Further, VX-765 clinical trial SpiC is involved in the AZD6244 chemical structure expression of

the fliC gene at the transcription level [16]. These results suggest the possibility that SpiC participates in flagellar phase variation or the fliC gene expression directly. However, in addition to the FliC protein, we newly identified a FliD flagella protein that was decreased in the spiC mutant using proteomic analysis with liquid chromatography-tandem mass spectrometry (K. Uchiya, unpublished result). Taken together, these results suggest that SpiC contributes to the flagellar system by mechanisms other than phase variation or direct expression of the fliC gene in S. enterica serovar Typhimurium. Flagella expression in S. enterica serovar Typhimurium is controlled in a hierarchical manner. At the top of the hierarchy is the class 1 flhDC operon that is essential for transcription of all of the genes in the flagellar cascade. The class 2 operons contain the genes encoding the hook-basal body-associated proteins, a few regulatory proteins, and a component of the type III export pathway. The class 3 operons contain genes involved in filament formation, flagella rotation and chemotaxis [17, 18]. As described above, proteomic analysis showed that the spiC

mutant had lower expression levels of FliC and FliD proteins, suggesting that SpiC is involved in the expression of the class 3 flagellar genes. Therefore, we first investigated the effect of the spiC mutation on the expression of the class 3 genes. The total RNA was isolated from bacteria grown to an OD600 of 1.6 in LB to induce the expression of the spiC gene (Fig. 1B). CB-839 We analyzed the transcript levels of the fliD and motA genes that encode the flagella cap and motor torque proteins [17], respectively, using quantitative real-time PCR (RT-PCR). The transcript levels of the fliD and motA genes in the spiC mutant

were reduced by approximately 15-fold and 6-fold compared to the wild-type strain, respectively (Fig. 2). Complementation of the spiC mutant with a plasmid carrying the wild-type Cyclin-dependent kinase 3 spiC gene (pEG9127) restored the fliD and motA transcripts to about 80% of the level of the wild-type strain. Further, to confirm the contribution of SpiC in the regulation of class 3 flagellar gene transcription, we constructed newly a deletion mutant of the spiC gene using the lambda Red mutagenesis technique and examined the motA mRNA level. The deletion mutant showed the same phenotype as the spiC mutant (EG10128) used in this study (data not shown). These data indicate that SpiC has an influence on the flagellar system. Figure 2 Expression of the class 3 fliD and motA genes in the spiC mutant. Bacteria were cultured in LB to an OD600 of 1.6, and the total RNA was extracted from the wild-type Salmonella (WT), spiC mutant strain, or spiC mutant strain carrying the spiC gene-containing plasmid pEG9127 (spiC +). Quantitative RT-PCR was conducted using a TaqMan probe.