The exchange of metabolites among members of microbial communities enables the catabolism of complex substrates and supports the growth of auxotrophic microbes. Many microbes depend on other organisms in their environment for the biosynthesis of essential metabolites such as corrinoids. Corrinoids are cobalt-containing tetrapyrroles that function as cofactors for enzymes that facilitate carbon skeleton rearrangements, methyl group transfers, and reductive dehalogenation. Members of all three domains of life use corrinoid cofactors, yet the complete biosynthesis of corrinoids, which requires approximately 30 enzymatic steps, is performed only by a subset of prokaryotes. Bioinformatic analyses show that while 76% of bacterial genomes contain corrinoid-dependent enzymes, only 39% of these contain the complete corrinoid biosynthesis pathway. As such, corrinoid cross-feeding is crucial for functionally integrated microbial communities.

Corrinoids are identified by the structure of their lower axial ligand, which can be a benzimidazole, purine, or phenolic compound. Corrinoids with different lower ligands do not function equivalently as cofactors. Given the diversity of corrinoid structures, microbes that require exogenous corrinoids must have mechanisms to acquire the specific corrinoids that function as cofactors for their corrinoid-dependent enzymes. However, the variety of corrinoids that can serve as cofactors for any one organism, the means by which microbes acquire specific corrinoids, and the diversity of corrinoids present in microbial communities have not been well studied.

In the first chapter, I summarize the strategies employed by corrinoid-dependent bacteria for fulfilling their corrinoid requirements and provide background on corrinoid structure, function, and biosynthesis.

Chapter 2 details my work examining the range of corrinoids that function as cofactors for the organohalide-respiring bacterium Dehalococcoides mccartyi, which has an obligate requirement for exogenously supplied corrinoids. D. mccartyi plays an important role in the bioremediation of chlorinated solvents in the environment, a process that relies on corrinoid-dependent enzymes. Together with my collaborators Shan Yi, Yujie Men, and Lisa Alvarez-Cohen, I showed that D. mccartyi can only use specific corrinoids containing benzimidazole lower ligands, but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base.

While Chapter 2 focuses on corrinoid metabolism in D. mccartyi in pure culture, my experiments presented in Chapter 3 focus on examining corrinoid cross-feeding in microbial communities containing D. mccartyi. Using a liquid chromatography-tandem mass spectroscopy method developed with my collaborators, I identified the specific corrinoids and free lower ligands present in microbial communities containing D. mccartyi, examined the contributions of different phylogenetic groups to the community corrinoid profile, and provided evidence that D. mccartyi engages in corrinoid remodeling in its natural environment. These findings provide novel insights into the roles played by different phylogenetic groups in corrinoid production and corrinoid cross-feeding within microbial communities, and may also have implications for optimizing chlorinated solvent bioremediation.

As a parallel to my work on how corrinoid cross-feeding supports the growth of D. mccartyi in a microbial community, and the strategies that D. mccartyi uses to obtain functional corrinoid cofactors, in Chapter 4, I explore how the cross-feeding of tetrapyrrole precursors allows microbes with an incomplete corrinoid biosynthesis pathway to fulfill their corrinoid requirements. Using a comparative genomics analysis, I identified 39 bacteria that lack genes required for the production of the universal tetrapyrrole precursors including 5-aminolevulinic acid (ALA), the first steps required for corrinoid biosynthesis, despite having otherwise complete corrinoid biosynthesis pathways. I tested the ability of three putative ALA auxotrophs to scavenge ALA from the growth medium and showed that in the presence of ALA, all three were capable of corrinoid production. My work showed that all 39 tetrapyrrole precursor auxotrophs are animal host-associated, raising the question of whether host-produced tetrapyrrole precursors might be scavenged by corrinoid-producing members of the microbiota.

The results of the experiments presented in this work provide insights into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment. Better understanding of corrinoid production, modification, and utilization in microbial communities will aid in the exploration of how nutrient exchange shapes these communities, and the ecological roles played by individual community members.

