The expression levels of both genes in the spiC Selleck Nutlin 3a mutant were greatly reduced compared to the wild-type strain. The spiC mutant is defective in flagella filament formation Because the flagella filament is made from the flagellin proteins FliC and FljB, we examined flagella of the respective Salmonella strains using electron microscopy. We found differences between

the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain (Fig. 3A), whereas the spiC mutant had few flagella (Fig. 3B). Additionally, the defective flagella filament formation in the spiC mutant was rescued by introducing pEG9127 (Fig. 3C). The data suggest that SpiC affects the formation of flagella filaments by controlling the expression of flagellar genes. We next examined the involvement of other SPI-2-encoded virulence factors in Crenolanib clinical trial flagella assembly. As expected, a mutation in the spiR gene [4], a two-component regulatory gene

involved in the expression of SPI-2-encoded genes, resulted in the defective formation of flagella filaments, similar to the spiC mutant (Fig. 3D); however, the defective phenotype was not seen in PF-02341066 datasheet the ssaV mutant that lacks a putative component of the SPI-2 TTSS (Fig. 3E) [32]. This suggests the specific involvement of SpiC in the assembly of flagella filaments. Further, we examined the effect of SpiC on formation of flagella filaments

using N-minimal medium containing low Mg2+ (pH 5.8) that is effective in inducing SPI-2 gene expression [29]. However, we did not observe flagella even in the wild-type strain (data not shown). Because the absence of SpiC leads to the reduction of class 3 genes expression including the motA gene, which is necessary for motor rotation, we next investigated the motility of the respective Salmonella strains using LB semisolid plates (Fig. 3F). Like the results for flagella formation, the wild-type strain, the ssaV mutant, and the spiC mutant carrying pEG9127 made large swarming rings, whereas the spiC and spiR mutant had weak swarming abilities. And the flhD mutant was non-motile. Figure 3 Transmission electron micrographs and motility assays of wild-type Salmonella and mutant Salmonella strains. A, wild-type Salmonella; B, spiC mutant strain; C, spiC mutant strain carrying almost pEG9127; D, spiR mutant strain; and E, ssaV mutant strain. The spiC mutant had no flagella or only a single flagellum, and the defective formation of flagella filaments in the spiC mutant could be restored to the wild-type phenotype by introducing pEG9127 into the spiC mutant. Bars represent 2 μm. (F) Motility assay of the wild-type Salmonella and mutant Salmonella strains. 1, wild-type Salmonella; 2, spiC mutant strain; 3, spiC mutant strain carrying pEG9127; 4, spiR mutant strain; 5, ssaV mutant strain; and 6, flhD mutant strain.