In hns mutants carrying the virF-lacZ reporter gene , the β-galactosidase activity under low osmotic conditions was 60.6% of that under physiological osmotic conditions (Fig. 7A). In the S. sonnei wild-type strain, it was 20.6% (see Fig. 1C, Graph 1). These results SBI-0206965 purchase indicated that the nucleoid protein H-NS is involved, at least in part, in the osmolarity-dependent regulation of virF expression. The level of H-NS protein and that of the two-component regulator CpxR, which is a critical activator of virF transcription , were similar under both low and physiological osmotic conditions
at 30°C and 37°C (Fig. 7B). Figure 7 A. Reporter assay of virF promoter activity in an hns mutant. An hns deletion mutant of S. sonnei strain MS4841 carrying virFTL-lacZ (striped bars) was grown in YENB media Ferrostatin-1 mw with or without 150 mM NaCl were subjected to the PF-01367338 mouse β-galactosidase assay. For a comparison of activities, the
data from Figure 1C, Graph 1, which was derived from simultaneous assays, is indicated by three solid bars on the left side of the graph. Strain and concentration of NaCl are indicated at the bottom of the graph as follows: Wt, wild-type strain (solid bars); hns, hns deletion mutant (striped bars); YENB medium, 0 (white bars); YENB medium with 150 mM NaCl, 150 mM (gray bars). B. Western blot analysis of H-NS and CpxR expression. An overnight LB culture of MS390 at 30°C was inoculated into fresh media and then the cells were cultured
until they reached mid-log phase (A 600 = 0.8). Media, temperature (YENB at 37°C; LB at 30°C and 37°C) and the concentration of NaCl are indicated on the top of the panel. Antibodies used for detection are indicated on the right side of the panels. A cross-reactive unknown protein detected by the anti-H-NS antiserum was used as a loding control. Discussion Virulence genes in Shigella are expressed in response to increases in temperature and/or osmolarity. Previously, we demonstrated that the temperature-dependent expression of virulence-related over genes is regulated mainly at the post-transcriptional level, and that the RNA chaperone Hfq is involved in the translational control of virulence gene mRNA expression . At that time, however, precise details on the mechanism of osmolarity-dependent regulation of virulence gene expression in Shigella were unavailable. The expression and synthesis of TTSS is controlled by the VirF-InvE regulator cascade. The expression of TTSS is markedly reduced by low osmolarity due to the repression of InvE synthesis. In the current study, several lines of evidence indicated that the repression of InvE occurs mainly at the post-transcriptional level: 1) there were significant, albeit low levels of invE mRNA in cells under low osmotic conditions, whereas InvE protein was barely detectable (Fig.