Quantitative analysis of fliA (B), flgB (C), or fliF (D) mRNA expression. Bacteria were

cultured in LB, and the total RNA was extracted from the wild-type Salmonella, spiC mutant strain, or spiC mutant strain carrying pEG9127 (spiC +) when the OD600 was 1.6. Quantitative RT-PCR was conducted using a TaqMan probe. Levels of each mRNA were normalized to the 16S rRNA concentration, and the results are shown relative to the expression in the wild-type strain. The expression levels of the fliA, flgB, or fliF gene in the spiC mutant were greatly reduced compared to the wild-type strain. SpiC is required for the post-transcriptional expression of the master regulator, FlhDC The class 1 genes products FlhD and FlhC form a heterotetramer that activates the σ70 promoter SRT2104 nmr in the class 2 genes by interacting with the RNA polymerase α subunit [41, 42]. flhDC check details expression is influenced at the transcription or post-transcription level by a number of global AZD2171 mouse regulatory factors. For example, cyclic AMP-CRP [43–46], H-NS [46, 47], QseBC [48], CsrA [49], and the heat shock-induced chaperones, DnaK, DnaJ, and GrpE [50], function as positive regulators, while negative regulation is mediated by OmpR [51], RcsCDB [52], LrhA [53], and ClpXP [54]. Because SpiC was found to affect

the expression of the class 2 genes including the fliA gene, we examined the involvement of SpiC in the flhDC operon expression using an flhDC-lacZ fusion (Fig. 5A), and measured the level using

quantitative RT-PCR (Fig. 5B). Although the spiC mutant showed a slight reduction in flhD expression compared to the wild-type strain, no significant difference in the flhD expression level was observed between the wild-type strain and the spiC mutant. Reports show that the flhD expression level is reduced approximately 2- to 3-fold by mutation to the regulatory genes affecting the flhD expression at the transcription level [46, 48, 51, 53]. Together with these findings, we concluded that the reduced level of the class 2 gene expression in the spiC mutant is not dependent on flhDC transcription. To investigate whether SpiC participates DOCK10 in flhD expression at the post-transcription level, we performed Western blot analysis with anti-FlhD peptide antibody. Although the detection level of FlhD was low, we found significant differences between the wild-type strain and the spiC mutant (Fig. 5C and 5D). The absence of spiC led to the reduced expression of the FlhD protein, indicating that SpiC is involved in flhD expression at the post-transcription level. Figure 5 Effect of the spiC mutation on flhDC expression. (A) β-galactosidase activity from flhD-lacZ transcriptional fusion expressed by wild-type Salmonella (WT) and the spiC mutant strain grown in LB to an OD600 of 1.6. β-galactosidase activity is expressed in Miller units. WT (pRL124) carries the vector with the promoterless lacZ. (B) Quantitative analysis of flhD mRNA expression.