methods, though faster, are limited not only by analytical sensitivity but also by the low number, density, and purity of live pathogens present in clinical specimens before culturing. Separating and concentrating pathogens from clinical specimen matrices and improving the analytic sensitivity of phenotypic measurement technologies remain active areas of research. However, to date, the literature lacks consensus over what is a reasonable goal for the minimum number of pathogens in a clinical specimen needed to accurately perform phenotypic AST.
\r\n\r\nI describe \"bulk filtration AST\" and \"digital filtration AST,\" two new filtration-based AST methods that improve an AST method previously published by others and myself. These methods use nucleic acid quantification to assess the activity of antibiotic classes (and only those classes) targeting peptidoglycan turnover, specifically the beta-lactams, which are the most frequently prescribed class of antibiotics. I use filtration AST to quantify the in vitro pharmacodynamics of beta-lactam antibiotics over time scales shorter than two hours, and I simultaneously validate the methods' accuracies on clinical isolates of Enterobacteriaceae. To analyze filtration AST results, either for fitting parameter values or for predicting susceptibility, I derive probabilistic models for the outcomes of each of the two filtration AST methods, then perform Bayesian parameter inference from my data.
\r\n\r\nI then propose a general mathematical framework for defining the concepts of the phenotypic assay and the ideal phenotypic assay. Within this framework, I calculate the ideal filtration AST performance as a function of the number of cells assayed, my fitted pharmacodynamic parameters, and other variables. Interestingly, the observed performance of my implementation of digital filtration AST is consistent with the implementation's approaching the ideal performance. I hope my demonstration of these new methods and my theoretical framework will help guide future research into rapid phenotypic AST.
", "doi": "10.7907/qhvg-7q92", "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: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:15173", "collection": "thesis", "collection_id": "15173", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152023-034200704", "primary_object_url": { "basename": "Sarai_Thesis.pdf", "content": "final", "filesize": 10850833, "license": "other", "mime_type": "application/pdf", "url": "/15173/2/Sarai_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Engineering Cytochrome P450BM3 for Oxidation and Silicon\u2013Carbon Bond Cleavage of Volatile Methylsiloxanes", "author": [ { "family_name": "Sarai", "given_name": "Nicholas Singh", "orcid": "0000-0002-4655-0038", "clpid": "Sarai-Nicholas-Singh" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "orcid": "0000-0002-6610-4414", "clpid": "Peters-J-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Directed evolution of enzymes can reveal activities that do not occur in the natural world. While most examples of directed evolution of new-to-nature chemistry have been applied in a synthetic direction, enzymatic biodegradation typically relies on wild-type enzymes. This thesis posits that directed evolution can generate enzymes capable of degrading non-biodegradable anthropogenic compounds, focusing on efforts to break silicon\u2013carbon bonds, which are not known to be cleaved by enzymes in Nature. Chapter I establishes background on how enzymes evolve to catalyze degradation of compounds over long timescales in Nature, highlighting the enzymatic depolymerization of lignocellulosic biomass. This sets the stage for a case study of rapid enzyme evolution in response to anthropogenic molecules such as plastics and agrochemicals. With this background, directed evolution of new-to-nature synthetic activities is presented to demonstrate how new enzymatic activities can be evolved in the laboratory. In Chapter II, the state of the art for biocatalytic reactions involving organosilicon compounds is reviewed, starting with a description of how biology uses silicon and concluding with a perspective on future opportunities in this nascent field. Finally, Chapter III describes the engineering of a novel siloxane oxidase based on a cytochrome P450, which conducts two reaction steps in tandem to cleave silicon\u2013carbon bonds. First, it hydroxylates the C\u2013H bonds of siloxanes\u2014the anthropogenic building blocks of silicone polymers\u2014to yield a carbinol species, an activity reminiscent of the parent enzyme\u2019s native hydroxylation of fatty acids. Via a function entirely different than its native activity, the enzyme converts this carbinol to a silanol species. In performing both of these steps, this is the first known enzyme that can cleave Si\u2013C bonds, an activity which is the first step toward enzymatic degradation of these persistent, man-made compounds. In sum, this thesis demonstrates that directed evolution can reveal enzymatic degradation chemistries that are not known in Nature by establishing new-to-nature Si\u2013C cleavage of siloxanes.", "doi": "10.7907/gn1j-fz77", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14338", "collection": "thesis", "collection_id": "14338", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08242021-212609828", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 36577433, "license": "other", "mime_type": "application/pdf", "url": "/14338/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Physical Biology of Cellular Information Processing", "author": [ { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-Manuel" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The state of matter that we define as life is different from anything else we have encountered so far in the universe. Living systems not only perpetuate their existence out of equilibrium against the will of the second law of thermodynamics, but they do so while keeping up with an ever-changing environment. A key part of this capacity to adapt to environmental changes is the ability of organisms to gather information from their surroundings to put together an adequate response to the challenges presented to them. This thesis presents an effort to understand, from first principles, this fundamental feature of information gathering that all life on earth shares. We dig into the physics behind one of the most pervasive mechanisms through which living systems sense and respond to the environment\u2013the ability to turn on and off genes. In doing so, we hope to uncover general principles of how organisms deal with the problem of collecting information about the world that surrounds them.
\r\n\r\nIn Chapter 1, we develop the theoretical and conceptual tools to navigate the rest of the thesis. I introduce the idea of gene regulation, as well as different theoretical models of this pervasive biological phenomenon. We also delve into the realm of information theory and learn how the plastic concept of information can be mathematically defined and quantified.
\r\n\r\nThe second stop in our exploration (Chapter 2) asks the following question: can we understand, from first principles, how it is that proteins allow cells to regulate their genes on-demand upon sensing environmental cues? For this, we explore the physics behind transcriptional control due to allosteric transcription factors. Using simple quasi-equilibrium models of the two processes involved in this type of regulation\u2014the regulation of the gene by the binding and unbinding of the transcription factor, and the regulation of the activity of the transcription factor itself by the binding and unbinding of an effector molecule\u2014we are able to predict the input-output function of a simple genetic circuit, and compare such predictions with experimental determinations of the mean response of a population of bacterial cells.
\r\n\r\nWe then expand on these insights to ask questions about the inescapable cell-to-cell variability that isogenic cells encounter. For this, we have to leave behind the pure thermodynamic framework and work in the language of chemical kinetics. This allows us to make predictions beyond the mean input-output gene expression response of cells by reconstructing full gene expression distributions. With these probabilistic input-output functions, in Chapter 3 we formalize the question of the amount of information that cells can gather from the environment. For this, we turn to information-theoretic concepts of maximal mutual information (otherwise known as channel capacity) between the state of the environment and the gene expression response from bacterial cells. Finally, we compare our predictions of the maximum amount of information\u2014measured in bits\u2014that cells can gather with single-cell inferences of this quantity.
", "doi": "10.7907/kpc2-b345", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14354", "collection": "thesis", "collection_id": "14354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09072021-015258902", "type": "thesis", "title": "Investigation of the Roles of Hopanoids in the Lifecycle of Bradyrhizobium diazoefficiens in the Context of Climate Change", "author": [ { "family_name": "Tookmanian", "given_name": "Elise M.", "clpid": "Tookmanian-Elise-M" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Ondrus", "given_name": "Alison E.", "orcid": "0000-0002-6023-3290", "clpid": "Ondrus-A-E" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhizobia are a group of bacteria that participate in plant-growth promoting symbioses with legumes, where the bacteria supply the plant with a source of useable nitrogen. In agriculture, crop rotation capitalizes on this symbiosis by planting legumes to restore the nitrogen content of depleted soils. The effects of climate change, such as increased temperature and changing precipitation patterns, threaten the future viability of agriculture. Rhizobia exemplify the role bacteria can play to improve agriculture\u2019s resilience to climate change and prevent land degradation and food insecurity. However, in order for bacteria to realize this potential, they need to survive the challenges of climate change. In my thesis, I detail the environments that rhizobia experience throughout their lifecycle and how the soil environment will likely change as the climate changes. Then, I connect these environmental parameters, especially hypo and hyperosmolarity, to the outer membrane. The outer membrane is the first line of defense for bacteria against external assaults. Rhizobia make many changes to their outer membrane compared to commonly studied enteric bacteria. For example, the ability to synthesize hopanoids, steroid-like lipids, is overrepresented in rhizobia.
\r\n\r\nHopanoids are known to help protect bacteria against a wide range of stresses \u2013 but, surprisingly, we found that the extended hopanoid class is not required for a moderately successful symbiosis between rhizobia strain Bradyrhizobium diazoefficiens and the tropical legume Aeschynomene afraspera. The main defect was in the initiation of the symbiosis, perhaps due to motility defects in the extended hopanoid\u2014deficient mutant. As we investigated this paradox, we discovered that hopanoids are conditionally essential in B. diazoefficiens depending on the medium in which the organism is grown. Specifically, we investigated the role of hypoosmolarity and divalent cation concentration, discovering that extended hopanoids confer robustness to the physicochemical environment. This property indicates that extended hopanoids may be important in the soil environment, which is prone to osmotic variability, especially as the climate changes. This work increases our understanding of the role of the outer membrane and hopanoids in bacterial resilience which may help with engineering or selection of better crop additives in the future.
", "doi": "10.7907/h0xe-jb65", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14388", "collection": "thesis", "collection_id": "14388", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10072021-173853251", "type": "thesis", "title": "Nucleic Acid Measurements for Antibiotic Susceptibility Testing and Early Detection of SARS-CoV-2", "author": [ { "family_name": "Savela", "given_name": "Emily Sue", "orcid": "0000-0001-9614-4276", "clpid": "Savela-Emily-Sue" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "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": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "abstract": "Nucleic-acid-amplification tests (NAATs) are widely used in microbial detection both in environmental characterization and human diagnostics. NAATs offer highly sensitive and specific detection of target molecules among the noise of complex samples. This thesis covers two important applications of nucleic-acid quantification techniques in human clinical samples. First, I co-developed a new phenotypic antibiotic susceptibility test that uses species-specific DNA detection to detect bacterial cell-wall damage following incubation with beta-lactam antibiotics. Second, I helped compile a longitudinal dataset of SARS-CoV-2 viral loads during a community-based COVID-19 study run by the Ismagilov Lab through October 2020 \u2013 April 2021 in the greater Los Angeles County area, USA. Sensitive and specific nucleic-acid tests allowed for robust detection of pathogenic microbes in both these applications. Designing and implementing NAATs for these applications required consideration of biological constraints of the microorganisms, molecular stability over the time of quantification, and the practical constraints of acquiring and transporting samples. Continued innovation of NAAT technologies will be critical to contain present and future pandemics and empower medical professionals with data to inform treatment options.
", "doi": "10.7907/vp9a-n206", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14497", "collection": "thesis", "collection_id": "14497", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02152022-081613451", "primary_object_url": { "basename": "Meirelles_LucasAndrade_2022_thesis.pdf", "content": "final", "filesize": 24486462, "license": "other", "mime_type": "application/pdf", "url": "/14497/1/Meirelles_LucasAndrade_2022_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Nuanced Effects of Redox-Active Metabolites on Bacterial Physiology and Antibiotic Susceptibility", "author": [ { "family_name": "Andrade Meirelles", "given_name": "Lucas", "orcid": "0000-0003-3194-7136", "clpid": "Andrade-Meirelles-Lucas" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The production of secondary metabolites is widespread throughout the tree of life. Bacteria, including many relevant opportunistic pathogens, can make redox-active secondary metabolites, both in the environment and while causing infections. Yet, their physiological consequences for the microbial communities exposed to them are much less understood. This thesis investigates the multifaceted and nuanced effects that such metabolites can have on their producers and other bacteria found in the producer's vicinity, focusing on the role these molecules play as modulators of antibiotic susceptibility. I start by presenting a literature review addressing the link between secondary metabolite production and resilience to clinical antibiotics in diverse opportunistic and enteric bacterial pathogens.
\r\n\r\nNext, using Pseudomonas aeruginosa (a widespread opportunistic pathogen) and its endogenously produced metabolite called pyocyanin, I explore the nuanced effects of the metabolite's production throughout the producer's lifecycle. Pyocyanin is part of a class of redox-active molecules made by P. aeruginosa called phenazines. I show that the production of pyocyanin, due to its self-poisoning effects, is a \"double-edged sword,\" where the ultimate consequences for the producer are directly dependent on the physiological and environmental conditions. Carbon source limitation plays a major role in the self-poisoning effect of pyocyanin, a process responsible for killing a subpopulation of cells that, through extracellular DNA release, seems critical for proper biofilm development.
\r\n\r\nDespite pyocyanin's toxicity, P. aeruginosa is remarkably tolerant to its harmful effects. For this reason, I then explore how P. aeruginosa handles the stress caused by the metabolite. I present results using a functional genomics approach (transposon-sequencing) to screen for genes involved in P. aeruginosa tolerance to pyocyanin. Defenses involved in pyocyanin tolerance are similar to ones involved in tolerance to clinical antibiotics. These shared mechanisms lead to testing the hypothesis that defenses induced by the production of or exposure to \"natural antibiotics\" (such as pyocyanin) may affect the efficacy of treatments with clinical antibiotics. Supporting this hypothesis, exposure to pyocyanin significantly induces tolerance and resistance to certain clinical drugs, both in P. aeruginosa and other opportunistic pathogens within the Burkholderia cepacia complex (Bcc). Pyocyanin and the drugs affected, such as fluoroquinolones, share molecular structure similarities, which is likely responsible for the shared protection.
