Flexible Cobamide Metabolism in Clostridioides (Clostridium) difficile 630 Δerm

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.