Cells actively sense and integrate information about their internal and external environments to execute adaptive physiological responses. This makes it possible to survive fluctuating and oftentimes harsh environmental conditions. Controlling the expression of genes is part of this vital cellular process. Indeed, since the birth of molecular biology theory, a multitude of cellular mechanisms have been found to control virtually every aspect of the flow of genetic information from DNA to RNA to functional proteins. The revelation that RNAs are more than just passive messengers between DNA and proteins was a turning point in the field of molecular biology. We now recognize that many types of RNA actively fold into complex three-dimensional structures to carry out important cellular functions with sophistication, precision, and efficiency that rivals proteins. A prime example are riboswitches – structured noncoding RNAs that sense specific small molecule effectors by direct binding and function as cis-regulatory genetic switches. My dissertation research focuses on the forms and functions of the cobalamin riboswitches and how they are impacted by the exceptional chemical diversity of naturally occurring variants of cobalamin known as corrinoids.

I begin the first chapter with a broad overview of bacterial gene regulation with specific emphasis on mechanisms of transcriptional and translational control. This sets the stage for delving into the distinctive elements of riboswitch structure, mechanism, and function. I also describe how various riboswitch classes connect to the cellular processes which they control. Lastly, I describe in detail the cobalamin riboswitch class and the roles that corrinoids play in bacterial physiology.

The second chapter describes the bulk of my endeavors as a graduate student researcher in the Taga Lab examining the corrinoid specificity of cobalamin riboswitches. The large number of bacterial metabolic pathways that involve corrinoids and the numerous types of corrinoid cofactors and intermediates present a puzzle as to how cells can effectively use cobalamin riboswitches to control their corrinoid-related physiology. The approach I took leveraged two strengths of the Taga Lab’s expertise: bacterial molecular genetics and biochemical production of commercially unavailable corrinoid molecules. I engineered an in vivo fluorescence reporter system to measure the responses of several cobalamin riboswitches to several corrinoids. From the patterns of corrinoid selectivity that I observed in my experiments, I developed a mechanistic hypothesis for corrinoid specificity of cobalamin riboswitch-based gene regulation in bacteria. Furthermore, I propose a regulatory strategy that attempts to explain how corrinoid specificities of gene regulation and bacterial physiology are functionally connected.

In the third chapter, I explore the functional versatility of cobalamin riboswitches. As the second most prevalent class of riboswitches, I speculated that novel functional variations should arise from the diversity of cobalamin riboswitch sequences. To increase the likelihood of finding uncommon functional variants, I focused on atypical cobalamin riboswitch regulon architectures among bacterial species that specialize in corrinoid metabolism. This rationale enabled me to successfully identify a novel activator cobalamin riboswitch, dissect modular functionalities of tandemly linked cobalamin riboswitches, and develop new hypotheses about corrinoid-specific physiology.

Together, my research studies constitute a step towards reconciling the apparent oversimplicity of current cobalamin riboswitch models with the intrinsic complexity of the cellular processes they control.

Many bacteria cannot synthesize all the nutrients they require. One type of nutrient that many bacteria must acquire from their environment is cobamides, a class of cofactors including vitamin B12 and related analogs. Less than half of the bacteria that use cobamides produce them, suggesting that cobamides are widely shared in microbial communities. Cobamides are used in a variety of bacterial enzymes for functions including methionine synthesis, deoxyribonucleotide synthesis, carbon utilization, reductive dehalogenation, and others. Cobamides consist of a corrin ring macrocycle with a cobalt center. Attached to the cobalt is the upper ligand, which is part of the reactive center of the cofactor. The lower ligand, which may also be attached to the cobalt, is structurally variable and is generally from one of three classes: benzimidazoles, purines, or phenolics. Most bacteria that use cobamides can only use a subset of all cobamides, referred to as cobamide selectivity.

In Chapter 1, I provide background on cobamide structure, function, and biosynthesis. I then review known processes that contribute to an organism’s observed cobamide selectivity. These mechanisms include strategies for making a cobamide: de novo biosynthesis, cobamide precursor salvaging, and cobamide remodeling. Uptake, regulation, and cobamide-dependent enzyme binding and activity also contribute to cobamide selectivity. Examples of cobamide sharing in a number of systems are also described.

