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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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" } ]