2A, α-IpaB). Figure 2 A. InvE expression in Δp invE:: p araBAD strain MS5512. ΔpinvE::paraBAD strain MS5512 and wild-type strain MS390 AZD2281 order were grown overnight in LB medium containing chloramphenicol and 50 μM arabinose, washed twice with fresh LB medium, and then inoculated into YENB media containing increasing concentrations of arabinose and cultured at 37°C with or without 150 mM NaCl, as indicated. Strains (ΔpinvE::paraBAD, MS5512; Wt, wild-type strain MS390), concentration of NaCl (0 mM or 150 mM) and concentration of arabinose (0, 0.2, 0.5, 1.0 mM) are indicated above the
panels. Primers and antibodies used in the experiments are indicated on the right side of the panels. B. Stability of InvE protein. ΔinvE strain MS1632 carrying the expression plasmid pBAD-invE was grown in YENB media containing ampicillin and 100 μM arabinose, with or without 150 mM NaCl, at 37°C. When cultures reached an A 600 of 0.8, rifampicin was added. Cells were harvested at 10 min intervals for a period of 40 min. Whole cell cultures (10 μl) were analysed by Western blot using anti-InvE and -IpaB antibodies. To determine whether the low level of InvE protein synthesis under conditions of low NaCl was due to decreased protein stability, we examined the metabolic stability of InvE in an invE deletion mutant strain Adriamycin chemical structure (strain
MS1632) carrying an expression plasmid for InvE (pBAD-invE) [11] at various times after treatment with rifampicin. The levels of InvE and IpaB were slightly lower in the AZD3965 solubility dmso absence Guanylate cyclase 2C of NaCl than in the presence of NaCl. Both
proteins gradually degraded over time after rifampicin treatment, but the rate of degradation was essentially the same in the presence or absence of NaCl (Fig. 2B). By comparison, invE mRNA decayed within 10 minutes (min) after rifampicin treatment, and the rate of decay was much faster in low NaCl than in 150 mM NaCl (see below). These results indicated that InvE protein is metabolically stable once it is synthesized. Involvement of Hfq in the post-transcriptional regulation of InvE synthesis Previously, we showed that the RNA-binding protein Hfq [15, 16] is involved in the temperature-dependent regulation of invE expression, and that this regulation occurs at the post-transcriptional level [11]. We next examined the expression of InvE in an hfq deletion mutant strain of S. sonnei (strain MS4831) under low osmotic conditions. As in the case of temperature-dependent regulation, the level of expression of InvE and IpaB in an hfq mutant strain in the absence of NaCl was approximately 33% of that seen in the presence of 150 mM NaCl (Fig. 3A lane 1), which suggested that Hfq is involved in the osmolarity-dependent post-transcriptional regulation of InvE and IpaB synthesis. Real-time analysis of virF mRNA in the hfq mutant in the absence of NaCl indicated that the level of expression of virF was 36.5 ± 4.