Starvation-induced mutagenesis in rhsC and ybfD genes extends bacterial tolerance to various stresses by boosting efflux function

Recent evidence showed that bacteria actively maintained a range of physiological functions to enhance survival fitness under adverse growth conditions. In this study, we investigated whether bacteria need to undergo active genetic changes for stress-protection purposes if environmental stress persists. Our results revealed that mutations became detectable at specific sites in several genes in E. coli after encountering starvation conditions for six days. This discovery is groundbreaking since bacteria are not known to undergo site-specific mutagenesis during prolonged starvation when most physiological activities are down-regulated. The genes in which mutations were consistently detected in the tolerant population were ybfD and rhsC within the ybf gene cluster, which are predicted to encode components of a transporter. To assess the impact of these mutations on bacterial survival, mutants with single or double mutations in these genes were generated and tested. The results demonstrated that these mutations caused significant increase in tolerance to antibiotics, heat, and oxidative stresses. Functional analysis indicated that the E. coli BW25113::ybfDrhsC double mutant exhibited elevated efflux activity, and that expression of the rhsC gene was suppressed in the E. coli BW25113:: ybfD mutant, suggesting that mutations in these two genes act synergistically to strengthen the stress tolerance phenotype. Consistently, deletion of the ybfD and rhsC genes resulted in significantly reduced tolerance under prolonged starvation conditions. Understanding the mechanisms of bacterial site-specific mutagenesis that enable bacteria to withstand multiple stresses over extended periods could aid development of innovative antimicrobial strategies.