\r\n\r\nFinally, based on these results, I explore the broader role of redox-active metabolites as modulators of antibiotic resilience in opportunistic pathogens. I show that pyocyanin, another phenazine called phenazine-1-carboxylic acid, and a non-phenazine redox-active molecule called toxoflavin can all modulate antibiotic susceptibility in Bcc species. Depending on the antibiotic's class, the metabolites' presence can either antagonize or potentiate the drug's efficacy. All the studied metabolites are produced by clinical isolates that infect cystic fibrosis and other immunocompromised patients. I demonstrate that the modulator effect of redox-active molecules in the pathogens is dependent on the transcription factor SoxR, which senses the presence of the metabolites and induces specific redox-regulated efflux systems that are effective in transporting both the metabolites and the structurally related drugs. To end, I provide a proof-of-principle that including such metabolites during clinical drug susceptibility tests may lead to a more accurate assessment of pathogens' resistance profile.
\r\n\r\nTaken together, the findings presented in this thesis demonstrate that redox-active secondary metabolites have profound effects on the physiology and antibiotic sensitivity levels of opportunistic pathogens. Their modulator effect on antibiotic susceptibility is likely a widespread phenomenon in polymicrobial communities that has been overlooked and may have direct consequences for the evolution of antibiotic resistance. Understanding the physiological roles of these metabolites at the molecular level is essential for accurate predictions of the drugs and pathogens affected, which may lead to more effective treatment strategies.
", "doi": "10.7907/67p2-q992", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14121", "collection": "thesis", "collection_id": "14121", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152021-173245433", "type": "thesis", "title": "Mechanisms and Consequences of Bacterial Resistance to Natural Antibiotics", "author": [ { "family_name": "Perry", "given_name": "Elena Kim", "orcid": "0000-0002-7151-1479", "clpid": "Perry-Elena-Kim" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Many bacteria secrete natural antibiotics\u2014toxic small molecules that can kill or inhibit the growth of other microorganisms. Several of these compounds have been commercialized as antimicrobial drugs, and the mechanisms and public health consequences of bacterial resistance to clinically-used antibiotics are well understood. By contrast, the role of bacterially-produced antibiotics in natural environments, where they have existed for millions of years, remains an open question. Besides potentially serving as tools of warfare between competing microbes, natural antibiotics have been proposed to serve less antagonistic functions ranging from the acquisition of nutrients to the transmission of signals between cells. Indeed, despite evidence that natural antibiotics can suppress sensitive microbes in environments such as the soil surrounding plant roots, the ecological significance of the toxicity of these molecules has sometimes been questioned. At the same time, for most natural antibiotics, the mechanisms and prevalence of resistance are either poorly characterized or entirely unknown.
\r\n\r\nThis thesis addresses the molecular mechanisms and consequences of bacterial resistance to a particular class of redox-active natural antibiotics called phenazines. Phenazines are produced by a major opportunistic human pathogen, Pseudomonas aeruginosa, during infections, as well as by several bacterial species that associate with the roots of crops such as wheat, where they serve to protect their plant hosts against fungal pathogens. Resistance to this family of natural antibiotics is therefore potentially relevant to multiple sectors of human society. I begin by investigating the intrinsic phenazine resistance of a common soil bacterium, Agrobacterium tumefaciens, that does not itself produce phenazines. Using a functional genetics approach, I find that the composition of the respiratory electron transport chain plays a critical role in mitigating phenazine toxicity, one that cannot be compensated by increased expression of efflux pumps that transport phenazines out of the cell or oxidative stress responses that neutralize the toxic byproducts of phenazine redox-cycling. Subsequently, we turn to P. aeruginosa, the phenazine-producing opportunistic pathogen, and demonstrate that the defenses it activates against its own toxic phenazine, pyocyanin, collaterally accelerate the acquisition of resistance to certain clinical antibiotics. Other bacteria known to form multispecies infections with P. aeruginosa can also benefit from exposure to pyocyanin in the presence of these clinical antibiotics; we show that in at least one strain isolated from a patient, the effect of pyocyanin on the frequency of spontaneous antibiotic-resistant mutants rivals that of disruptions in DNA repair machinery. Importantly, a growing body of reports suggests that, besides pyocyanin, other metabolites produced by bacterial pathogens can also affect the efficacy of clinical antibiotics. We review the evidence for which types of bacterial metabolites alter susceptibility to antimicrobial drugs, as well as the mechanisms underlying this phenomenon. Finally, I examine the prevalence of bacterial resistance to an agriculturally-relevant phenazine in a wheat field where the use of native phenazine producers to control crop diseases has been studied for decades. I discover that while Gram-positive bacteria are generally more susceptible to this phenazine compared to Gram-negative bacteria, the sharpness of this distinction is pH-dependent; moreover, I uncover surprising heterogeneity in phenazine resistance within certain taxonomic groups. Taken together, these findings illuminate recurring themes in mechanisms of phenazine resistance and point to an underappreciated role for natural antibiotics in the resilience of opportunistic pathogens to clinical antibiotics. This thesis also lays the groundwork for developing a predictive model of phenazine resistance across diverse bacteria, with potential implications for optimizing the use of clinical antibiotics and improving agricultural sustainability.
", "doi": "10.7907/tv8n-kr43", "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: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:13667", "collection": "thesis", "collection_id": "13667", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04022020-212557295", "type": "thesis", "title": "Mechanisms of Phenazine-Mediated Extracellular Electron Transfer by Pseudomonas aeruginosa", "author": [ { "family_name": "Saunders", "given_name": "Scott Harrison", "orcid": "0000-0003-4224-9106", "clpid": "Saunders-Scott-Harrison" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Extracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in individual organisms, a potentially widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. Here, I first review general aspects of the electron shuttling strategy, such as the chemical diversity and potential distribution of electron shuttle producers and users, and the costs associated with electron shuttle biosynthesis. Then I address the long-standing question: how do these electron shuttles catalyze electron transfer within biofilms without being lost to the environment? I show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox-active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.
", "doi": "10.7907/P4Z5-5445", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13767", "collection": "thesis", "collection_id": "13767", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022020-102020436", "primary_object_url": { "basename": "GriffinChure_Thesis.pdf", "content": "final", "filesize": 96078878, "license": "cc_by", "mime_type": "application/pdf", "url": "/13767/2/GriffinChure_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Molecular Biophysics of Evolutionary and Physiological Adaptation", "author": [ { "family_name": "Chure", "given_name": "Griffin Daniel", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-Daniel" } ], "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": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Central to any definition of Life is the ability to sense changes in one\u2019s environment and respond in kind. Adaptive phenomena can be found across the biological scales ranging from the nanosecond-scale conformational changes of proteins, to temporary rewiring of metabolic networks, to the 3.5 billion years of evolution that produced the enormous biodiversity we see today. This thesis presents a body of work which attempts to examine the overlap between these three scales of adaptation through the quantitative lens of statistical physics. Namely, we examine how molecular, physiological, and evolutionary adaptation governs a feature common to all life \u2013 the regulation of gene expression.
\r\n\r\nWe begin by examining the phenomenon of molecular adaptation in the context of allostery, specifically in the context of allosteric transcriptional repressors. Using simple tools of quasi-equilibrium thermodynamics, we derive and experimentally dissect a quantitative model of how such a repressor adapts to different concentrations of an extracellular inducer molecule, modulating the repressors activity and thereby gene expression. While the model is relatively simple, it is remarkably powerful in its ability to draw concrete, quantitative predictions about not only the level of gene expression at a given concentration of inducer, but details of how the repressor responds to changes in the inducer concentration. With a few lines of simple mathematics, we are able to use this model to derive a state variable of the simple repression motif which we term the free energy of the regulatory architecture. This permits us to collapse nearly 500 distinct measurements of the level of gene expression onto a master curve defined by this free energy.
\r\n\r\nWe leverage this feature of the model and use data collapse as a method to identify the effects of mutation, a strong evolutionary force responsible for much of the genetic diversity in bacteria. In Chapter 3, we examine how mutations within the allosteric repressor itself can be mapped to changes in the free energy. The precise value of these free energy shifts and their dependence on the inducer concentration reveal different classes of mutations with one class affecting only the DNA-repressor interaction and another class governing the allosteric nature of the repressor. We test these pen-and-paper predictions experimentally and illustrate that given sufficient knowledge of how single mutants behave, the complete phenotypic response of pairwise double mutants can be predicted with quantitative accuracy.
\r\n\r\nWith this framework in hand, we turn to exploring how changes in the physiological state of the cell influence the molecular biophysics of the regulatory architecture. We hypothesize that changes in the source of carbon in the growth medium or changes in the growth temperature can be accounted for by the existing model without any additional parameters. We experimentally show that the parameter values determined in one physiological state are inherited when the available carbon source is verified, but changes in the growth temperature require some additional considerations. Chapter 4 as a whole reveals that, while there remains work to be done both theoretically and experimentally when it comes to temperature variation, thermodynamic models can remain powerful tools to draw predictions of gene expression in different physiological contexts.
\r\n\r\nFinally, in Chapter 5, we explore physiological adaptation and cellular decision making where it counts \u2013 in the survival of cells to environmental insults. We turn our focus beyond transcriptional regulation and consider the relationship between osmotic shocks, the abundance of mechanosensitive channels, and cellular survival with single cell resolution. Using a combination of quantitative microscopy and tricks of statistical inference, we infer how the probability of a cell surviving an osmotic shock scales as a function of the cell\u2019s number of mechanosensitive channels.
", "doi": "10.7907/q8h6-xr92", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13800", "collection": "thesis", "collection_id": "13800", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082020-163907557", "primary_object_url": { "basename": "Silva_RebekahMB_2020.pdf", "content": "final", "filesize": 21105463, "license": "other", "mime_type": "application/pdf", "url": "/13800/2/Silva_RebekahMB_2020.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme", "author": [ { "family_name": "Silva", "given_name": "Rebekah Miriam Brawer", "orcid": "0000-0002-9144-4939", "clpid": "Silva-Rebekah-Miriam-Brawer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein-bound iron sulfur clusters are critical in cells and allow proteins to carry out many essential functions as electron carriers, catalysts for challenging organic reactions, and sensors of cellular environments. A wide range of protein families are known to coordinate iron sulfur clusters, and a growing category includes proteins involved in maintenance of the genome. Within the last three decades, iron sulfur clusters have been demonstrated to be important for enzymes that function in DNA repair, DNA replication, and transcription pathways. To date, iron sulfur clusters in the cubane [4Fe4S] geometry with all cysteine ligands have been exclusively reported for DNA repair and replication enzymes. In contrast to enzymes where the cofactor is necessary for active site chemistry or directly-linked to protein function, the [4Fe4S] cluster in the overwhelming majority of repair and replication enzymes is not involved in the catalytic modification of DNA substrates. Rather, the role of the cofactor appears to vary in function from protein to protein, and has been demonstrated to be important for protein stability, in the assembly of multisubunit proteins, and for substrate recognition, among other roles. Through investigations of the redox chemistry of the cofactor, our group has found that these enzymes participate in DNA-mediated charge transport chemistry, the process through which electrons rapidly migrate through well-stacked, duplex DNA. Long-range, DNA-mediated redox signaling provides a means of rapid communication among DNA-processing proteins for organizing repair and replication activities across the nucleus.
\r\n\r\nNotably, the first observations of the [4Fe4S] cofactor associated with repair and replications enzymes has consistently occurred well after the first biochemical studies of these enzymes. In some cases, the demonstration of a [4Fe4S] center has taken place decades later after initial work. Some proteins have required use of anaerobic methods in order to detect the cofactor, perhaps explaining why in some cases the metal center had eluded observation. Analysis of protein sequences might be expected to help accelerate identification of new iron sulfur centers in repair and replication enzymes. However, even with the abundance of sequencing data available in the post-genomic era, prediction of a metal center based on sequences alone has been challenging. This is in large part because the spacing of the coordinating cysteine residues can be quite irregular, leading to a weak bioinformatic signature.
\r\n\r\nIdentifying proteins with overlooked [4Fe4S] cofactors poses an exciting challenge, and there are some elegant examples in the literature where data from genetics assays has been used in combination with careful sequence analysis to predict and discover iron sulfur centers in repair and replication enzymes. Described here is the evolution of our studies on one well-known repair enzyme from Escherichia coli, UvrC. UvrC is part of the nucleotide excision repair pathway in the Bacteria domain which is responsible for addressing the wide class of bulky, helix-distorting lesions that can form after exposure to sources such as ultraviolet light, cigarette smoke, chemotherapeutics, and protein-DNA crosslinks. UvrC, an excision nuclease with two distinct active sites that incise the phosphodiester backbone on either side of the site of damage, has been historically challenging to study. Given how essential UvrC is in repairing damaged substrates, new insight has been greatly needed.
