The osmotic pressure of YENB medium without and with 150 mM NaCl was 96 ± 3 and 397 ± 3 mOsm/kg• H2O, respectively. When
150 mM NaCl was replaced with 155 mM KCl, the osmotic pressure was 391 ± 2 mOsm/kg• H2O, whereas when NaCl was replaced with 260 mM sorbitol, osmotic pressure was 384 ± 1 mOsm/kg• H2O. To monitor the expression of TTSS, we measured the expression of the effector protein IpaB and the regulatory molecule InvE. The expression of IpaB and InvE was tightly repressed in low osmotic conditions, whereas in the presence of either 150 mM NaCl or 155 mM KCl, the level of both proteins increased to a similar HKI-272 clinical trial extent (Fig. 1A). A linear relationship was observed between salt concentration and the levels of InvE and IpaB (data not shown), which indicated that there is no threshold for the effective induction of TTSS synthesis. In the presence of 260 mM sorbitol, the levels of both InvE and IpaB were approximately 50% lower than in the presence of NaCl and Selleckchem IWP-2 KCl (Fig. 1A). When the concentration of sorbitol was increased to 520 mM, InvE and IpaB levels increased to the level of the NaCl and KCl growth conditions. These results indicated that in addition to salt concentration, osmolarity regulates the expression of TTSS, although the optimum concentration for maximum induction differed among osmolytes (see discussion). Figure 1 A. InvE
and IpaB expression in different C59 nmr osmotic conditions. An overnight culture of strain MS390 at 30°C was inoculated into fresh YENB medium with or without osmolytes and then incubated at 37°C until mid-log phase (A 600 = 0.8). Medium, osmolyte, and concentration are indicated at the top of the panel. Antibodies used for detection are indicated on the right of the panels. A cross-reactive unknown protein detected by the anti-InvE antiserum was used as a loading control for InvE Staurosporine Western blot analysis throughout this study. B. Expression of > invE and virF
mRNA and InvE and IpaB protein expression in S. Sonnei. Total RNA (100 ng) and 10 μl of the indicate culture were subjected to analysis of mRNA and protein levels, respectively. The 6S RNA ssrS gene was used as control for RT-PCR. Primers and antibodies are indicated on the right side of the panels. Concentration of NaCl in the medium is indicated at top of the panel. C. Expression of invE and virF >promoter-driven reporter genes. Wild-type S. sonnei strain MS390 carrying the indicated reporter plasmids were subjected to a β-galactosidase assay: Graph 1, virFTL-lacZ translational fusion plasmid pHW848; Graph 2, invETx-lacZ transcriptional fusion plasmid pJM4320; Graph 3, invETL-lacZ translational fusion plasmid pJM4321. Concentration of NaCl is indicated at the bottom of the graphs. Details of the control experiments, indicated by black bars (NC)are described in methods.