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. Bacteria were cultured in LB, and the total RNA was extracted from the wild-type Salmonella (WT), spiC mutant strain, or spiC mutant strain carrying pEG9127 (spiC+) when the culture OD600 was 1.6. Levels of flhD mRNA were normalized to the 16S rRNA concentration, and the results are shown relative to the expression in the wild-type strain. In both assays, no significant difference in the expression levels of the flhD gene was observed between the wild-type strain and the spiC mutant. (C) Western blot analysis of FlhD expression. Whole-cell lysates from the wild-type Salmonella (WT), spiC mutant strain, or flhD mutant strain were prepared and were analyzed using Western blot with an anti-FlhD peptide antibody or an anti-DnaK specific antibody. The black arrowhead indicates FlhD protein. Molecular masses are indicated on the left. (D) Densitometric analysis of the amount of FlhD normalized to the amount of DnaK, a bacterial heat shock protein, in the same samples. The spiC mutant showed a reduced expression level in FlhD protein compared to the wild-type strain. *P < 0.001, significantly different from the wild-type strain.