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

pathogens cause cholera, an acute diarrheal disease resulting in significant morbidity and mortality worldwide. Biofilm formation by enhances its survival in natural ecosystems and facilitates transmission during cholera outbreaks. Critical components of the biofilm matrix are the polysaccharide (VPS) produced by the -1 and -2 gene clusters, and biofilm matrix proteins encoded in the 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 but also in phylogenetically distant Vibrio species. Additionally, -1 clusters tend to co-locate with genes, while -2 clusters are often adjacent to genes in various species, which helps explain the separation of these clusters by the cluster in well-characterized 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. genes are more prevalent, while likely resulted from an ancient duplication event of and is only present in a major clade of species containing counterparts. Notably, a novel loop-less Bap1 variant, identified in two subspecies clades of , was found to be associated with altered biofilm formation and the loss of antibiotic efflux pumps and chemotaxis. Another cluster gene, , involved in biofilm dispersal, was found to share a common ancestor with 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.