To identify the level at which IpaB and InvE expression was regulated in response to changes in osmolarity, we analyzed the expression of virF. In the absence of salt, virF mRNA was detectable by RT-PCR (Fig. 1B, virF mRNA), although the level of mRNA expression was approximately 29.0 ± 4.6% of the maximum level observed in the presence of 150 mM NaCl. In an attempt to determine GSK1210151A price the mechanism of regulation of virF transcription, we performed a reporter gene assay in which the expression of lacZ

was driven by the virF {Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|buy Anti-diabetic Compound Library|Anti-diabetic Compound Library ic50|Anti-diabetic Compound Library price|Anti-diabetic Compound Library cost|Anti-diabetic Compound Library solubility dmso|Anti-diabetic Compound Library purchase|Anti-diabetic Compound Library manufacturer|Anti-diabetic Compound Library research buy|Anti-diabetic Compound Library order|Anti-diabetic Compound Library mouse|Anti-diabetic Compound Library chemical structure|Anti-diabetic Compound Library mw|Anti-diabetic Compound Library molecular weight|Anti-diabetic Compound Library datasheet|Anti-diabetic Compound Library supplier|Anti-diabetic Compound Library in vitro|Anti-diabetic Compound Library cell line|Anti-diabetic Compound Library concentration|Anti-diabetic Compound Library nmr|Anti-diabetic Compound Library in vivo|Anti-diabetic Compound Library clinical trial|Anti-diabetic Compound Library cell assay|Anti-diabetic Compound Library screening|Anti-diabetic Compound Library high throughput|buy Antidiabetic Compound Library|Antidiabetic Compound Library ic50|Antidiabetic Compound Library price|Antidiabetic Compound Library cost|Antidiabetic Compound Library solubility dmso|Antidiabetic Compound Library purchase|Antidiabetic Compound Library manufacturer|Antidiabetic Compound Library research buy|Antidiabetic Compound Library order|Antidiabetic Compound Library chemical structure|Antidiabetic Compound Library datasheet|Antidiabetic Compound Library supplier|Antidiabetic Compound Library in vitro|Antidiabetic Compound Library cell line|Antidiabetic Compound Library concentration|Antidiabetic Compound Library clinical trial|Antidiabetic Compound Library cell assay|Antidiabetic Compound Library screening|Antidiabetic Compound Library high throughput|Anti-diabetic Compound high throughput screening| promoter [8]. In wild-type S. sonnei carrying the virF-lacZ reporter gene, the level of β-galactosidase activity in the absence of salt was 20.6% of that in the presence of 150 mM NaCl (Fig. 1C, Graph 1), which indicated that the virF promoter is partially active even in the absence of NaCl. We examined VirF-dependent expression of invE by Western blot and RT-PCR. The production of InvE protein was almost completely repressed under conditions of low osmolarity (Fig. 1B, α-InvE),

whereas under the same conditions, there was a significant level of invE mRNA detectable by RT-PCR (Fig. 1B, invE mRNA). Real-time RT-PCR analysis indicated that the amount of invE mRNA in the absence of NaCl was 9.5 ± 1.6% of the level in the presence of 150 mM NaCl. We carried out a reporter gene assay to examine the expression of invE at both the transcriptional and translational levels [13]. In low osmolarity, β-galactosidase activity Diflunisal in wild-type S. sonnei that expressed the transcriptional fusion gene invETx-lacZ was moderately decreased, to 28.9% of that seen in the presence of 150 selleckchem mM NaCl (Fig. 1C, Graph 2). In contrast, β-galactosidase activity in cells that expressed the translational fusion gene invETL-lacZ was 7.3% of the level in the presence of 150 mM NaCl (Fig. 1C, Graph 3). These results indicated

that the expression of InvE protein is repressed in the absence of salt, a condition under which genes for at least two regulatory proteins are still transcribed, albeit at reduced levels. Thus, the repression of InvE synthesis occurs primarily at the post-transcriptional level. Post-transcriptional regulation of invE To examine the mechanism of post-transcriptional regulation of invE expression more directly, we replaced the native invE promoter with a promoter cassette containing the E. coli araC repressor and the araBAD promoter region [14]. In this system, we were able to examine VirF-independent expression of InvE under the control of the AraC-dependent araBAD promoter. Strain MS5512 carrying ΔpinvE::paraBAD [11] was cultured in the presence or absence of 150 mM NaCl, and the synthesis of InvE protein was induced by increasing the concentration of arabinose. Similar levels of invE mRNA were detected in the presence of 0.2 and 1.0 mM arabinose, independently of the presence or absence of NaCl (Fig. 2A, invE mRNA). However, the synthesis of InvE protein was significantly decreased in the absence of NaCl (Fig.