High Stability of the Genome of Akkermansia Muciniphila MucT under Long-Term Culturing Conditions

ABSTRACT To advance our understanding of the genomic stability of Akkermansia muciniphila, its type strain MucT was grown for over 1,000 generations at five different culturing conditions, followed by the isolation of single colonies and their subsequent genomic DNA sequencing, physiological, and functional analysis. Notably, not a single mutation was found in the genomes using our sequencing approach of the dozen strains isolated after growth for 1,000 generations in media containing porcine gastric mucin, either grown with or without shaking. Similarly, strains isolated from cultures grown in minimal medium containing high or low N-acetylglucosamine, or low N-acetylglucosamine containing 1% ox bile, exhibited very low mutation rates between 2.1 × 10−10 and 8.6 × 10−11 per nucleotide per generation. From these, a total of five unique strains were obtained that contained one or more nucleotide variations. Subsequent analysis and characterization revealed that two of the five strains included a G duplication in a nonanucleotide homopolymer G region in the gene with the locus tag Amuc_1413, resulting in a frameshift and a subsequent loss of mucin binding capacity. Analysis of published genomes of Akkermansia spp. confirmed the instability of this nonanucleotide G region in this Amuc_1413 gene, predicted to be involved in exopolysaccharide export. These findings provide valuable insight into the stability of the A. muciniphila genome and identify phase variation as a mechanism that can explain some of the earlier reported heterogeneity. We conclude that A. muciniphila MucT has large genomic stability under long-term culturing conditions and identified the Amuc_1413 protein as essential for mucus binding.IMPORTANCEAkkermansia muciniphila MucT has emerged as a next-generation beneficial microbe due to its capacity to improve gut barrier function in mouse models and humans. To assess the potential of A. muciniphila MucT for industrial applications, we studied the genomic stability by cultivating different growth conditions for over 1,000 generations. We found that the genome of A. muciniphila MucT is highly stable when grown on mucin medium and relatively stable when grown in industrial media. Additionally, we characterized the obtained mutants that identified phase variation as a mechanism operating in A. muciniphila, which allowed us to identify the gene with the locus tag Amuc_1413, encoding a protein involved in exopolysaccharide production, to be involved in mucus binding.