\r\n\r\nThrough integration of several key reports from the literature regarding the sequence of UvrC and evidence that pointed to a cofactor from genetics assays, our group predicted that UvrC is a [4Fe4S] protein. Development of a new overexpression system and an anaerobic purification method allowed for isolation of UvrC in holo form. We used spectroscopic techniques to confirm that the cluster type was [4Fe4S], and a combination of spectroscopy and chromatography to demonstrate that the UvrC-bound cofactor is susceptible to oxidative degradation. We also found that loss of the cofactor, either through aerobic degradation or mutation of coordinating cysteines, is associated with aggregation of apoprotein. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 \u00b1 20 nM and 80 \u00b1 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with DNA-bound midpoint potential of 90 mV vs. NHE.
\r\n\r\nThe work detailed in this dissertation has highlighted how critical the [4Fe4S] center is for UvrC, where the cofactor has been implicated in protein stabilization, substrate binding, and redox signaling on DNA. Handling an apo form of UvrC may have led to the previous challenges catalogued by researchers. Through the development of entirely new methods to study UvrC under anaerobic conditions, many opportunities are now available to study UvrC and the NER pathway anew in vitro and in vivo. Such work will contribute additional insight on how iron sulfur clusters are essential for enzymes that maintain genomic integrity.
\r\n\r\n", "doi": "10.7907/r0j6-jk09", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13728", "collection": "thesis", "collection_id": "13728", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282020-112303076", "primary_object_url": { "basename": "Xinran Liu Thesis 2020.pdf", "content": "final", "filesize": 12082731, "license": "other", "mime_type": "application/pdf", "url": "/13728/20/Xinran Liu Thesis 2020.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Cell-Selective Proteomic Profiling in Complex Biological Systems", "author": [ { "family_name": "Liu", "given_name": "Xinran", "orcid": "0000-0003-0045-8823", "clpid": "Liu-Xinran" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cells within biological systems are constantly adjusting their protein synthesis in response to various environmental changes. To study the rapid cellular regulations in complex biological systems, global proteomic profiling provides important information on system-level regulations, yet physiological properties characteristic of individual cellular subpopulations could be hidden under the characterization. Instead, cell-selective proteomic profiling allows researchers to reveal the heterogeneities in biological systems with phenotypically and even genetically distinct subpopulations under different microenvironments.
\r\n\r\nChapter 1 describes the development of bioorthogonal noncanonical amino acid tagging (BONCAT) for proteomic profiling with resolution in both space and time: its initial role is protein labeling with temporal resolution via pulse-addition of noncanonical amino acid, which could be recognized by endogenous aminoacyl tRNA-synthetase (aaRS), into systems of interest; later on, mutant aaRSs are identified through mutant synthetase library screening, which allows for efficient incorporation of various types of noncanonical amino acids that could hardly be activated by endogenous machineries. The identification and exploitation of mutant aaRSs allow sensitive cellular selectivity during protein labeling. With unprecedented spatiotemporal resolution of BONCAT, and the advancement in high-resolution mass spectrometry and computational algorithms, BONCAT is a powerful technique for selective proteomic profiling to study physiological regulations in a wide range of complex biological systems.\r\nChapter 2 describes the application of the BONCAT method in cell-selective proteomic profiling in Pseudomonas aeruginosa biofilms. In this work, we targeted an iron-starved subpopulation in biofilms and compared its proteomic profile with that of the entire system. Key gene and pathway regulations in the subpopulation are found through the analysis of the proteomic data, which suggest that iron-starved cells shift their priority towards housing keeping pathways, adapt an energy- and resources-saving mode to cope with their harsh local environmental conditions, and get prepared to disperse for better survival. Analysis of poorly studied proteins highly upregulated in the subpopulation led to the discovery of a previously uncharacterized protein (PA14_52000) that is potentially related to iron acquisition. The transposon insertion mutant PA14_52000::tn showed significantly enhanced pyoverdine production in rich medium and reduced biofilm formation.
\r\n\r\nChapter 3 describes the study of physiological regulations in Bacillus subtilis K-state subpopulation via BONCAT. A subset of B. subtilis cells, typically 10% - 20% of the entire population, enter K-state in a stochastic manner. With the low level of K-state entry rate and high randomness, we challenged BONCAT to specifically capture gene and pathway regulations in K-state cells and compared the proteomic profiling with that of the entire population. Regardless of the difficulties of selective protein labeling inherent in the system, our results indicate that BONCAT has high specificity and resolution in proteomic profiling for minor subpopulations and proteins with low overall absolute abundance. We found multiple pathways and genes characteristic of K-state regulated differentially from the entire population, either significantly up- or down-regulated. Proteins that are uncharacterized or previously known for functions irrelevant of K-state are highly abundant in the subpopulation, providing new insight toward their alternative functions critical for K-state cells and future investigation directions of K-state study.
", "doi": "10.7907/p18t-5j69", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11562", "collection": "thesis", "collection_id": "11562", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292019-144516804", "type": "thesis", "title": "Structural and Biochemical Studies of Enzymes in Bacterial Glycobiology", "author": [ { "family_name": "Yun", "given_name": "Hyun Gi", "orcid": "0000-0002-3508-5791", "clpid": "Yun-Hyun-Gi" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The speed that bacterial pathogens gain resistance to antibiotics is alarming. Designing new antibacterial agents is urgent, but it requires understanding their bacterial targets at the molecular level to achieve high specificity and potency. In this thesis, I discuss the structural and biochemical investigations of three potential protein targets for antibiotics. The first is a UDP-Glc/GlcNAc 4-epimerase, called Gne, from the human pathogen Campylobacter jejuni. This enzyme is the sole source of N-acetylgalactosamine (GalNAc) in C. jejuni, which is a common component in three major glycoconjugates decorating the cell surface and is critical for pathogenesis. The second target protein is an integral membrane protein, called MraY, which catalyzes the transfer of phospho-N-acetylmuramyl (MurNAc) pentapeptide to a lipid carrier, undecaprenyl phosphate (C55-P), producing Lipid I in the peptidoglycan biosynthesis pathway. In the following step, a peripheral protein called MurG catalyzes transferring N-acetylglucosamine (GlcNAc) to Lipid I and produces Lipid II, which provides the first building block of the peptidoglycan layer. Peptidoglycan is uniquely bacterial, with MraY and MurG both being essential for cell viability; therefore, they are attractive targets for the development of antibacterial agents and work toward their structures is presented. Finally, MraY from Escherichia coli is the target for the lysis protein E from phage \u03a6X174.Efforts toward elucidating the EcMraY-E complexstructure are demonstrated here. In total, this thesis provides important data toward a full mechanistic understanding of these important antibacterial targets.", "doi": "10.7907/VKH3-XX98", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11243", "collection": "thesis", "collection_id": "11243", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10232018-150005837", "primary_object_url": { "basename": "AngelesAlbores_David_2019.pdf", "content": "final", "filesize": 8920493, "license": "other", "mime_type": "application/pdf", "url": "/11243/1/AngelesAlbores_David_2019.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "A Theory of Genetic Analysis Using Transcriptomic Phenotypes", "author": [ { "family_name": "Angeles-Albores", "given_name": "David", "orcid": "0000-0001-5497-8264", "clpid": "Angeles-Albores-David" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "WormBase" }, { "literal": "div_bbe" } ], "abstract": "This thesis deals with the conceptual and computational framework required to use transcriptomes as effective phenotypes for genetic analysis. I demonstrate that there are powerful theoretical reasons why Batesonian epistasis should feature prominently in transcriptional phenotypes. I also show how to compute and interpret the aggregate statistics for transcriptome-wide epistasis and transcriptome-wide dominance using whole-organism transcriptomic profiles of C. elegans mutants. Finally, I developed the WormBase Enrichment Suite for enrichment analysis of genomic data.
\r\n\r\nRNA-seq as a tool has enormous potential because it relies on protocols that are fast, simple and increasingly cheap. In spite of their potential, transcriptomes have seen their use largely limited to single-factor experiments. Even when many transcriptomes are collected, the main analytic approach is to apply clustering algorithms that correlate responses but do not have any power to identify causal mechanisms.
\r\n\r\nI demonstrate that if a complete genetic experimental design is used (in the form of a full two-factor matrix), transcriptomes can establish genetic interactions between a pair of genes without the need for clustering algorithms. Surprisingly, when we performed epistasis analyses of hypoxia pathway mutants in C. elegans we did not simply observe a generalized epistatic interaction between the mutants. In fact, the transcriptomes recapitulated the same Batesonian epistatic relationship that had been observed using classical phenotypes. In other words, we observed that the transcriptomic phenotype of one gene can be masked by the transcriptomic phenotype of a second gene, such that a double mutant of these two genes has exactly the same phenotype as a single mutant of the epistatic gene. Motivated by this observation, we developed methods to recognize and interpret Batesonian epistasis at the transcriptomic level. This method relies on the calculation of a single aggregate coefficient that we named the transcriptome-wide epistasis coefficient.
\r\n\r\nThe observation that Batesonian epistasis could be reproduced on a transcriptomic level was surprising. To explain how transcriptome-wide epistasis can arise, I studied a simplified model of transcriptional regulation using statistical mechanics. These studies demonstrate that epistatic analysis is equivalent to a perturbative analysis of the partition function of a promoter. Moreover, these studies revealed that a sufficient condition for Batesonian epistasis to occur is if the two genes encode variables that are transformed and multiplied together to form an effective single compound variable. Finally, these studies clearly demonstrate the connection between statistical (or generalized) epistasis and Batesonian epistasis and establish a physical basis for genetic logic.
\r\n\r\nGenetic analyses of gene functional units can also be carried out using allelic series in tandem with complementation (also known as dominance) tests. I developed a statistical coefficient known as transcriptome-wide dominance to enable analyses of allelic series using expression profiles. A crucial aspect of allelic series is the ability to enumerate the independent phenotypes associated with an arbitrary set of alleles. I developed the concept of phenotypic classes as a transcriptomic analogue of classical phenotypes for this purpose. Briefly, a phenotypic class is a set of transcripts that are differentially expressed in a specific set of genotypes. Thus, an allelic series consisting of two mutant alleles (and a wild-type) can at most result in 7 phenotypic classes. However, some of these phenotypic classes may be artifactual as a result of the significant false positive and false negative rates that are associated with RNA-seq. I developed a simple algorithm that tries to identify phenotypic classes that are artifactual, though often these classes may also be identified through a critical evaluation of their biological implications. I applied these concepts to a small allelic series of the dpy-22 gene, which encodes a Mediator subunit in C. elegans, and identified 3\u20134 functional units along with their sequence requirements.
\r\n\r\nFinally, I developed the WormBase Enrichment Suite by implementing a hypergeometric test on the tissue, gene and phenotype ontology for C. elegans. The importance of this tool derives mainly from its integration to WormBase, the repository of all C. elegans knowledge, which means that the databases that are tested will undergo continuous improvement and curation, and thus will yield the most accurate results.
", "doi": "10.7907/JRNS-NS05", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11540", "collection": "thesis", "collection_id": "11540", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242019-115311540", "primary_object_url": { "basename": "Ferdinand_Huber_PhD_Thesis.pdf", "content": "final", "filesize": 112541653, "license": "other", "mime_type": "application/pdf", "url": "/11540/1/Ferdinand_Huber_PhD_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular Basis for Ribosomal Protein Protection from Cellular Degradation", "author": [ { "family_name": "Huber", "given_name": "Ferdinand Michael", "clpid": "Huber-Ferdinand-Michael" } ], "thesis_advisor": [ { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ribosomes are large macromolecular machineries composed of both protein and RNA constituents with a species-dependent molecular mass of at least ~3.3\u00a0MDa for the fully assembled eukaryotic 80S ribosome. Their catalytic activity is dependent on ribosomal RNA; therefore, ribosomes are bona fide ribozymes, and as such they mediate the final step of gene expression from DNA to RNA to protein by peptide bond formation between amino acids. Importantly, spatial separation of ribosome function and biogenesis into distinct cellular compartments allows for intricate regulatory mechanisms and rigorous quality control. Ribosome biogenesis occurs predominantly in the nucleolus and nucleus of the cell with final cytoplasmic maturation and quality control steps. Briefly, nucleolar ribosomal RNA together with ~200 trans-acting assembly factors co-transcriptionally forms the 40S and 60S pre-ribosomal subunits into which ~80 ribosomal proteins are incorporated in a hierarchical fashion.
\r\n\t\r\nRecent studies, including this thesis, have identified a novel class of dedicated ribosome assembly chaperones, in addition to the ~200 trans-acting ribosome assembly factors, which facilitate ribosomal protein shuttling. Ribosomal proteins are generated in the cytoplasm, and with only few exceptions they all have to enter the nucleus for incorporation into the pre-ribosomal subunits. Assembly chaperones can bind and protect unassembled ribosomal proteins either co-translationally or following nuclear import and shuttle them in a timely fashion to their destination sites at the maturing pre-ribosomal subunits. The first chapter of this thesis describes the identification and characterization of a dedicated assembly chaperone for the large ribosomal subunit protein RpL4, termed Acl4. Interestingly, Acl4 and likely also other dedicated assembly chaperones not only interact with ribosomal proteins to avoid aggregation and to shield them from unfavorable interactions, but also protect their client proteins from cellular degradation by the ubiquitin-proteasome machinery.
