Expressing, Mutation Of RpoS Gene And Characterization Of RpoS Mutanted Vibrio Anguillarum

V. anguillarum is a halophilic, gram negative comma-shaped rod bacterium, which causes vibriosis, a lethal hemorrhagic septicemia affecting a variety of fish species and other aquatic animals, causing important economic losses throughout the world. Several virulence-related factors have been identified in V. anguillarum, including an iron uptake system, extracellular metalloprotease, haemolysin, lipopolysaccharide and cytotoxin. However, the mechanisms of the pathogenicity are not completely understood and the causes of disease occurrence are still elusive in V. anguillarum.Bacteria encounter various stresses, including high temperatures, periods of nutrient starvation, osmotic changes and oxidative stress in its natural environment. There are many regulators involved in this complex process.Bacteria develope strategies to survive in harsh environments. The alternate sigma factor RpoS, which is a regulator during the stationary phase, plays an important role in surviving under these stressful situations in some gram-negative bacteria. The rpoS of Escherichia coli is involved in survival under conditions of famine, oxidative stress, osmotic shock, and low pH. About 200 genes of E. coli are regulated by the RpoS regulon under cellular starvation.The rpoS was cloned by PCR amplification from the chromosomal DNA of V. anguillarum W-1 .The nucleotide sequence was determined and the ORF of the gene consists of 1002 bp , which encoded a polypeptide of 333 amino acids. The similarity of the nucleotide sequence with that of other V. anguillarum strain was 100 %. The encoded protein showed 93 %, 86 %, 85 %, 85 %, 82 %, and 73 % of identity to the corresponding amino acid sequence of rpoS in V. parahaemolyticus (ZP01991880), V. cholerae (NP230185), V. harveyi (ZP01988417), V. alginolyticus (ZP01262007), V. vulnificus (NP760484) and E. coli (YP542094) respectively. The gene was subcloned to pET-24d (+) and expressed in E. coli JM109. The molecular weight of the expressed protein was about 43 kDa when detected by SDS-PAGE after purified by Ni2+-affinity chromatography.A 486-bp fragment of rpoS gene was subcloned into the corresponding sites of the suicide vector pNQ705. The resulting suicide plasmid was transferred into E. coli SY327 and then to E. coli S17-1. An rpoS mutant was constructed by integration of the suicide plasmid containing the 486 bp fragment of rpoS gene into the chromosomal rpoS gene of V. anguillarum W-1 by homologous recombination.The response of the rpoS mutant to environmental stresses such as hydrogen peroxide, high temperature, UV irradiation and high osmotic condition was examined by determining the viable plate counts after they were exposed to various stresses. The rpoS mutant was more sensitive to oxidative stress than the wild-type counterparts, and the survival of the rpoS mutant was determined to be 10-fold less than that of the wild type when they were incubated in nutrient-free ASW containing 5 mM of H2O2 for 60 min (p < 0.05). The sensitivity of the rpoS mutant to heat shock was also increased, when the stationary pHase cells were incubation at 45°C for 45 min, the survival of the rpoS mutant was approximately 33-fold less than that of the wide type (p < 0.05). The resistance of the rpoS mutant to UV-irradiation for 20 s was estimated to be almost 10-fold less than that of the wild-type counterparts (p < 0.05). The sensitivity of the rpoS mutant to osmotic stress (2.4 M NaCl) did not change obviously (p > 0.05). The response to prolonged starvation at low temperature did not change obviously, after a period of 40 day starvation at 4°C in ASW, the direct viable counts of the rpoS mutant declined from 5.73×106 to 2.20×104 cells mL-1, whereas the wild strain reduced from 5.00×107 to 5.30×104 cells mL-1 (p > 0.05)The production of extracellular enzymes of the rpoS mutant was studied. The catalase activity of the rpoS mutant decreased to 32.14% compared with that of the wild-type strain (p < 0.01). The protease activity of the mutant cells decreased to 75.15% when determines with azocasein as the substrate (p < 0.05). Clearing zones are observed on the agar plates for phospholipase, lipase, diastase, caseinase and hemolytic activity when the rpoS mutant strain and wild-type strain were spotted on 2216E agar plates containing various substrates, but the enzyme activities of the rpoS mutant were decreased to 35.71 % (p < 0.01), 40.00 % (p < 0.05), 66.70 % (p < 0.05), 38.03 % (p < 0.05) and 16.00 % (p < 0.05) when compared with those of the wild-type strain respectively.Virulence of the rpoS mutant strain was also decreased when they were intraperitoneally inoculated into zebra fish, the LD50 of the wild-type and the mutant were 8.66×104 CFU per fish and 2.55×106 CFU per fish respectively.These results indicates that the RpoS of V. anguillarum playes important roles in bacterial adaptation to environmental stresses and its pathogenicity.