The vitamin B 12 family of cofactors known as cobamides are essential for a variety of microbial metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, but only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis and use vary at the phylum level. While 57% of Actinobacteria are predicted to biosynthesize cobamides, only 0.6% of Bacteroidetes have the complete pathway, yet 96% of species in this phylum have cobamide-dependent enzymes. The form of cobamide produced by the bacteria could be predicted for 58% of cobamide-producing species, based on the presence of signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 17% of bacteria have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. Bacteria with a partial cobamide biosynthesis pathway include those in a newly defined, experimentally verified category of bacteria lacking the first step in the biosynthesis pathway. These predictions highlight the importance of cobamide and cobamide precursor salvaging as examples of nutritional dependencies in bacteria.

The stability and function of a microbial community depends on nutritional interactions among community members such as the cross-feeding of essential small molecules synthesized by a subset of the population. In this review, we describe examples of microbe–microbe and microbe–host cofactor cross-feeding, a type of interaction that influences the forms of metabolism carried out within a community. Cofactor cross-feeding can contribute to both the health and nutrition of a host organism, the virulence and persistence of pathogens, and the composition and function of environmental communities. By examining the impact of shared cofactors on microbes from pure culture to natural communities, we stand to gain a better understanding of the interactions that link microbes together, which may ultimately be a key to developing strategies for manipulating microbial communities with human health, agricultural, and environmental implications.

dCorrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism in Dehalococcoides mccartyi strain 195. Dehalococcoides bacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolated D. mccartyi strains require exogenous corrinoids such as vitamin B 12 for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterized D. mccartyi strains has been shown to be capable of synthesizing corrinoids de novo. In this study, we investigate the ability of D. mccartyi strain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification by D. mccartyi provides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.

Cobamides are members of the vitamin B12 family of cofactors that function in a variety of metabolic processes and are synthesized only by prokaryotes. Cobamides produced by different organisms vary in the structure of the lower axial ligand. Here we explore the molecular factors that control specificity in the incorporation of lower ligand bases into cobamides. We find that the cobT gene product, which activates lower ligand bases for attachment, limits the range of lower ligand bases that can be incorporated by bacteria. Furthermore, we demonstrate that the substrate specificity of CobT can be predictably altered by changing two active site residues. These results demonstrate that sequence variations in cobT homologs contribute to cobamide structural diversity. This analysis could open new routes to engineering specific cobamide production and understanding cobamide-dependent processes.

Cobalamin and other corrinoids are essential cofactors for many organisms. The majority of microbes with corrinoid-dependent enzymes do not produce corrinoids de novo, and instead must acquire corrinoids produced by other organisms in their environment. However, the profile of corrinoids produced in corrinoid-dependent microbial communities, as well as the exchange and modification of corrinoids among community members have not been well studied. In this study, we applied a newly developed LC/MS/MS–based corrinoid detection method to examine relationships between corrinoids, their lower ligand bases, and specific microbial groups in microbial communities containing Dehalococcoides mccartyi that has an obligate requirement for benzimidazole-containing corrinoids for trichloroethene-respiration. We found that p-cresolylcobamide ([p-Cre]Cba) and cobalamin were the most abundant corrinoids in the communities. It suggests that members of the family Veillonellaceae are associated with the production of [p-Cre]Cba. The decrease of supernatant-associated [p-Cre]Cba and the increase of biomass-associated cobalamin were correlated with the growth of D. mccartyi by dechlorination. This supports the hypothesis that D. mccartyi is capable of fulfilling its corrinoid requirements in community through corrinoid remodeling, in this case, by importing extracellular [p-Cre]Cba and 5,6-dimethylbenzimidazole (DMB) (the lower ligand of cobalamin), to produce cobalamin as a cofactor for dechlorination. This study also highlights the role of DMB, the lower ligand produced in all of the studied communities, in corrinoid remodeling. These findings provide novel insights on roles played by different phylogenetic groups in corrinoid production and corrinoid exchange within microbial communities. This study may also have implications for optimizing chlorinated solvent bioremediation.

dCorrinoids are essential cofactors of reductive dehalogenases in Dehalococcoides mccartyi, an important bacterium in bioremediation, yet sequenced D. mccartyi strains do not possess the complete pathway for de novo corrinoid biosynthesis. Pelosinus sp. and Desulfovibrio sp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growing D. mccartyi strain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)-and lactateamended medium in cocultures with Desulfovibrio vulgaris Hildenborough (DvH) or Pelosinus fermentans R7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.C hlorinated solvents such as tetra-and trichloroethene (PCE/ TCE) have been among the most common subsurface contaminants in the United States for decades (1, 2). In situ bioremediation is an effective, economical, and environmentally friendly technology for treating chlorinated solvents (2, 3). Dehalococcoides mccartyi is the only known bacterium capable of carrying out respiratory reductive dechlorination of chloroethenes to nontoxic ethene and plays a key role in the bioremediation of contaminated groundwater (4, 5).As cofactors of reductive dehalogenases (RDases), the enzymes responsible for reductive dechlorination, corrinoids (e.g., cobalamin) are essential nutrients for supporting D. mccartyi growth and dechlorination. Genomic analyses of sequenced D. mccartyi strains reveal a lack of complete corrinoid biosynthesis pathways, rendering D. mccartyi incapable of synthesizing corrinoids de novo (6, 7). Exogenous cobalamin (a specific corrinoid also known as vitamin B 12 ) is generally added to grow D. mccartyi in isolation (8). Cobalamin, 5-methylbenzimidazolylcobamide ([5-MeBza]Cba), and 5-methoxyb...

Regulation of iron acquisition genes is critical for microbial survival under both iron-limiting conditions (to acquire essential iron) and iron-replete conditions (to limit iron toxicity). In fungi, iron acquisition genes are repressed under iron-replete conditions by a conserved GATA transcriptional regulator. Here we investigate the role of this transcription factor, Sre1, in the cellular responses of the fungal pathogen Histoplasma capsulatum to iron. We showed that cells in which SRE1 levels were diminished by RNA interference were unable to repress siderophore biosynthesis and utilization genes in the presence of abundant iron and thus produced siderophores even under iron-replete conditions. Mutation of a GATA-containing consensus site found in the promoters of these genes also resulted in inappropriate gene expression under iron-replete conditions. Microarray analysis comparing control and SRE1-depleted strains under conditions of iron limitation or abundance revealed both ironresponsive genes and Sre1-dependent genes, which comprised distinct but overlapping sets. Iron-responsive genes included those encoding putative oxidoreductases, metabolic and mitochondrial enzymes, superoxide dismutase, and nitrosative-stressresponse genes; Sre1-dependent genes were of diverse functions. Genes regulated by iron levels and Sre1 included all of the siderophore biosynthesis genes, a gene involved in reductive iron acquisition, an iron-responsive transcription factor, and two catalases. Based on transcriptional profiling and phenotypic analyses, we conclude that Sre1 plays a critical role in the regulation of both traditional iron-responsive genes and iron-independent pathways such as regulation of cell morphology. These data highlight the evolving realization that the effect of Sre1 orthologs on fungal biology extends beyond the iron regulon.

Vitamin B 12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.