\r\n\t\r\nRibosomes are built by assembling equimolar amounts of ribosomal proteins, which generates a challenge for the cell to ensure stoichiometric quantities of ribosomal proteins. Recent studies have demonstrated that stoichiometric levels of ribosomal proteins are established by cellular degradation of excess protein via ubiquitination of unassembled components. The second chapter of this thesis describes a conserved degradation pathway, which is dependent on the E3 ubiquitin ligase Tom1 to mark unprotected and unassembled ribosomal proteins and target them for degradation. Moreover, it is demonstrated in the third chapter for the first time how an assembly chaperone protects its client ribosomal protein from ubiquitination and proteasome-mediated degradation. High resolution structures of the Acl4\u2022RpL4 complex as well as RpL4 in complex with the nuclear transport factor Kap104 visualize the molecular interactions of those proteins and uncover the molecular mechanism of protecting conserved Tom1-target sites within RpL4. Together, the reported results identify and characterize both a novel degradation pathway as well as a protection mechanism for ribosomal proteins and advance the understanding of the intricate regulation of ribosome biogenesis.
", "doi": "10.7907/4S7C-V170", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11549", "collection": "thesis", "collection_id": "11549", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282019-090132145", "primary_object_url": { "basename": "BastaDavid2019thesis.pdf", "content": "final", "filesize": 17391825, "license": "other", "mime_type": "application/pdf", "url": "/11549/17/BastaDavid2019thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases", "author": [ { "family_name": "Basta", "given_name": "David Wagdi", "orcid": "0000-0003-4176-6566", "clpid": "Basta-David-Wagdi" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Growth arrest is the dominant mode of microbial existence on the planet, yet the molecular mechanisms that underpin survival during growth arrest remain far less studied than other growth states. A better understanding of these mechanisms would provide valuable insight into the activity of microbial communities in both biogeochemical and clinical contexts, including the treatment of chronic infections. This thesis investigates the genetic requirements for survival of the bacterium Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen that thrives in diverse environments in which growth arrest is often caused by energy limitation. After reviewing our current knowledge of the strategies used by growth-arrested bacteria to adjust metabolism, regulate transcription and translation, and maintain the chromosome, I perform a functional genomic screen to identify genes that promote fitness of P. aeruginosa during growth arrest caused by carbon or oxygen starvation. I find that P. aeruginosa can survive for days to weeks in these energy-starved conditions by maintaining a reduced steady-state level of ATP, and that many functional classes of genes are required for fitness. Intriguingly, a majority of genetic fitness determinants differ between carbon and oxygen starvation, despite the common endpoint of reduced ATP levels in these two conditions. Among the few genes generally required for fitness are the stress response sigma factor encoded by rpoS and the heat shock protease encoded by ftsH. Using independently-generated deletion strains, I show that mutants in distinct functional categories exhibit temporal fitness dynamics during oxygen starvation: regulatory genes generally manifest a phenotype early during growth arrest, whereas genes involved in cell wall metabolism are required later. Building on these findings, I investigate the functional role of FtsH during growth arrest more deeply and find a surprising negative genetic interaction between ftsH and rpoS, with mutations in rpoS alleviating the fitness defects of \u0394ftsH during growth arrest. I also find that FtsH functions coordinately with the other conserved heat shock proteases to maintain cellular integrity and delay aging of P. aeruginosa during growth arrest. Finally, I investigate the role of FtsH and the other heat shock proteases in a novel N-terminal protein degradation pathway and find that the molecular details of this pathway likely differ between E. coli and P. aeruginosa. Together, these findings uncover essential molecular processes that promote fitness of an important bacterial pathogen during growth and survival.
", "doi": "10.7907/K6X1-GS91", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "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:11703", "collection": "thesis", "collection_id": "11703", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-035956740", "primary_object_url": { "basename": "WenChen_2019_Caltech-thesis.pdf", "content": "final", "filesize": 23199748, "license": "other", "mime_type": "application/pdf", "url": "/11703/1/WenChen_2019_Caltech-thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Proteomics Profiling and Functional Characterization of Caenorhabditis elegans Excreted/Secreted Proteins", "author": [ { "family_name": "Chen", "given_name": "Wen", "orcid": "0000-0001-8056-5711", "clpid": "Chen-Wen-Biochemistry-Molecular-Biophysics" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "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_chem" } ], "abstract": "Excretory-secretory products (ESPs) are first characterized and defined in parasitic nematode proteomics studies as the combination of various biomolecules that are continuously excreted or secreted into the environment throughout the whole life cycle. ESPs are particularly interesting to many scientists as anti-parasitic vaccine candidates and\r\nas promising drug targets since large portions of ESPs are active enzymes that potentially function directly at the parasite-host or worm-environment interfaces. However, majority of the parasites lack whole genome sequence knowledge and genome-editing tools. Thus, the number of ESPs identified is limited and many functions of ES proteins cannot be elucidated. Therefore, we use the most studied nematode, Caenorhabditis elegans, as the model to characterize the composition of excreted/secreted proteins with the help of nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). We characterized more than 509 excreted/secreted proteins with mix-staged worms, including many metalloproteases, cysteine proteases, and lysozymes. Proteases and proteases inhibitors are a major group in C. elegans ESPs. We performed stable isotope dimethylvlabeling quantitative proteomics and compared C. elegans ESPs on different bacteria diets. Lysozymes are not only enriched in C. elegans ESPs but are also up-regulated in response to pathogen and bacteria.
\r\n\r\nComparative studies of expression profiles of developmental life stages and pathogen infections elucidate the dynamics in regulating ESP components. We successfully identified stage-specific ESP groups associated with L1, L3, adult, L2 dauer, and postdauer. We demonstrated that proteases activities are down regulated by increased protease inhibitor expressions, while during dauer exit proteases expressions are increased. The comparison between dauer excretome/secretome and RNA-seq dauer expression profiles revealed 91 ESP encoding genes that are highly expressed in dauers. We performed dauer formation assay to these dauer-associated gene mutants. The great prediction rate confirmed that our comparative method is the simplest way to quickly pick out candidates for functional assays. Similarly, we employed this comparative method to pathogeninduced transcriptomes. We reported a group of genes that are associated with Serratia marcescens infection and a group of bacterial pathogens responding genes. We confirmed the roles of C. elegans ESPs in immuoregulation by infection assays with various pathogens. Lysosomes and cysteine protease inhibitor are among the most important genes in innate immune response pathway of C. elegans defending pathogen infection.
\r\n\r\nThe recent discovery of a C. elegans sibling species, Caenorhabditis inopinata, allows the deeply comparative study for evolutional interpretation. The excretome/secretome of C. inopinata has not been characterized. We took advantage of the sensitive and highthroughput technique of nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) to directly characterize the protein components of C. inopinata excretome/secretome. Functional annotations reveal several protein families, including C-type lectins, Cathepsin Z, Cathepsin B family, transthyretin, and saposin-like families, suggesting ESPs play critical roles in regulating innate immune response. We compared C. inopinata excretome/secretome with C. elegans. The structures are highly conserved across species, suggesting the sibling species share common mechanism to respond to environmental stimuli.
", "doi": "10.7907/WTMK-7M75", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10958", "collection": "thesis", "collection_id": "10958", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-133205686", "primary_object_url": { "basename": "barnes_stephanie_2018.pdf", "content": "final", "filesize": 61047841, "license": "other", "mime_type": "application/pdf", "url": "/10958/1/barnes_stephanie_2018.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Decoding the Regulatory Genome: Quantitative Analysis of Transcriptional Regulation in Escherichia coli", "author": [ { "family_name": "Barnes", "given_name": "Stephanie Loos", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-Stephanie-Loos" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "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" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Over the past decades DNA sequencing has become significantly cheaper and faster, which has enabled the accumulation of a huge amount of genomic data. However, much of this genomic data is illegible to us. For noncoding regions of the genome in particular, it is difficult to determine what role is played by specific DNA sequences. Here we focus on regions of DNA that play a role in transcriptional regulation. We develop models and techniques that allow us to discover new regulatory sequences and better understand how DNA sequence determines regulatory output.
\r\n\r\nWe start by considering how quantitative models serve as a powerful tool for testing our understanding of biological systems. We apply a statistical mechanical framework that incorporates the Monod-Wyman-Changeux model to analyze the effects of allostery in simple repression, using the lac operon as a test case. By fitting our model to experimental data, we are able to determine the values of the unknown parameter values in our model. We then show that we can use the model to accurately predict the induction responses of an array of simple repression constructs with a variety of repressor copy numbers and repressor binding energies.
\r\n\r\nNext, we consider how the DNA sequence of a promoter region can provide details about how the promoter is regulated. We begin by describing an approach for discovering regulatory architectures for promoters whose regulation has not previously been studied. We focus on six promoters from E. coli including three well-studied promoters (rel, mar, and lac) to serve as test cases. We use the massively parallel reporter assay Sort-Seq to identify transcription factor binding sites with base-pair resolution, determine the regulatory role of each binding site, and infer energy matrices for each binding site. Then, we use DNA affinity chromatography and mass spectrometry to identify each transcription factor.
\r\n\r\nWe conclude with an in vivo approach for analyzing the sequence-dependence of transcription factor binding energies. Again using Sort-Seq, we show that we can represent transcription factor binding sites using energy matrices in absolute energy units. We then show that these energy matrices can be used to accurately predict the binding energies of mutated binding sites. We provide several examples of how understanding the relationship between DNA sequence and transcription factor binding provides us with a foundation for addressing additional scientific topics, such as the co-evolution of transcription factors and their binding sites.
", "doi": "10.7907/D13T-7868", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "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:10175", "collection": "thesis", "collection_id": "10175", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162017-130844945", "type": "thesis", "title": "Structure and Function of the Mycobacterial Mechanosensitive Channel of Large Conductance, MscL\r ", "author": [ { "family_name": "Herrera", "given_name": "Nadia", "orcid": "0000-0003-4157-9429", "clpid": "Herrera-Nadia" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "MscL is a ubiquitous channel found in bacterial membranes. It provides a protective response to osmotic downshock by opening and closing in response to tension in the membrane. A number of studies have aimed to develop a mechanism for the gating of MscL in E. coli, but structural details describing the process have remained elusive. A few structures of non-conducting states of MscL have been solved using X-ray crystallography, Mycobacterium tuberculosis (Mt) MscL and Staphylococcus aureus (Sa) MscL with a C-terminal domain truncation. In addition, the structure of the E. coli (Ec) MscL C-terminal cytoplasmic domain has been solved.
\r\n \r\nThe goals of the studies presented in this thesis are as follows: (i) capturing a C-terminal domain truncation of MtMscL using X-ray crystallography, and (ii) analyzing the functional regulation of MscL channels in mycobacteria. To achieve the latter goal, we generated a knockout of the mscL gene in a fast-growing mycobacteria species, Mycobacterium smegmatis. This strain was used to analyze the role of MscL in the cell during antibiotic entry. Structural studies of MtMscL are focused on identifying the role of the C-terminal domain by studying a channel with a truncation at the C-terminal domain. The motivation for this goal comes from the structure of SaMscL, which showed that truncation of the C-terminal domain resulted in crystallizing the protein as a tetramer, an alternative oligomeric state to the pentameric state observed for the MtMscL structure. Studies on an MtMscL C-terminal domain truncation aimed to further establish that correlation. This protein was overexpressed in E. coli BL21 DE3 mscL-, purified, and crystallized by sitting drop vapor diffusion. Native crystals diffracted to 6.5 \u00c5, and heavy atom derivative crystals diffracted to 5.8 \u00c5. The structure of the MtMscL C-terminal truncation has been solved, and is presented in this thesis. Our studies on the structure show that the pentameric state of the channel remains intact upon truncation of the C-terminal domain. To analyze the function of our mutant, we utilized patch clamp electrophysiology studies using our expression strain as the giant spheroplast platform. The findings from the electrophysiology studies indicate that MtMscL C-terminal domain truncation results in a channel that has gating tension requirements similar to EcMscL, whereas full-length MtMscL has much higher gating tension requirements than our construct. In addition, the role of MscL in mycobacterial antibiotic susceptibility is being tested in Mycobacterium smegmatis. We have created a strain of M. smegmatis with the mscL gene knocked out, MC2155 mscL- and we have observed that upon deletion of mscL an increase in tolerance to spectinomycin is observed in our knockout strain.
", "doi": "10.7907/Z9JH3J77", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10230", "collection": "thesis", "collection_id": "10230", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312017-133325449", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 4208773, "license": "other", "mime_type": "application/pdf", "url": "/10230/1/THESIS.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Physiological and Biochemical Mechanisms of Phenazine-Mediated Survival in Pseudomonas aeruginosa", "author": [ { "family_name": "Glasser", "given_name": "Nathaniel Robert", "orcid": "0000-0002-2833-5166", "clpid": "Glasser-Nathaniel-Robert" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The opportunistic pathogen Pseudomonas aeruginosa secretes a class of colorful redox-active small molecules known as phenazines. Numerous functions have been proposed for phenazines, including antibiotic activity, virulence, cell-to-cell signaling, iron acquisition, and survival. This thesis delves into mechanisms of the latter role, that of long-term survival under oxidant-limiting conditions. Using a diverse array of methods, I investigated how phenazines support survival and how cells transfer electrons to phenazines, as well as the downstream effects that phenazines have on P. aeruginosa.