To explore cobamide biosynthesis and dependence patterns in bacteria, I have taken a genomics-centered approach in Chapter 2. I assessed the potential of publically available genomes for use of cobamides and for cobamide biosynthesis based on the presence and absence of genes that have been experimentally validated, creating predictions for the phenotype of an organism. I analyzed 11,000 representative species from a dataset of all bacteria, and observed trends of cobamide biosynthesis and cobamide dependence potential at the phylum level.

To further validate the genomic prediction of cobamide phenotypes, the cobamide requirements of the human pathogen Clostridioides (Clostridium) difficile were interrogated in Chapter 3. This bacterium has seven cobamide dependent pathways encoded in its genome, and was validated to be an ALA salvaging bacterium that produces pseudocobalamin. How cobamides affect the ability of C. difficile to grow and cause disease in vivo remains to be discovered, but the in vitro experiments revealed different cobamide preferences of methionine synthase and its cobamide-dependent ribonucleotide reductase. Its methionine synthase could surprisingly support growth with all nine cobamides and two cobamide precursors tested. C. difficile also exemplifies the strategy of metabolic flexibility, whereby it can use many cobamides and precursors to use cobamide-dependent metabolism, and also has options to bypass these pathways and not use cobamides.

The observation of the patchy distribution of the cobamide biosynthesis pathway across bacterial genomes led to the hypothesis that cobamide biosynthesis capability has been gained and lost multiple times in the domain Bacteria. In Chapter 4, I analyzed the cobamide biosynthesis genes in the order Clostridiales in the phylum Firmicutes by annotating the presence and absence of these genes onto phylogenetic trees. The Clostridiales in particular have many instances of putative gain or loss of some but not all genes in the biosynthesis pathway, possibily leading to the observed ALA-salvaging bacteria observed in the clade.

My work used comparative genomics to generate testable hypotheses about cobamide biosynthesis, cobamide precursor salvaging, and cobamide dependence in bacteria. Characterization of cobamide biosynthesis and dependence in C. difficile validated some of the genomic predictions and filled a knowledge gap in understanding the metabolism of this opportunistic pathogen. My final chapter summarizes some of the major findings of my PhD thesis, and describes a few aspects of cobamide sharing that remain to be investigated.

Microbes exist in complex, multispecies communities where they participate in nutritional interactions that shape microbial community structure, function and stability. Nutritional interactions can range from competition for shared nutrients, to mutualisms, where nutrients are reciprocally exchanged in ways that benefit both partners. Not all microbes are able to produce all of their required nutrients, and as a result, rely on receiving nutrients from neighboring microbes. I am interested in understanding mechanisms of nutrient release and its implications in supporting microbial communities.

In Chapter 1, I provide background on how microbes interact in communities, and discuss examples of different types of microbial interactions. I then review how microbes impact their surroundings by releasing by-products and metabolites into their environment, that can then be used by other microbes as nutrients. Next, I explore cooperative and mutualistic behaviors, and theories for how we think cooperative interactions evolve overtime, before discussing auxotrophy, and how this trait may have evolved via adaptive evolution. I end Chapter 1 by discussing cell death, different forms of cell lysis, including bacteriophage-mediated lysis, and review what is known about how these processes are related to nutrient release and nutrient cycling in microbial communities.

In Chapter 2, I discuss two theories for how I think metabolites can be released into the environment to facilitate interdependent metabolite sharing. Microbial communities are composed of complex networks of metabolically interdependent organisms. But it is unclear how these nutritional networks evolve. In particular, the incentive for releasing metabolites, such as amino acids, vitamins and nucleobases is not obvious. I discuss that nutrient release could be a by-product of processes, like cell lysis and regulated metabolite efflux, that could facilitate the emergence of interdependent metabolite sharing.

