Comprehensive Genomic and Evolutionary Analysis of Biofilm Matrix Clusters and Proteins in the Vibrio Genus

Vibrio cholerae pathogens cause cholera, an acute diarrheal disease resulting in significant morbidity and mortality worldwide. Biofilm formation by V. cholerae enhances its survival in natural ecosystems and facilitates transmission during cholera outbreaks. Critical components of the biofilm matrix are the Vibrio polysaccharide (VPS) produced by the vps-1 and vps-2 gene clusters, and biofilm matrix proteins encoded in the rbm cluster. However, the biofilm matrix clusters and associated matrix proteins in other Vibrio species remain under investigated, and their evolutionary patterns are largely unknown. In this study, we systematically annotated the biofilm matrix clusters across 6,121 Vibrio genomes, revealing their distribution, diversity, and evolution. We found that biofilm matrix clusters not only exist in V. cholerae but also in phylogenetically distant Vibrio species. Additionally, vps-1 clusters tend to co-locate with rbmABC genes, while vps-2 clusters are often adjacent to rbmDEF genes in various Vibrio species, which helps explain the separation of these clusters by the rbm cluster in well-characterized V. cholerae strains. Evolutionary analysis of RbmC and Bap1 reveals that these two major biofilm matrix proteins are sequentially and structurally related and have undergone domain/modular alterations during their evolution. RbmC genes are more prevalent, while bap1 likely resulted from an ancient duplication event of rbmC and is only present in a major clade of species containing rbmC counterparts. Notably, a novel loop-less Bap1 variant, identified in two subspecies clades of V. cholerae, was found to be associated with altered biofilm formation and the loss of antibiotic efflux pumps and chemotaxis. Another rbm cluster gene, rbmB, involved in biofilm dispersal, was found to share a common ancestor with Vibrio prophage pectin lyase-like tail proteins, indicating its functional and evolutionary linkages to Vibriophage proteins. In summary, our findings establish a foundational understanding of the proteins and gene clusters that contribute to Vibrio biofilm formation from an evolutionary perspective across a broad taxonomic scale. This knowledge paves the way for future strategies aimed at engineering and controlling biofilms through genetic modification.