\r\n\r\nDirect measurements of NAD(H), ATP, the membrane potential, and fermentation products revealed that phenazines promote redox homeostasis and subsequently ATP synthesis. The ATP is used to maintain a membrane potential through the reverse action of the ATP synthase complex. Even though P. aeruginosa does not ferment on sugars, phenazines enable the anaerobic oxidation of glucose to acetate, suggesting P. aeruginosa may have previously under-appreciated metabolic flexibility in the absence of terminal electron acceptors. Activity assays with proteins purified natively from P. aeruginosa showed that glucose oxidation might be enabled in vivo by the pyruvate dehydrogenase complex, which can directly reduce phenazines using pyruvate as an electron donor. Liquid chromatography and mass spectrometry of culture supernatants showed that phenazines alter the chain length distribution of secreted quinolones, which may have indirect downstream signaling effects. Based on this result, combined with data from survival experiments, I hypothesize that phenazine-mediated redox homeostasis promotes \u03b2-oxidation and that fatty acid metabolism contributes to long-term survival. Further analysis also showed that P. aeruginosa cultures contain several previously-unreported sulfonated phenazines. In its natural environment, P. aeruginosa undoubtedly encounters other microbial species that consume or modify its phenazines. At least one of these, a Mycobacterium, contains a pyocyanin demethylating enzyme. The X-ray crystal structure of this protein revealed a novel reaction mechanism wherein the substrate is its own electron acceptor. Together, this work illuminates some of the many ways phenazines shape microbial communities in both clinical and environmental contexts.
\r\n", "doi": "10.7907/Z9SN070S", "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:9704", "collection": "thesis", "collection_id": "9704", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05062016-131543245", "primary_object_url": { "basename": "babin_thesis_final.pdf", "content": "final", "filesize": 10129687, "license": "other", "mime_type": "application/pdf", "url": "/9704/39/babin_thesis_final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Bioorthogonal Noncanonical Amino Acid Tagging for Selective Analysis of the Pseudomonas aeruginosa Proteome", "author": [ { "family_name": "Babin", "given_name": "Brett Michael", "orcid": "0000-0002-4133-6665", "clpid": "Babin-Brett-Michael" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA\u2019s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.", "doi": "10.7907/Z94X55S1", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9063", "collection": "thesis", "collection_id": "9063", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07192015-214603085", "primary_object_url": { "basename": "DNA CT signaling within the cell_MAG_2015.pdf", "content": "final", "filesize": 4584274, "license": "other", "mime_type": "application/pdf", "url": "/9063/1/DNA CT signaling within the cell_MAG_2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Signaling Within the Cell", "author": [ { "family_name": "Grodick", "given_name": "Michael Andrew", "clpid": "Grodick-Michael-Andrew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA possesses the curious ability to conduct charge longitudinally through the \u03c0-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.
\r\n\r\nHerein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.
\r\n\r\nIn separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?
\r\n\r\nUvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.
\r\n\r\n\r\n", "doi": "10.7907/Z9F769GX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9692", "collection": "thesis", "collection_id": "9692", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292016-193643737", "type": "thesis", "title": "A Core Mitophagic Machinery Promotes Selective Degradation of Paternal Mitochondria in Mouse Embryos and MEF Cells", "author": [ { "family_name": "Rojansky", "given_name": "Rebecca Bloom", "orcid": "0000-0002-3735-8159", "clpid": "Rojansky-Rebecca-Bloom" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "orcid": "0000-0001-6597-2036", "clpid": "Stathopoulos-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The maternal mode of mitochondrial inheritance is conserved across mammalian species; however, little is known about how mitochondria from the sperm are eliminated from early mammalian embryos. Mitophagy, the regulated degradation of mitochondria in the lysosome, has been proposed as a possible mechanism. Mitophagy is an important means by which the cell responds to changes in mitochondrial fitness, and has been observed under a number of physiological and non-physiological circumstances, including, but not limited to, hypoxia, mitochondrial depolarization, mitochondrial fission, and erythrocyte differentiation.
\r\n\r\nHere we examine the core component of mitophagy proteins involved in three physiological states: respiration-induced mitophagy in cultured mouse fibroblasts, mitophagy of dysfunctional mitochondria in the absence of mitochondrial fusion, and degradation of paternal mitochondria in pre-implantation mouse embryos. We find that a common pathway is used for elimination of mitochondria, involving mitochondrial depolarization, and the E3 ubiquitin ligases PARKIN and MUL1. We find that PARKIN and MUL1 play partially redundant roles in elimination of paternal mitochondria that is also dependent on PINK1 kinase, the fission factor, FIS1, and the autophagy receptor, p62. We find that p62 is specifically recruited to defective mitochondria in fusion deficient cells by a mechanism independent of ubiquitin binding. Our results elucidate the molecular mechanism of strict maternal transmission of mitochondria and uncover a collaboration between MUL1 and PARKIN in mitophagy.
\r\n", "doi": "10.7907/Z96Q1V75", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "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: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:8903", "collection": "thesis", "collection_id": "8903", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-153534197", "primary_object_url": { "basename": "Jessica Ricci Thesis 2015 FINAL.pdf", "content": "final", "filesize": 4770646, "license": "other", "mime_type": "application/pdf", "url": "/8903/1/Jessica Ricci Thesis 2015 FINAL.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Constraining the Interpretation of 2-Methylhopanoids through Genetic and Phylogenetic Methods", "author": [ { "family_name": "Ricci", "given_name": "Jessica Nicole", "clpid": "Ricci-Jessica-Nicole" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record. ", "doi": "10.7907/Z9MC8X0S", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "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:8777", "collection": "thesis", "collection_id": "8777", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03092015-135659710", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 19472312, "license": "other", "mime_type": "application/pdf", "url": "/8777/1/Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Ferrous Iron Sensing and Responding in Pseudomonas aeruginosa", "author": [ { "family_name": "Kreamer", "given_name": "Naomi N.", "clpid": "Kreamer-Naomi-N" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "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" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator\u2019s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa\u2019s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future.", "doi": "10.7907/Z9DN4324", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8456", "collection": "thesis", "collection_id": "8456", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312014-143352981", "type": "thesis", "title": "Development of Integrated Parylene Fluidic Devices for Use as a Microbial Monitoring System in Wastewater Treatment", "author": [ { "family_name": "Satsanarukkit", "given_name": "Penvipha", "clpid": "Satsanarukkit-Penvipha" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" }, { "family_name": "Abu-Mostafa", "given_name": "Yaser S.", "clpid": "Abu-Mostafa-Y-S" }, { "family_name": "Doyle", "given_name": "John Comstock", "orcid": "0000-0002-1828-2486", "clpid": "Doyle-J-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "[Abstract Embargoed]", "doi": "10.7907/Z98G8HPQ", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "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:8456", "collection": "thesis", "collection_id": "8456", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312014-143352981", "type": "thesis", "title": "Development of Integrated Parylene Fluidic Devices for Use as a Microbial Monitoring System in Wastewater Treatment", "author": [ { "family_name": "Satsanarukkit", "given_name": "Penvipha", "clpid": "Satsanarukkit-Penvipha" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" }, { "family_name": "Abu-Mostafa", "given_name": "Yaser S.", "clpid": "Abu-Mostafa-Y-S" }, { "family_name": "Doyle", "given_name": "John Comstock", "orcid": "0000-0002-1828-2486", "clpid": "Doyle-J-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "[Abstract Embargoed]", "doi": "10.7907/Z98G8HPQ", "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:7710", "collection": "thesis", "collection_id": "7710", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-213725125", "type": "thesis", "title": "Single-Cell Analysis of the Physiology of Mechanosensation in Bacteria", "author": [ { "family_name": "Bialecka-Fornal", "given_name": "Maja I.", "clpid": "Bialecka-Fornal-Maja-I" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.", "doi": "10.7907/7SRD-WS94", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6277", "collection": "thesis", "collection_id": "6277", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04032011-125158842", "primary_object_url": { "basename": "Complete_Thesis.pdf", "content": "final", "filesize": 6687006, "license": "other", "mime_type": "", "url": "/6277/12/Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases", "author": [ { "family_name": "Romano", "given_name": "Christine Anne", "clpid": "Romano-Christine-Anne" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work performed herein describes three proteins: Uracil DNA glycosylase (UDG) from Archaeoglobus fulgidus, MutY, and Endonuclease III (EndoIII) from Escherichia coli. They are DNA repair glycosylases that contain [4Fe-4S] clusters. While the catalytic mechanisms of many BER enzymes have been studied in detail, questions remain about how these enzymes search the vast amount of cellular DNA to find their substrates, and why some require a [4Fe-4S] cluster. The iron-sulfur cluster is not necessary for catalysis, and it only displays a physiologically relevant midpoint potential when bound to DNA. We have proposed that UDG, MutY, and EndoIII use their [4Fe-4S] clusters to participate in DNA-mediated charge transport (CT), and that these proteins mediate long-range electrochemical signaling in order to detect DNA damage.
\r\n\r\nThis scheme for DNA damage detection assumes that CT occurs efficiently between the DNA helix and the [4Fe-4S] cluster of the bound protein. In order for efficient CT to occur, a pathway of amino acids must be present that facilitates CT between the DNA and the iron-sulfur cluster. For each of the enzymes mentioned, this pathway was explored through mutagenesis. In UDG, MutY, and EndoIII, several amino acids thought to be important for CT were mutated and the resulting proteins were characterized biochemically. Their CT capabilities were analyzed by cyclic voltammetry on DNA-modified electrodes. In these experiments, the mutants\u2019 signal intensities were quantified and compared to those of wild-type enzyme. An attenuated signal relative to wild-type protein may indicate that the mutant is deficient in CT and that the targeted amino acid is part of the protein-DNA CT pathway in the native enzyme. Many mutants were also screened by enzymatic assays and circular dichroism spectroscopy to further characterize their DNA-binding properties and structural stability.
\r\n\r\nThe A. fulgidus UDG mutants examined, C17H, C85S, and C101S, all contained mutations in the cysteine residues that ligate the [4Fe-4S] cluster. These mutants were designed to determine how the iron-sulfur cluster coordination environment affects protein-DNA CT. The mutants exhibited varying signal strengths relative to WT UDG on DNA-modified electrodes. C85S produced a weaker signal, indicating a CT deficiency. The signal intensity from C101S was within error of that of WT, and the signal from C17H was larger than that of WT, possibly indicating that this mutant is less structurally stable than WT UDG.
\r\n \r\nIn E. coli MutY, position Y82 aligns with Y165 in MUTYH, a residue in which mutations have been found in many colorectal cancer patients. To better understand the correlation between protein-DNA CT and colorectal cancer, the MutY mutants Y82C and Y82L were prepared and characterized. Y82C exhibited a CT deficiency relative to WT MutY, whereas Y82L did not. These data indicate that Y82 forms part of the CT pathway in native E. coli MutY, but that other long-chain amino acids, such as leucine, can also mediate CT efficiently at this position.
\r\n \r\nSeveral different mutants of E. coli EndoIII were examined. First, the Y82 position was targeted, since the aligning MUTYH residue has been found mutated in colorectal cancer patients and because this residue is located near the protein-DNA interface. Five mutations were made at or near the Y82 position, and their cyclic voltammetry signals demonstrated that aromatic amino acids best mediate CT at this position. Other residues towards the interior of the protein, Y75, Y55, and F30 were also mutated to alanines. These mutants exhibited CT deficiencies, implicating the residues as part of a potential CT pathway. Residues W178 and Y185, located near the [4Fe-4S] cluster of EndoIII, were also mutated to alanines. The resulting mutants produced larger signals than that of WT EndoIII. These mutants were later shown by circular dichroism spectroscopy to be less stable structurally than WT EndoIII. All of the mutants mentioned exhibited enzymatic properties similar to those of WT, suggesting that they are able to bind DNA and excise damage nucleobases as well as the native enzyme. Several of these mutants were also used in a mutagenesis-based experiment to assay how EndoIII variants help MutY search for DNA lesions, although data from these experiments showed no significant differences in mutation rate between strains expressing different EndoIII variants.
\r\n \r\nIn total, the mutagenesis studies performed here helped determine the characteristics of BER enzymes that enable them to mediate DNA-protein CT. All these enzymes must contain a stable, well-protected metallocluster that charge can access through a series of CT-facilitating amino acids. In discovering several residues important for protein-DNA CT in UDG, MutY, and EndoIII, we have strengthened support for the hypothesis that these enzymes facilitate DNA-mediated CT in vivo. These enzymes may in fact be part of a much larger array of redox-active DNA-binding proteins that communicate electrochemically to help each other detect and repair DNA lesions inside the cell.
\r\n", "doi": "10.7907/63TC-FN74", "publication_date": "2011-06-10", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:674", "collection": "thesis", "collection_id": "674", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02182009-100346", "primary_object_url": { "basename": "pricewhelan_21809.pdf", "content": "final", "filesize": 7043779, "license": "other", "mime_type": "application/pdf", "url": "/674/1/pricewhelan_21809.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Physiology and Mechanisms of Pyocyanin Reduction in Pseudomonas aeruginosa", "author": [ { "family_name": "Price-Whelan", "given_name": "Alexa Mari", "orcid": "0000-0001-7587-7534", "clpid": "Price-Whelan-Alexa-Mari" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The opportunistic pathogen Pseudomonas aeruginosa excretes redox-active small molecules called phenazines. This thesis addresses the possibility that the phenazine pyocyanin acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined by comparing the physiological status of the wild type to a mutant defective in phenazine production. Quantification of intracellular NADH and NAD+ pools revealed a more reduced intracellular redox state in the phenazine-null mutant compared to the wild type, consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of culture metabolites showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways.