I experimentally test these predictions in Chapter 3 by hypothesizing that bacteriophage-mediated lysis is a dominant mechanism of nutrient release that can support amino acid auxotrophs. I use bacterial growth assays to investigate how well supernatants, mechanical cell lysates, and phage-generated lysates are able to support a set of amino acid auxotrophs. I found that supernatants and mechanical lysates minimally support auxotrophs, and phage lysates release a significant amount of bioavailable nutrients, suggesting that in nature, phage are likely to play a large role in providing auxotrophs with their required nutrient.

Chapter 4 explores secretion as a mechanism of nutrient provisioning. More specifically, I test how nutrient overproduction can occur as result of auxotrophic mutations, and the implications of this nutrient overproduction on co-culture growth. To test this I developed an obligate mutualistic synthetic co-culture using two engineered E. coli that reciprocally exchange vitamin B12 and methionine. I show that co-culture growth is limited by methionine secretion, and reveal how specific auxotrophic mutations are able to increase flux through the methionine biosynthesis pathway to improve co-culture growth.

In addition to exploring mechanisms of nutrient provisioning, I also conducted a sociological project about doctoral students in the biological sciences. In Chapter 5, I investigate key elements of socialization that doctoral students in the biological sciences experience as they navigate their graduate programs. From interviews with over 30 doctoral students, I highlight how informal interactions affect students access to scientific help and expertise.

The cobamide class of small molecules includes the essential micronutrient cobalamin (Vitamin B12). Cobamides catalyze a variety of reactions involving one-carbon transfers and radical mediated molecular rearrangements. These reactions are vital parts of biochemical pathways found in both humans (one-carbon metabolism, fatty acid metabolism) and prokaryotes (methanogenesis, acetogenesis, fermentation and bioremediation).

These chemically difficult reactions are made possible by the complex structure of cobamides. Cobamides are tetrapyrroles related to heme, chlorophyll and Factor F430, but are unique in containing a cobalt atom that binds an upper and lower ligand. The upper ligand of cobamide cofactors is made up of a cobalt-carbon covalent bond and varies depending on the metabolic role of the cofactor. The lower ligand can vary widely and is largely responsible for the diversity of cobamides found in nature. Lower ligands can take the form of benzimidazoles (5,6-dimethylbenzimidazole in the case of cobalamin), purines (such as adenine), or phenolics (such as p-cresol).

The first chapter of my thesis will provide more background into the functions and structures of cobalamin and other cobamides. I will focus on the known diversity of cobamides in nature and how that diversity is the result of the metabolisms of cobamide producing organisms.

The next three chapters detail a series of experiments that I have performed to expand our knowledge of how cobamide diversity is maintained. The first set of experiments (Chapter 2) describes how the CobT enzyme, the first enzyme in the lower ligand attachment pathway, is responsible for determining which lower ligands are incorporated into cobamides. I examined this at the organismal and genetic levels and collaborated on a companion project examining these mechanisms at the enzymatic level. I also demonstrated that altering this specificity can have deleterious effects on the host organism. In Chapter 3, building on the previous chapter, I and a collaborator in the laboratory (Amrita Hazra) conducted a series of experiments to show that some CobT enzymes also specify the orientation of the cobamide lower ligand. Organismal, genetic, and biochemical analyses showed that CobT enzymes and cobamide producing bacteria can synthesize pairs of products as structural isomers. These findings add a new potential source of structural variability in cobamides and reveal that CobT specificity extends to the orientation as well as the identity of the lower ligand. Finally, Chapter 4 describes a bioassay I developed to detect benzimidazole lower ligands in the environment and the results of its application to a variety of samples. I found that benzimidazoles are ubiquitous in the environment, a finding that has important implications for cobamide diversity in microbial communities.

Across these experiments I have explored the means by which cobamide diversity is maintained in nature through the actions of CobT and the availability of lower ligand bases. The final chapter summarizes my findings and places them in the context of the field. This section also contains novel hypotheses generated by my results about the significance of cobamide diversity in nature as well as future experiments to test these hypotheses.