UC Berkeley

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.