\r\n\r\nWe set out to identify mechanisms allowing P. aeruginosa to catalyze pyocyanin redox cycling. Through a genetic screen, we found two loci required for full pyocyanin-dependent ferric citrate reduction activity in P. aeruginosa PA14: (1) the gene gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) the operon fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA had oxidized cytoplasms and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, mutants lacking a functional cytochrome bc1 complex produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction.
\r\n\r\nPyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form wrinkled (rugose) colonies, while the wild type forms smooth colonies. Using this colony biofilm assay, we showed that the \u0394gpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in stimulating smooth colony formation. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results imply that rugosity is an adaptation to electron acceptor limitation.
\r\n\r\nThe work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa, mechanisms controlling intracellular redox state in bacteria, and mechanisms that may contribute to P. aeruginosa virulence.
\r\n", "doi": "10.7907/N42E-M534", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1444", "collection": "thesis", "collection_id": "1444", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04212009-104756", "primary_object_url": { "basename": "Brenner-CompleteThesis.pdf", "content": "final", "filesize": 1967823, "license": "other", "mime_type": "application/pdf", "url": "/1444/10/Brenner-CompleteThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Synthetic Biofilm-Forming Microbial Consortia", "author": [ { "family_name": "Brenner", "given_name": "Sarah Katherine", "clpid": "Brenner-Sarah-Katherine" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "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": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Most bacteria on earth live in heterogeneous surface-bound congregations called biofilms, and vast reaches of the earth are coated in these living films. In many cases, the microorganisms comprising this ubiquitous coating form complex, interactive communities called consortia. Microbial consortia are implicated in processes of great importance to humans, from environmental remediation and wastewater treatment to assistance in food digestion. Synthetic biologists are honing their ability to program the behavior of individual microbial populations, forcing the microbes to focus on specific applications, such as the production of drugs and fuels. Given that microbial consortia can perform even more complicated tasks and endure more changeable environments than monocultures can, they represent an important new frontier for synthetic biology. This thesis describes two engineered microbial consortia that live and perform their designed functions in biofilms. The biofilm consortium elucidated in Chapter 2 serves as a proof of concept for the development of the symbiotic biofilm consortium of Chapter 3. To provide a context for these two consortia, the first chapter highlights the salient features of microbial consortia that are of interest to synthetic biologists and reviews recent efforts to engineer synthetic microbial consortia, while the final chapter suggests challenges associated with and future directions for engineering microbial consortia.\r\n", "doi": "10.7907/PMZ3-4Z96", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "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:1398", "collection": "thesis", "collection_id": "1398", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04162007-092010", "primary_object_url": { "basename": "JJH_Thesis.pdf", "content": "final", "filesize": 3818751, "license": "other", "mime_type": "application/pdf", "url": "/1398/1/JJH_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Acyl-Homoserine Lactone Quorum Signal Degradation by Soil and Clinical Pseudomonas sp.", "author": [ { "family_name": "Huang", "given_name": "Jean Jing", "clpid": "Huang-Jean-Jing" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "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": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Acyl-homoserine lactones (AHLs) are signaling molecules that are used by several species of Proteobacteria in a process of cell-to-cell communication known as quorum sensing. The production, secretion, and detection of these signaling molecules are used to regulate a variety of microbial group behaviors, such as motility, the production of extracellular enzymes, antibiotics, and virulence factors. This thesis describes the ability for two Pseudomonas sp., a soil - isolate strain PAI-A and a clinical - isolate Pseudomonas aeruginosa strain PAO1, to degrade long chain acyl-homoserine lactone quorum signaling molecules, and explores the implications for this degradation activity. P. aeruginosa is an opportunistic pathogen that engages in quorum sensing with a long and a short chain AHL: 3OC12HSL and C4HSL and regulates the production of its virulence genes in this way. The soil isolate does not accumulate AHLs, and there is no evidence for its engagement in quorum sensing. Both species degrade long chain AHL via an acylase mechanism in which the molecule is cleaved at the amide bond. Two enzymes, PvdQ and QuiP, encoded by the genes PA2385 and PA1032 of P. aeruginosa, were found sufficient for the degradation of long chain AHL, but only the PA1032 gene is necessary for this process. PA1032 is transcribed and its protein product is present during degradation of long chain AHL. Studies of PAO1 lagless, a variant of P. aeruginosa that always degrades long chain AHL, indicate that this strain is broken in the regulation of PA1032. PAO1 lagless was found to express the PA1032 gene throughout planktonic and biofilm growth states, but wild type PAO1 expressed PA1032 locally in the center of biofilm microcolonies. This finding suggests PAO1 may use its ability to degrade one of its two AHLs during this dynamic growth state. Degenerate primers designed from PA1032 of PAO1 enabled the determination of a 2.5 kb putative AHL acylase of the soil isolate. Collectively, these studies of how Pseudomonas soil and clinical isolates degrade AHL suggest the diverse ways in which the degradation of acyl-homoserine lactone molecules may be used.", "doi": "10.7907/fjr6-5f51", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1365", "collection": "thesis", "collection_id": "1365", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04122007-135320", "primary_object_url": { "basename": "01Kopp2007-PhDThesis.pdf", "content": "final", "filesize": 6486656, "license": "other", "mime_type": "application/pdf", "url": "/1365/1/01Kopp2007-PhDThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Identification and Interpretation of Microbial Biogeomagnetism", "author": [ { "family_name": "Kopp", "given_name": "Robert Evans, III", "orcid": "0000-0003-4016-9428", "clpid": "Kopp-Robert-Evans-III" } ], "thesis_advisor": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" } ], "thesis_committee": [ { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" }, { "family_name": "Rossman", "given_name": "George Robert", "orcid": "0000-0002-4571-6884", "clpid": "Rossman-G-R" }, { "family_name": "Grotzinger", "given_name": "John P.", "orcid": "0000-0001-9324-1257", "clpid": "Grotzinger-J-P" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Microbial activity plays a major role in the sedimentary iron cycle. Some microbes gain energy by reducing or oxidizing iron and thus induce changes in the sedimentary iron mineral assemblage. Magnetotactic bacteria engage in controlled, intracellular precipitation of magnetic iron minerals. These biological transformations are frequently a major influence on the magnetic properties of sediments. Understanding the biogeochemical iron cycle therefore facilitates the interpretation of sedimentary paleomagnetism; conversely, magnetic tools provide a non-destructive and rapid way of analyzing the biogeochemical iron cycle in modern and ancient environments.
\r\n\r\nFerromagnetic resonance (FMR) spectroscopy, a form of microwave spectroscopy, provides a rapid means of assessing internal fields generated in magnetic particles by interparticle interactions and particle anisotropy. It can therefore assess particle shape, arrangement, and heterogeneity. Because magnetotactic bacteria typically produce chains of crystals with narrow distributions of size and shape, FMR spectroscopy is well suited as a screening tool for identifying fossil magnetotactic bacteria (magnetofossils).
\r\n\r\nApplication of FMR and other techniques to modern carbonate sediments of the Triple Goose Creek region, Andros Island, Bahamas, reveals the contributions of magnetotactic bacteria, iron metabolizing bacteria, and sulfate reducing bacteria to the magnetization of carbonate sediments. In sediments above mean tide level, magnetofossils dominate sediment magnetism. Although stable remanent magnetization is preserved throughout the sediments, the quantity of biological magnetite diminishes by an order of magnitude in the iron reduction zone. Below this zone, the development of a sulfate reduction interval can lead to the authigenesis of magnetic iron sulfides. Supratidal portions of shallowing-upward parasequences in carbonate rocks therefore likely provide the most accurate record of syndepositional paleomagnetism.
\r\n\r\nAnomalous magnetic properties of clay deposited in the Atlantic Coastal Plain, New Jersey, during the Paleocene/Eocene Thermal Maximum (PETM) led previous authors to speculate that an extraterrestrial impact triggered the PETM. Reexamination of the clay using FMR and transmission electron microscopy reveals instead that the clay hosts abundant magnetofossils. The first identification of ancient biogenic magnetite using FMR indicates that the anomalous magnetic properties of PETM sediments were not produced by an impact, but instead reflect paleoenvironmental changes along the western North Atlantic margin.
", "doi": "10.7907/83R3-VB23", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1044", "collection": "thesis", "collection_id": "1044", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03212007-235559", "primary_object_url": { "basename": "WangThesis.pdf", "content": "final", "filesize": 3801652, "license": "other", "mime_type": "application/pdf", "url": "/1044/1/WangThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies on Environmental Relevance of Quorum Sensing Signal Decay", "author": [ { "family_name": "Wang", "given_name": "Ya-Juan (Megan)", "clpid": "Wang-Ya-Juan-Megan" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hering", "given_name": "Janet G.", "orcid": "0000-0002-0865-1946", "clpid": "Hering-J-G" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show for the first time that acyl-HSLs are biodegraded in soils sampled from diverse US sites. When amended to soil samples at physiologically relevant concentrations, \u00b9\u2074C-labeled acyl-HSLs were mineralized to \u00b9\u2074CO\u2082 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 \u00b5M acyl-HSL, ca. 1000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol . h\u207b\u00b9 . g of fresh weight soil\u207b\u00b9. An MPN of 4.6 \u00d7 105 cells . g of turf soil\u207b\u00b9 degraded physiologically relevant amounts of hexanoyl-[1-\u00b9\u2074C]HSL to \u00b9\u2074CO\u2082. The results implicate a real-world challenge for acyl-HSL-producing bacteria to outpace biological acyl-HSL degradation and thus to successfully engage in cell-cell communications in soils and other environments. Furthermore, high-affinity acyl-HSL degraders were enriched in oligotrophic biofilm reactors that were inoculated with the turf soil. When supplied at physiological concentration, acyl-HSL was observed to be >95% consumed through the reactors. Six bacterial strains were isolated belonging to Variovorax, Mesorhizobium, Bradyrhizobium and Labrys genera. Unambiguous growth of the six isolates on physiological amount of acyl-HSLs under oligotrophic condition was demonstrated. A Variovorax strain SOD31 exhibited acyl-HSL-limiting growth kinetics with a half-saturation constant of 1.7 \u00b5M, which is in high agreement with what we observed from the turf soil. The results suggest that strain SOD31 may be an active contributor to the soil\u2019s activity of degrading acyl-HSLs. This research reinforces the importance of signal decay as intrinsic to bacterial cell-cell communication, as well as providing bases for further studies towards how signal decay may influence community function and structure in naturally occurring microbial communities.", "doi": "10.7907/C00R-DG30", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2343", "collection": "thesis", "collection_id": "2343", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05312007-190155", "primary_object_url": { "basename": "Teal_thesis.pdf", "content": "final", "filesize": 42290423, "license": "other", "mime_type": "application/pdf", "url": "/2343/1/Teal_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies of the Spatial Organization of Metabolism in Shewanella oneidensis and Pseudomonas aeruginosa Biofilms", "author": [ { "family_name": "Teal", "given_name": "Tracy Kristin", "orcid": "0000-0002-9180-9598", "clpid": "Teal-Tracy-Kristin" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Adami", "given_name": "Christoph Carl", "orcid": "0000-0002-2915-9504", "clpid": "Adami-C-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Bacteria grow in the environment as surface-attached microbial communities. These communities are pervasive and resilient in the face of changing and challenging environmental conditions. Because of their community organization and three-dimensional structure, conditions within a biofilm are heterogenous, exposing the bacterial cells to individual microenvironments depending on their location in the biofilm and the biomass of the structure. Communities are therefore thought to be metabolically stratified. To understand how communities are organized with regard to growth activity and metabolic state and what role endogenous compounds might play in this organization, this thesis explores the spatiometabolic organization and dynamics of Shewanella oneidensis biofilms and the roles that acyl-homeserine lactones and phenazines might have in Pseudomonas aeruginosa communities.
\r\n\r\nUsing unstable fluorescent reporters to measure growth activity and protein synthesis and conducting quantitative image analysis, domains of activity were determined for developing S. oneidensis biofilms. Biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of structure size. Within domains of growth-inactive cells, genes upregulated under anaerobic conditions are expressed demonstrating that cells in the nutrient-limited regions of the biofilm are not dead, but are capable of generating enough energy to persist.
\r\n\r\nTo determine if these growth-inactive cells are able to respond dynamically to changes in environmental conditions and what types of nutrients affect growth activity profiles, S. oneidensis biofilms were exposed to increased concentrations of an electron acceptor and an electron donor. Cells in the growth-inactive regions were able to respond to nutrient changes, but were more affected by a change in electron acceptor than electron donor.
\r\n\r\nTo investigate the role of small molecules in biofilm community organization, the degradation of acyl-homoserine lactone (AHL), was studied. This molecule is an important part of the quorum sensing signaling network in P. aeruginosa, where the bacteria both produce and sense this molecule. When bacteria sense a specific concentration of the AHL, they are induced to form a biofilm or initiate a community wide response. To determine what role AHL degradation has on the community response, a mutant that constitutively degrades the compound was characterized and expression profiles for degradation were compared between this strain and wild type communities. Genes for AHL degradation were expressed in the middle of biofilm colonies suggesting that degradation may be an important part of the community response network. It was also shown that AHLs can be used as a substrate for growth, so nutrient-limited cells might also be able to use AHLs to generate energy.
