Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains

Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (Aml to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B-12) biosynthesis within the Amll and AmIll phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B-12 biosynthesis genes via PCR. Two Amll strains were positive for the presence of cobalamin biosynthesis genes, while all 9 Aml strains tested were negative. To demonstrate vitamin B-12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the Aml and Amll phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the Amll strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B-12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the Aml strains when vitamin B-12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B-12 production by the Amll phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome. IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila. However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B-12. The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.