OBJECTIVETo study the role of RpoE and RpoS on the influence of the metabolism and growth of bacterial under hyperosmotic stress.

METHODSThe rpoS/rpoE double deletion mutant of Salmonella enterica serovar typhi (S. typhi) was prepared by homologous recombination through the suicide plasmid mediated. The recombination was visualized by PCR. Growth curves were drawn by using photometric value A600 as the ordinate and cultivation time as abscissa. The survival abilities of bacterial were compared under hyperosmotic stress. Statistical differences of early logarithmic growth stage (4 h) and laters logarithmic growth stage (12 h) were analyzed by one-way ANOVA. The expression difference of metabolism related genes of wild-type and mutant strains of S. Typhi incubated under hyperosmotic stress were investigated by Salmonella genomic DNA microarray. Real-time quantitative PCR (qRT-PCR) was performed to validate the results of microarray assay in some selected genes.

RESULTSThe rpoS/rpoE double deletion mutant of S. Typhi was successfully generated. The analysis of growth curve showed that the 4-hour and 12-hour A600 values were separately 0.503 ± 0.018 and 2.060 ± 0.112 in rpoS deletion mutant strains, 0.293 ± 0.053 and 1.933 ± 0.115 in rpoE deletion mutant strains, and 0.051 ± 0.007 and 0.963 ± 0.111 in rpoS/rpoE double deletion mutant strains; all of which were lower than the values of wild-type strains, who were 0.725 ± 0.097 and 2.496 ± 0.171, respectively. The difference were statistically significant (P < 0.05). The genomic DNA microarray revealed that 42 genes relevant with bacterial metabolism were influenced by RpoE and RpoS. Results of qRT-PCR showed that the expression values of rpsE, rbsK, nusG and etuB in rpoS deletion mutant strains were (1.86 ± 0.14)×10(6), (1.37 ± 0.11)×10(6), (2.72 ± 0.58)×10(6) and (8.27 ± 1.01)×10(6) copies/µg, respectively; while those in rpoE deletion mutant strains were (2.19 ± 0.17)×10(6), (1.51 ± 0.12)×10(6), (2.73 ± 0.57)×10(6) and (9.63 ± 1.42)×10(6) copies/µg, respectively. Compared with the values in wild-type strains, which were separately (1.94 ± 0.10)×10(6), (1.52 ± 0.11)×10(6), (2.39 ± 0.52)×10(6) and (10.83 ± 1.52)×10(6) copies/µg, the differences was not statistical significance (P > 0.05). However, compared with the values in rpoS/rpoE double mutant strains, which were separately (5.64 ± 0.59)×10(6), (4.17 ± 0.40)×10(6), (9.44 ± 1.22)×10(6) and (2.95 ± 0.88)×10(6) copies/µg, the difference was significant (P < 0.05).

CONCLUSIONRpoE and RpoS could influence the expression of lots of metabolism genes. Together, they regulated the metabolism and growth of S. Typhi under hyperosmotic stress.