\r\n\r\nFinally, to investigate whether endogenously produced redox-active small molecules could potentially play a role in energy maintenance in communities, the SoxR sensing system was studied. This system is typically thought to regulate the response to superoxide radicals. In P. aeruginosa and other organisms outside the class of enterics, however, recent evidence suggested that they may instead play a role in the sensing of redox-active small molecules produced under conditions of low nutrients and high cell density. To determine the ubiquity of this response mechanism, bioinformatic analyses were conducted to discover SoxR binding sites across all genomes containing SoxR. Predictions for binding sites and the mechanism of regulation, redox-active molecule induction, were confirmed in the Gram-positive bacterium Streptomyces coelicolor.
\r\n\r\nThis work brings us closer to understanding how cells persist and retain the capacity to dynamically regulate their metabolism in biofilm communities. Using reporter assays and quantitative analyses, studies can be done to determine metabolic organization within communities and further investigate the role that endogenous small molecules can play in community organization.
\r\n", "doi": "10.7907/2gdf-yh13", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2205", "collection": "thesis", "collection_id": "2205", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282007-225441", "primary_object_url": { "basename": "keathesis75.pdf", "content": "final", "filesize": 2877981, "license": "other", "mime_type": "application/pdf", "url": "/2205/1/keathesis75.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Fundamental Mechanisms and Biological Applications of DNA-Mediated Charge Transport", "author": [ { "family_name": "Augustyn", "given_name": "Katherine Emily", "clpid": "Augustyn-Katherine-Emily" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Collier", "given_name": "C. Patrick", "orcid": "0000-0002-8198-793X", "clpid": "Collier-C-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Pi-stacked array of heterocylic aromatic DNA base pairs provides an intriguing medium for facilitating the transport of migrating charges. The mechanism of hole transport through this dynamic molecule has been extensively investigated using a wide range of techniques. In particular, our group has taken advantage of the octahedral metal complexes of rhodium (III) and ruthenium (II) to probe charge transport reactions through DNA at long range. These intercalating photooxidants, which are extremely well coupled to the DNA ?-stack, can provide us with mechanistic information through a variety of biochemical and spectroscopic techniques. Here we continue to investigate the mechanism of DNA-mediated charge transport on fast time scales using a variety of hole traps and photooxidants and examine this interesting chemistry in a biological context. DNA-mediated charge transport across three different adenine tracts lengths is monitored using a probe interior to the bridge, N6-cyclopropyladenine, CPA. Upon oxidation, the cyclopropylamine-subsituted deoxynucleoside decomposes rapidly, and the efficiency of decomposition can be used as a kinetically fast measurement of hole occupancy. This trap, incorporated serially across the bridge, can be oxidized by a distally bound photooxidant, [Rh(phi)2(bpy\u2019)]3+ (phi = 9,10-phenanthrenequinone diimmine) without significant attenuation in yield over a distance of 5 nm. These results are consistent with complete delocalization across the DNA bridge. Photooxidation of N2-cyclopropylguanine, CPG, within duplex DNA is used to probe DNA charge transport reactions initiated by the covalently bound photooxidants, [Rh(phi)2(bpy\u2019)]3+ and anthraquinone. Duplexes containing the photooxidant separated from the CPG trap by an increasing number of intervening bases are examined in order to probe DNA charge transport reactions with this kinetically fast hole trap as a function of distance and sequence. Charge transport events through sequences containing various length adenine tracts as well as most mixed sequence bridges do not simply decay exponentially nor geometrically as a function of distance. In particular, for variable-length A-tracts, decomposition decreases in a periodic fashion with increasing distance between the photooxidant and the trap; the period is ~4-5 base pairs. Results obtained from charge injection studies using 2-aminopurine as a fluorescent probe have shown a similar periodic distance dependence. These periodicities are not observed in measurements of oxidative DNA damage using double guanine sites as a slow, irreversible hole trap. Thus, CT through DNA must be probed on multiple time scales to provide mechanistic information. These results are consistent with our model for DNA CT through transient delocalized DNA domains defined by sequence-dependent base pair dynamics. While mechanistic investigations are critical for a fundamental understanding of how charges migrate through DNA, it is important to consider the biological consequences of this process. A biological role for DNA-mediated CT has been investigated in the context of the transcription factor, p53, a tumor suppressor protein involved in myriad cellular pathways such as apoptosis and growth arrest. DNA assemblies containing an anthraquinone photooxidant tethered to the 5\u2019 end of sequences containing p53 binding sites were constructed to examine the binding affinity as a function of photooxidation. We demonstrate that through photoinduced DNA-mediated CT, the p53 protein becomes oxidized and exhibits differential binding for various promoter sequence including Gadd45, p21, and Mdm2. Additionally, insertion of a mismatch intervening between the photooxidant and the p53 binding site serves to attenuate this change in binding affinity associated with photooxidation. MALDI-TOF mass spectrometric analysis of p53 tryptic digests following irradiation of the DNA bound protein provides further evidence that a chemical change occurs, consistent with oxidation of a cysteine residue in the DNA binding domain. Dipyridophenazine complexes of ruthenium (II) have been used extensively to spectroscopically investigate DNA-mediated charge transport. A novel tris heteroleptic dipyridophenazine complex of ruthenium (II), [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentaammine quencher coordinated through a bridging histadine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Capable of undergoing intramolecular photoinduced electron transfer, the steady-state and time-resolved luminescence measurements indicate that the tethered-quencher complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy\u2019]2+ and [Ru(phen)(dppz)(bpy\u2019-his)]2+ in DNA and acetonitrile. Intercalated into guanine containing DNA, [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex as evidenced by EPR detection of guanine radicals. DNA-mediated charge transport is also evidenced using a kinetically fast cyclopropylamine-substituted base as a hole trap that undergoes irreversible oxidative ring opening on the picosecond time scale. Guanine oxidation is not observed in measurements using guanine radical as a slow, irreversible hole trap indicating that back electron transfer reactions are competitive with hole injection into the duplex. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+ in the presence of DNA, competitive with the intermolecular flash-quench process. These results illustrate the remarkable redox chemistry occurring within a bimolecular ruthenium complex intercalated in duplex DNA.", "doi": "10.7907/AYHS-7Q13", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "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:5043", "collection": "thesis", "collection_id": "5043", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12182006-102819", "primary_object_url": { "basename": "Campbell_Thesis_Complete.pdf", "content": "final", "filesize": 5706066, "license": "other", "mime_type": "application/pdf", "url": "/5043/13/Campbell_Thesis_Complete.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Biogeochemical Mechanisms of Arsenic Mobilization in Haiwee Reservoir Sediments", "author": [ { "family_name": "Campbell", "given_name": "Kate Marie", "orcid": "0000-0002-8715-5544", "clpid": "Campbell-Kate-Marie" } ], "thesis_advisor": [ { "family_name": "Hering", "given_name": "Janet G.", "orcid": "0000-0002-0865-1946", "clpid": "Hering-J-G" } ], "thesis_committee": [ { "family_name": "Hering", "given_name": "Janet G.", "orcid": "0000-0002-0865-1946", "clpid": "Hering-J-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Rossman", "given_name": "George Robert", "orcid": "0000-0002-4571-6884", "clpid": "Rossman-G-R" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Naturally-occurring arsenic (As) in the Los Angeles Aqueduct, a major drinking water source for the City of Los Angeles, is removed by precipitating an amorphous iron (Fe) oxyhydroxide floc in the aqueduct waters. The floc is removed via sedimentation at Haiwee Reservoir, where the Fe- and As-rich sediments provide a unique field site for studying the mechanisms of As mobilization to sediment porewater.
\r\n\r\nA gel probe equilibrium sampler was developed to measure the porewater concentrations and As sorption behavior in Haiwee Reservoir sediments. The gels consisted of a polyacrylamide polymer matrix and were 92% water. Undoped gels (clear gels) were used to determine porewater composition, and hydrous ferric oxide (HFO)-doped gels were used to measure in situ As adsorption chemistry. Gels were placed in a plastic holder, covered with a membrane filter, and allowed to equilibrate with the sediment porewaters. This study combined data from the gel probe samplers, gravity cores, and laboratory studies, to elucidate the biogeochemical processes governing As partitioning between the solid and aqueous phases. The gel probe device allowed for in situ observation of the effect of porewater chemistry on As adsorption.
\r\n\r\nArsenic was reduced from As(V) to As(III) in the upper layers of the sediment, but the change in redox state did not cause As to be mobilized into the porewaters. Arsenic mobilization occurred during reductive dissolution of Fe(III) oxides. Arsenate and Fe(III) reduction were probably microbially mediated. Arsenic sorption onto the HFO-doped gels was inhibited at intermediate depths, probably due to dissolved carbonate produced from organic carbon mineralization. The partitioning of As onto the sediment in this region may be primarily controlled by porewater chemistry, rather than sorption site availability. Deeper in the sediment column, the Fe(III) phase was partially transformed to carbonate green rust, possibly sequestering dissolved carbonate. In this region, As adsorption onto HFO-doped gels was controlled by dissolved phosphate. The accumulation of As in the porewater in this region may be due to lack of available surface adsorption sites on the sediment. Arsenic partitioning between solid and aqueous phases depends on microbially driven diagenetic processes, as well as porewater composition.
\r\n", "doi": "10.7907/T3WC-DA42", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:721", "collection": "thesis", "collection_id": "721", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02232007-132917", "primary_object_url": { "basename": "ThesisFormatted12Revise.pdf", "content": "final", "filesize": 5201647, "license": "other", "mime_type": "application/pdf", "url": "/721/1/ThesisFormatted12Revise.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular and Environmental Studies of Bacterial Arsenate Respiration", "author": [ { "family_name": "Malasarn", "given_name": "Davin", "clpid": "Malasarn-Davin" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Arsenate [As(V)]-respiring bacteria that reduce As(V) to arsenite, As(III), for energy production have been implicated as possible catalysts for arsenic mobilization into drinking water supplies. To understand how this metabolism contributes to arsenic geochemistry, this thesis explores the dynamics of As(V)-respiratory gene expression, the impact of As(V) respiration on microbial ferric [Fe(III)] reduction, and biochemical properties of the arsenate respiratory reductase, ARR.
\r\n\r\nUsing sequences for arrA, a gene encoding the terminal reductase involved in As(V) respiration, degenerate PCR primers were designed to amplify a diagnostic region of the gene in multiple As(V)-respiring isolates. These primers were used to track arrA transcription in microcosm studies involving synthetic sediments. arrA was required for As(V) reduction in this context, and the gene was expressed in contaminated sediments at Haiwee Reservoir in Olancha, CA.
\r\n\r\nTo understand the impact of As(V) respiration on Fe(III) reduction, native microbial consortia from Haiwee Reservoir and pure cultures of the genetically tractable Shewanella sp. strain ANA-3 were incubated with As-sorbed hydrous ferric oxide (HFO), and rates of As(V) and Fe(III) reduction were determined. As(V) reduction occurred simultaneously with or prior to Fe(III) reduction, consistent with the idea that electron acceptor utilization is determined by thermodynamic favorability. Furthermore, the presence of sorbed As(III) increased rates of Fe(III) reduction, potentially by increasing HFO surface area.
\r\n\r\nLastly, the expression, assembly, and kinetic properties of ARR from ANA-3 were characterized. ARR is a soluble periplasmic heterodimer that is expressed during early exponential growth and persists into late stationary phase. The enzyme contains molybdenum, Fe, and sulfur cofactors. It has a Km of 5 \u00b5M, a Vmax of 11,111 \u00b5mol As(V) reduced . min-1 . mg protein-1, and reduces only As(V). Mutational analysis of the residues corresponding to the diagnostic region of arrA mentioned above resulted in loss of enzyme activity.
\r\n\r\nThis work brings us closer to being able to quantify and predict the contribution of As(V) respiration to the solubilization of arsenic from sediments. Structural studies, the development of probes to detect ARR, and comparisons of ARR from different bacterial species are now possible.
", "doi": "10.7907/6PAA-PF90", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3201", "collection": "thesis", "collection_id": "3201", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08232005-174620", "primary_object_url": { "basename": "00-Tobias_Whole_Thesis.pdf", "content": "final", "filesize": 5273145, "license": "other", "mime_type": "application/pdf", "url": "/3201/1/00-Tobias_Whole_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Directed Evolution of Biosynthetic Pathways to Carotenoids with Unnatural Carbon Backbones", "author": [ { "family_name": "Tobias", "given_name": "Alexander Vincent", "orcid": "0000-0002-5866-5254", "clpid": "Tobias-Alexander-Vincent" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Over the course of evolution, nature continually discovers new small molecules through the alteration of biosynthetic enzymes and pathways by mutation and gene transfer. Hundreds of these natural products have proven indispensable to medicine, culture, and technology, greatly contributing to increases in the length and quality of human lives. Chemists have found that the \"chemical space\" surrounding natural products is especially rich in functional molecules, and synthesis of natural product analogs has uncovered many with new or improved properties.
