Construction And Characterization Of RpoS Mutant Of Vibrio Alginolyticus

Vibrio alginolyticus was an opportunistic pathogen of human being and marine animals. The bacterium was able to adapt to a variety of environmental stresses and enter into the viable but nonculturable (VBNC) state. The alternate sigma factor RpoS, which was a regulator during the stationary phase, played an important role in surviving under these stressful situations in many bacteria. The aim of this study was to evaluate the roles of RpoS of V. alginolyticus in adapting to environmental stresses and entering into the VBNC state under starvation stresses. The rpoS gene of V. alginolyticus VIB283 was cloned and an rpoS mutant was constructed by homologous recombination. The mutant strain was more sensitive to environmental stresses such as high osmolarity and long-term starvation. Both wild-type and the mutant strains of V. alginolyticus grew well in 2216E broth at 28℃, with a little decrease of the viable plate count in the rpoS mutant (2.5×10~9 CFU/mL ) compared with the wild-type strain (5×10~9 CFU/mL) during the stationary phase. The mutant strain was more sensitive to oxidative stress than the wild-type counterparts. When the wild type and the mutant cells were exposed to hyperosmolarity (2.4 M NaCI), there were no difference between the mutant and the wild type. The viable counts of the wild-type strain maintained to 53.50%, whereas the viable counts of the mutant strain reduced to 1.96% after 12 days of carbon starvation. The mutant V. alginolyticus entered into the VBNC state within 37 days at 4℃, whereas the wild-type strain entered the VBNC state within 69 days under the same conditions. Resuscitation of the rpoS mutant in VBNC state could also be obtained. Disruption of rpoS in V. alginolyticus VIB283 did not obviously affect its virulence to zebra fish. These results suggested that rpoS of V. alginolyticus is important for adaptation to environmental stresses.