Anguillarum Rpos Function

The sigma factor RpoS has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. In this study ,we cloned the whole rpoS gene and the flanking rigions from the Fosmid library of Vibrio anguillarum strain M3. Sequence analysis revealed a 999-bp open reading frame which is predicted to have 71% identity to Escherichia coli and 78% identity to Vibrio cholerae. To elucidate the role of RpoS in physiology and pathogenesis of V.anguillarum,we constructed a non-polar rpoS mutant by in-frame deletion technology .A complemented srtain was also constructed by using low copy plasmid pSUP202 into which the rpoS gene of M3 was introduced.The rpoS mutant strain was subjected to various environmental stresses, and the resistance phenotypes of the this mutant strain was investigated. When cultured in TSB, the rpoS mutant strain grew slower in the phase of logarithm than the wild type . In addition, the rpoS mutant in logarithmic phase was more sensitive to1M sucrose or 5M NaCl(osmotic stress), 5%(v/v)ethanol , 42℃heat stress and 15mM H2O2 compared with the wild type strain,there was no difference in stationary phase.When the rpoS mutant strain was complemented with pSUP202 carried the rpoS gene,the mutant restored the phenotypes almost similar to those of the wild type strain.Interestingly, the rpoS mutant strain in stationary phase had no difference with the wild type when exposed to 15mM H2O2.The role of rpoS was also assessed in the motility and the activities of several extracellular proteases. The motility of the rpoS mutant strain on the soft agar was severely diminished.In addition, the mutant strain exhibited a diminished protease activities in extracellular products (ECP). The gelatinase and casinase activies of ECPs of the rpoS mutant strain decrease by 73.8% and 69%, respectively, when compared with to those of the wild type strain. The activity of ECP of the mutant against azocasin was only 35.5% of that of the wild type. The complemented strain restored the proteases activities almost to the wild-type.Infection experiments showed that the rpoS mutant increased at least twenty times in LD50 compared to the wild-type in turbot fish.Overall, these studies suggested that RpoS in Vibrio anguillarum is important for adaptation to environmental changes and maybe have a role in virulence.