\r\n\r\nInspired by nature's search algorithm, we and others have conducted our own evolution of carotenoid biosynthetic pathways in the laboratory. Chapter 1 comprehensively reviews the motivations, accomplishments, and challenges of this research area as of early 2005, and describes in detail how biosynthetic routes to dozens of new carotenoids have been established.
\r\n\r\nTo expand the number of carotenoid backbones beyond the C30 and C40 carbon scaffolds that give rise to the ~700 known natural carotenoids, we subjected a carotenoid synthase, the enzyme responsible for carotenoid backbone synthesis, to directed evolution. Chapter 2 describes the evolution of the C30 carotenoid synthase CrtM from Staphylococcus aureus for the ability to synthesize C40 carotenoids. This work also resulted in novel carotenoids with C35 backbones. We later found that some of the CrtM mutants generated in this laboratory evolution experiment, as well as several second-generation variants, are also capable of synthesizing unnatural C45 and C50 carotenoid backbones when supplied with appropriate prenyl diphosphate precursors.
\r\n\r\nChapter 3 describes the creation of full-fledged pathways to carotenoid pigments based on the C45 and C50 scaffolds. Coexpression of the carotenoid desaturase CrtI from Erwinia uredovora resulted in the biosynthesis of at least 10 new C45 and C50 carotenoids with different systems of conjugated double bonds. We also present evidence of an unnatural asymmetric C40 carotenoid pathway beginning with the condensation of farnesyl diphosphate (FPP, C15PP) and farnesylgeranyl diphosphate (FGPP, C25PP). In addition to clarifying how CrtM and CrtI achieve their product specificities, this work also sheds light on the molecular mechanisms used by evolution to access new chemical diversity and the selective pressures that have shaped natural product biosynthesis.
", "doi": "10.7907/WF0Q-2J98", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "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" }, { "id": "thesis:2471", "collection": "thesis", "collection_id": "2471", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062005-011632", "primary_object_url": { "basename": "LRC_Thesis.pdf", "content": "final", "filesize": 2170851, "license": "other", "mime_type": "application/pdf", "url": "/2471/13/LRC_Thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Fe(II) Oxidation by Anaerobic Phototrophic Bacteria: Molecular Mechanisms and Geological Implications", "author": [ { "family_name": "Croal", "given_name": "Laura Rosemary", "clpid": "Croal-Laura-Rosemary" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Rossman", "given_name": "George Robert", "orcid": "0000-0002-4571-6884", "clpid": "Rossman-G-R" }, { "family_name": "Kirschvink", "given_name": "Joseph L.", "orcid": "0000-0001-9486-6689", "clpid": "Kirschvink-J-L" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In this thesis, the hypothesis that photoautotrophic Fe(II)-oxidizing bacteria catalyzed the deposition of Banded Iron Formations (BIFs), an enigmatic class of ancient sedimentary rocks is explored. Ecophysiological, geochemical, genetic and biochemical approaches are taken to elucidate the molecular mechanism of photoautotrophic Fe(II) oxidation in an effort to identify molecular biosignatures that are unique to this metabolism and capable of being preserved BIFs. In an ecophysiological approach, we show that Fe(II) oxidation by these phototrophs proceeds at appreciable rates in the presence of high concentrations of H2 when CO2 is abundant. These findings substantiate a role for the involvement of these phototrophs in BIF deposition under the presumed geochemical conditions of the Archean. In a geochemical approach, we find that although phylogenetically distinct phototrophs fractionate Fe isotopes in a way that is consistent with Fe isotopic values found in Precambrian BIFs, it is unlikely that this fractionation can be used as a biosignature for this metabolism given its similarity to fractionations produced by abiotic Fe(II) oxidation reactions. In two distinct genetic approaches, we identify genes involved in Fe(II) oxidation in Rhodopseudomonas palustris TIE-1 and Rhodobacter SW2. Genes identified in TIE-1 encode a predicted integral membrane protein that appears to be part of an ABC transport system and a putative CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis. Candidate genes on a cloned fragment of the Rhodobacter SW2 genome that confer Fe(II) oxidation activity to a non-oxidizing strain include those predicted to encode permeases and a protein with potential redox capability. Finally, in a preliminary biochemical approach, c-type cytochromes and other proteins that are exclusive or more highly expressed under Fe(II) growth conditions in TIE-1 and SW2 are identified in SDS-PAGE gels. The work described here furthers our search for a biosignature unique to photoautotrophic Fe(II) oxidation by providing mechanistic information on this metabolism.", "doi": "10.7907/PW00-W724", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2422", "collection": "thesis", "collection_id": "2422", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06032005-113204", "primary_object_url": { "basename": "Meinhold_Thesis.pdf", "content": "final", "filesize": 4426788, "license": "other", "mime_type": "application/pdf", "url": "/2422/2/Meinhold_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Cytochrome P450 BM-3 for Selective Hydroxylation of Alkanes", "author": [ { "family_name": "Meinhold", "given_name": "Peter", "clpid": "Meinhold-Peter" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Labinger", "given_name": "Jay A.", "orcid": "0000-0002-1942-9232", "clpid": "Labinger-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cytochromes P450 are of potential synthetic value because they hydroxylate a large array of substrates, often with high regio- and enantioselectivity. In contrast to most P450s, the BM-3 variant from Bacillus megaterium is soluble, easily expressed in E. coli, and does not require additional electron transfer proteins. A highly efficient enzyme for its preferred reaction, hydroxylation of medium-chain (C12 to C18) fatty acids, BM-3 is a good candidate for engineering for applications requiring activity on other substrates.
\r\n\r\nUsing iterations of random mutagenesis, recombination, and high throughput screening, we engineered P450 BM-3 mutants to hydroxylate linear alkanes as short as propane. Activity towards linear alkanes was further increased by changing two key active site residues. The resulting mutants hydroxylate linear alkanes with high regioselectivity and, notably, enantioselectivity.
\r\n\r\nWe further engineered these enzymes with guidance from the crystal structure of substrate-bound P450 BM-3. Eleven active-site residues were chosen for saturation mutagenesis, and the resulting mutants were screened for improved activity towards alkanes, as measured by total product formation. Substitutions at these positions generally did not affect correct folding of the enzyme, and a large fraction of folded proteins retained similar levels of activity as their predecessor. Moreover, several of the 11 selected amino acid substitutions yielded mutants that were both more active and produced various combinations of product regioisomers.
\r\n\r\nRecombination of these beneficial active-site mutations generated BM-3 variants that catalyze: (a) regio- and enantioselective hydroxylation of linear alkanes; (b) terminal hydroxylation of linear alkanes; (c) regio- and enantioselective hydroxylation of heterocyclic compounds; and (d) ethane hydroxylation.
\r\n\r\nThe selective conversion of ethane to ethanol, not previously reported for any P450, is catalyzed by the most active mutant from this library. In nature, this reaction is solely observed for methane monooxygenases (MMOs) and related enzymes in alkane-assimilating bacteria.
\r\n\r\nAdditionally, we have found that the reductase domain can be engineered to increase the efficiency of these reactions. Our progress in converting BM-3 from a fatty-acid hydroxylase into an enzyme able to selectively hydroxylate smaller alkanes, including ethane, is an important step towards our ultimate goal, achieving selective BM-3 catalyzed conversion of methane to methanol.
", "doi": "10.7907/aats-ca30", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2433", "collection": "thesis", "collection_id": "2433", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06042004-011242", "primary_object_url": { "basename": "01_Title.pdf", "content": "final", "filesize": 50214, "license": "other", "mime_type": "application/pdf", "url": "/2433/1/01_Title.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Mechanisms of Indirect Mineral Reduction by Bacteria", "author": [ { "family_name": "Hern\u00e1ndez", "given_name": "Maria Eugenia", "clpid": "Hern\u00e1ndez-Maria-Eugenia" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "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": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "This thesis concerns the biological process of iron reduction mediated by microbially produced extracellular redox-active organic molecules. Two different iron reducing bacteria were studied: Shewanella oneidensis and Pseudomonas chlororaphis. S. oneidensis can grow by reducing ferric iron [Fe(III)] as a terminal electron acceptor in anaerobic respiration (i.e. dissimilatory iron reduction). Previous studies had suggested that it produces extracellular electron shuttles as a strategy for reducing poorly crystalline iron (hydr)oxides, however this had not been shown. To investigate this, a new method was developed to measure iron reduction at a distance using Fe-coated porous glass beads. Given this assay, it was shown that Fe(III) reduction at a distance is an active process that requires anaerobic conditions and coincides with biofilm formation. The possibility that S. oneidensis excretes a soluble quinone derived from the menaquinone biosynthetic pathway as a mediator was ruled out, but it was shown that such molecules are present in culture fluids and can be used by the cells to make menaquinone. Regardless of the nature of the mediator, it appears to act locally within the biofilm-bead environment for S. oneidensis. P. chlororaphis is a plant root isolate that cannot respire iron but produces redox active secondary metabolites (e.g. phenazine carboxamide, PCN) that promote microbial mineral reduction. P. chlororaphis can reductively dissolve poorly crystalline iron and manganese oxides whereas a mutant in one of the phenazine biosynthetic genes (phzB) cannot. PCN functions as an electron shuttle rather than an iron chelator. Multiple phenazines and the glycopeptidic antibiotic, bleomycin, can also stimulate mineral reduction by S. oneidensis MR-1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and thermodynamic calculations suggest that phenazines have lower redox potentials than poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), it seems likely that natural products like phenazines promote microbial mineral reduction in the environment. Whether cycling of microbially produced extracellular redox-active organic molecules serves a physiological function remains to be determined.", "doi": "10.7907/G5P3-ER69", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2980", "collection": "thesis", "collection_id": "2980", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07252005-142630", "primary_object_url": { "basename": "Ching_WK_2001.pdf", "content": "final", "filesize": 8660436, "license": "other", "mime_type": "application/pdf", "url": "/2980/1/Ching_WK_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Disinfection by Pulsed Power Discharges", "author": [ { "family_name": "Ching", "given_name": "Weng Ki", "clpid": "Ching-Weng-Ki" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Nealson", "given_name": "Kenneth H.", "orcid": "0000-0001-5189-3732", "clpid": "Nealson-K-H" }, { "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" } ], "local_group": [ { "literal": "GALCIT" }, { "literal": "div_eng" } ], "abstract": "We study the disinfection of Escherichia coli in a pulsed power discharge reactor. The pulsed power discharge process is an electrohydraulic phenomenon characterized by a rapid release of electrical energy across a submerged electrode pair.
\r\n\t\r\nThe survival kinetics of pure strains of E. coli suspensions exposed to 5.5 kV, 90 kA electrohydraulic discharges (EHD) is investigated. The probability of survival Pn of a 2 x 10\u2077 E. coli CFU mL\u207b\u00b9 population after 50 consecutive EHD's follows a logit distribution that corresponds to lethal doses of LD\u2085\u2080 = 2.2 and L:D\u2089\u2080 = 10.8 EHDs. Variation of the initial cell concentration produced nearly constant values of LD\u2085\u2080 = 0.9\u00b10.1 in the range 2 x 10\u00b3 \u2264 E. coli/CFU mL\u207b\u00b9 \u2264 2 x 10\u2076. Beyond 10\u2076 CFU mL\u207b\u00b9, the LD\u2085\u2080 values increase exponentially due to nonlinear light absorbance with increasing E. coli concentrations. Qualitatively similar initial cell concentration dependence is observed for survival under low intensity 254 nm irradiation, in contrast with lower values of LD\u2085\u2080 obtained in denser colonies to 20 kHz power ultrasound exposure.
\r\n\r\nThe high intensity (3.3 x 10\u00b9\u2070 W m\u207b\u00b2) ultraviolet radiation emitted by the electrohydraulic discharge is completely suppressed in the presence of less than 100 mg L\u207b\u00b9 2,2'-dihydroxy-4,4'-dimethoxybenzophenone-5,5'-disulfonic acid (BP9), a well-known sunscreen agent. Concentrations of BP9, in the range (0 - 100) mg L\u207b\u00b9, are varied to measure the sterilization kinetics of ~3 x 10\u2077 CFU mL\u207b\u00b9 suspensions to varying degrees of high intensity UV exposure. The slope of the logit plots of E. coli as function of BP9 concentration is consistent with the screening of radiation as the sole lethal agent. Computed values of biologically available light fluences are 5.0 x 10\u00b9\u00b9 photons CFU\u207b\u00b9 for high intensity, high power, pulsed EHD experiments and 6.6 x 10\u2078 photons CFU\u207b\u00b9 for separate low power continuous UV experiments. The net availability of 3 orders of magnitude more photons during high intensity UV exposure suggests the possibility of a multiphotonic disinfection mechanism at play in the EHD process relative to low intensity case.
\r\n\r\nThe overall resistance to long term exposure to EHD is also investigated. Selective pressure experiments with E. coli exposed to 11 cycles of 50 consecutive EHDs each show a weak kinetic change in the dose-response curves reflected in the nearly constant values of LD\u2085\u2080 - 0.24 \u00b1 0.03. A greater than 98% metabolic similarity in carbon source consumption between initial and final E. coli populations are enzymatically related. In addition, the results indicate that no bacterial contaminants are propagated throughout the experiment.
", "doi": "10.7907/RV3M-0849", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" } ]