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.

Microbial communities are essential to fundamental processes on Earth. Underlying the compositions and functions of these communities are nutritional interdependencies among individual species. One class of nutrients, cobamides (the family of enzyme cofactors that includes vitamin B12), is widely used for a variety of microbial metabolic functions, but these structurally diverse cofactors are synthesized by only a subset of bacteria and archaea. Advances at different scales of study—from individual isolates, to synthetic consortia, to complex communities—have led to an improved understanding of cobamide sharing. Here, we discuss how cobamides affect microbes at each of these three scales and how integrating different approaches leads to a more complete understanding of microbial interactions.

Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile. IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

11Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to 12 colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino 13 acid metabolism have been investigated, but its cobamide (vitamin B 12 and related cofactors) 14 metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent 15 metabolisms encoded in its genome in addition to a nearly complete cobamide biosynthesis 16 pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, 17we studied C. difficile 630 ∆erm and mutant derivatives under cobamide-dependent conditions in 18 vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for 19 methionine and deoxyribonucleotide synthesis, and can use alternative metabolites or enzymes, 20 respectively, to bypass these cobamide-dependent processes. C. difficile 630 ∆erm produces the 21 cobamide pseudocobalamin when provided the early precursor 5-aminolevulinc acid or the late 22 metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show 34 that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of 35 cobamides and has alternative functions that can bypass some of its cobamide requirements. 36 Furthermore, C. difficile does not synthesize cobamides de novo, but produces them when given 37 cobamide precursors. Better understanding of C. difficile cobamide metabolism may lead to new 38 strategies to treat and prevent C. difficile-associated disease. 383This work was supported by grants NIH DP2AI117984 and R01GM114535 to M.E.T. 384 385We thank Aimee Shen for her extensive advice and protocols for working with C. difficile, Craig

20 21The vitamin B 12 family of cofactors known as cobamides are essential for a variety of microbial 22 metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent 23 . CC-BY-NC 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/342006 doi: bioRxiv preprint first posted online Jun. 8, 2018; 2 and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in 24 this data set have at least one of 15 cobamide-dependent enzyme families, yet only 37% are 25 predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis varies at 26 the phylum level, with 57% of Actinobacteria, 45% of Proteobacteria, and 30% of Firmicutes, 27and less than 1% of Bacteroidetes containing the complete biosynthetic pathway. Cobamide 28 structure could be predicted for 58% of cobamide-producing species, based on the presence of 29 signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 30 was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/342006 doi: bioRxiv preprint first posted online Jun. 8, 2018; 3 Introduction 41Microorganisms almost universally reside in complex communities where individual 42 members interact with each other through physical and chemical networks. A major type of 43 chemical interaction is nutrient crossfeeding, in which microbes that lack the ability to synthesize 44 particular required nutrients (termed auxotrophs) obtain these nutrients from other organisms in 45 their community (Seth and Taga, 2014). By understanding which organisms require nutrients and 46 which can produce them, we can predict specific metabolic interactions between members of a 47 microbial community (Abreu and Taga, 2016). With the development of next-generation 48 sequencing, the genome sequences of tens of thousands of bacteria from diverse environments 49 are now available, leading to the possibility of predicting community interactions based on the 50 genomes of individual members. However, the power to predict the metabolism of an organism 51 by analyzing its genome remains limited. 52The critical roles of cobamides (the vitamin B 12 family of enzyme cofactors) in the 53 metabolism of humans and diverse microbes has long been appreciated. Only recently, however, 54 has cobamide-dependent metabolism been recognized as a potential mediator of microbial 55 interactions (Degnan et al., 2014b;Seth and Taga, 2014). Cobamides are used in a variety of 56 enzymes in prokaryotes, including those involved in central metabolic processes such as carbon 57 metabolism and the biosynthesis of methionine and deoxynucleotides (Fig. 1). Some of the 58 functions carried out by cobamide-depend...