Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.

霍乱弧菌（Vibrio cholerae）已知可在营养受限的水生环境中持续存活。鉴于此前多项研究表明，rpoS编码的替代σ因子在多种其他细菌的营养匮乏存活过程中发挥关键作用，为探究该因子是否参与霍乱弧菌的环境适应，研究人员通过野生型基因组文库对大肠杆菌（Escherichia coli）突变株进行功能互补克隆，成功获得霍乱弧菌的rpoS同源基因。对该互补克隆的序列分析显示，其包含一段长1008 bp的开放阅读框，预测可编码一个含336个氨基酸的蛋白质，与已报道的其他rpoS基因编码产物的整体同源性为63%~71%。为明确rpoS在霍乱弧菌中的功能，研究人员通过同源重组技术敲除了rpoS基因。缺失rpoS的霍乱弧菌菌株，在应对多种环境胁迫（包括过氧化氢暴露、高渗透压及碳源饥饿）时的存活能力显著受损。上述结果表明，rpoS可能是霍乱弧菌在水生栖息地持续存活的必需基因。此外，rpoS突变会导致血凝素/蛋白酶（hemagglutinin/protease）的产生与分泌量降低。但通过乳鼠肠道竞争定植实验证实，rpoS并非霍乱弧菌体内存活所必需的基因。