Flagella-related gene mutations in Vibrio cholerae during extended cultivation in nutrient-limited media impair cell motility and prolong culturability

Understanding the survival strategies of pandemic cholera pathogens in aquatic environments is important for preventing their dissemination. Here, we report a phenomenon wherein long-term cultivation of Vibrio cholerae under nutrient-limited condition (M9 minimal medium supplemented with 0.2% glucose) causes mutations in flagella-related genes, thus inhibiting motility. Moreover, the motility defect reduced the transition of the viable but nonculturable state of the organisms. Maintenance of proliferative capability allows for the rapid population growth and prolonged survival in environments with nutrient sources. Diverse flagella-related genes, including flrA, flrC, flrD, flhA, flhF, fliD, fliF, fliG, fliH, fliI, fliP, fliQ, flgF, flgL, flgK, motX, and pomA, underwent mutations during the cultivation. However, only one gene (site) was mutated in a single V. cholerae. Longer cultivation for up to 300 days yielded further mutations in metabolism-related genes and the loss of virulence factors (e.g., CTX phage) and large DNA regions (~35 kb). These mutations were detected in most organisms of V. cholerae, accompanying the loss of genomic integrity. Motility-defective variants with mutations in the acetate kinase gene had become predominant in culturable cells after long-term cultivation in independently replicated experiments. These findings shed light on a survival mechanism in which V. cholerae adapts to an environmental niche by accumulating mutations. IMPORTANCE: Vibrio cholerae undergoes a transition to a viable but non-culturable (VNC) state when subjected to various environmental stresses. We showed here that flagellar motility was involved in the development of the VNC state of V. cholerae. In this study, motility-defective isolates with mutations in various flagella-related genes, but not motile isolates, were predominantly obtained under the stress of long-term batch culture. Other genomic regions were highly conserved, suggesting that the mutations were selective. During the stationary phase of long-term culture, V. cholerae isolates with mutations in the acetate kinase and flagella-related genes were predominant. This study suggests that genes involved in specific functions in V. cholerae undergo mutations under certain environmental conditions.