Comparative Genomics Guides Elucidation of Vitamin B ₁₂ Biosynthesis in Novel Human-Associated <i>Akkermansia</i> Strains

Abstract

<i>Akkermansia muciniphila</i> 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 <i>Akkermansia</i> 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 (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B₁₂) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 <i>Akkermansia</i> strains were isolated from adults and screened for vitamin B₁₂ biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B₁₂ biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B₁₂ biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B₁₂ was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B₁₂ production by the AmII phylogroup. This novel physiological trait of human-associated <i>Akkermansia</i> strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.<b>IMPORTANCE</b> There is significant interest in the therapeutic and probiotic potential of the common gut bacterium <i>Akkermansia muciniphila</i> 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 <i>Akkermansia</i> strains that were able to synthesize vitamin B₁₂ The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated <i>Akkermansia</i> strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.