| Microbes respond to different stresses with the synthesis or activation of auxiliary sigma factors that direct the transcription of regulons whose gene products mitigate the effects of the stresses.σS, encoded by rpoS, is the master regulator of the general stress response in bacterial species. This factor controls the expression of a large number of genes involved in the stationary phase and the mitigation of a diverse number of stresses. However, its roles in regulating the expression of genes vary in different bacterial species.Pseudomonas stutzeri A1501 is an associative nitrogen-fixing bacterium which has a property of high salt resistance. In order to investigate the response mechanism of RpoS to multiple stresses and to detect the link between RpoS and the nitrogen metabolism factor RpoN, a rpoS disruption mutant was constructed and used to detect physiological and biochemical traits. The interaction between RpoS and its target DNA fragments was also studied by electrophoresis mobility and shift primer extension assays. The major findings from this study were:1. Multiple sequence alignment revealed that RpoS in A1501 had an identity of 83%~91% with the reported sequence of RpoS from other Pseudomonas and 75% with E. coli RpoS. The rpoS mutant had no influence on the growth of A1501 under normal conditions. However, the mutant showed a decreased viability in LB medium with 0.8M NaCl. The sensitivity of the rpoS mutant to high temperature (48℃) and prolonged starvation (ASW 4℃) increased.2. The nitrogenase activity of the rpoS mutant was similar to that of the wild-type A1501 under microaerobic conditions in the free-living state. However, nitrogenase activity decreased by approximately 50% in the rpoS mutant compared to that of the wild type in the presence of 0.2M NaCl. Also, there was no new ethylene generated after 5 hours. The rpoS-deletion might have affected the growth of the strain or rpoS might have an interaction with RpoN.3. After an analysis of the recently published A1501 genome sequence, at least 100σS-dependent genes were identified by the method of bioinformatics and the genome-wide expression profiling data of E.coli. We selected 10 genes and wanted to examine differences in expression at the transcription level after salt shock with 1.0M NaCl. According to the results, the expression of 9 genes was down-regulated in the rpoS mutant, indicating that the regulation of RpoS in A1501 was very similar with that in E.coli.4. The RpoS protein was expressed in Escherichia coli BL21 (DE3) in this study and a two-intein purification system was developed for the affinity purification of RpoS. Fragments of the upstream regions (250bp) of the otsA, osmC and yohF genes were amplified. Electrophoresis mobility shift assay (EMSA) showed that they could all bind to RpoS. Further progressive deletion assay found that the promoter region of otsA recognized by RpoS was located 50bp upstream of the transcription start site. The conserved sequence CTA***T recognized byσS in E.coli was found in this region. In some other genes, this conserved sequence might appear in other regions or even disappear, indicating that Pseudomonas stutzeri A1501 was strain-specific in recognizing target genes. This preliminary study revealed the response mechanism of RpoS to multiple stresses in Pseudomonas stutzeri A1501 and laid a theoretical foundation for the genome-wide analysis of RpoS-dependent gene expression.
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