Assembling Flagella in Salmonella Mutant Strains Producing a Type III Export Apparatus without FliO

The type III export apparatus of the Salmonella flagellum consists of six transmembrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) and three soluble proteins (FliH, FliI, and FliJ). Deletion of the fliO gene creates a mutant strain that is poorly motile; however, suppressor mutations in the fliP gene can partially rescue motility. To further understand the mechanism of suppression of a fliO deletion mutation, we isolated new suppressor mutant strains with partially rescued motility. Whole-genome sequence analysis of these strains found a missense mutation that localized to the clpP gene [clpP(V20F)], which encodes the ClpP subunit of the ClpXP protease, and a synonymous mutation that localized to the fliA gene [fliA(+36T→C)], which encodes the flagellar sigma factor, σ28. Combining these suppressor mutations with mutations in the fliP gene additively rescued motility and biosynthesis of the flagella in fliO deletion mutant strains. Motility was also rescued by an flgM deletion mutation or by plasmids carrying either the flhDC or fliA gene. The fliA(+36T→C) mutation increased mRNA translation of a fliA′-lacZ gene fusion, and immunoblot analysis revealed that the mutation increased levels of σ28. Quantitative real-time reverse transcriptase PCR showed that either the clpP(V20F) or fliA(+36T→C) mutation rescued expression of class 3 flagellar and chemotaxis genes; still, the suppressor mutations in the fliP gene had a greater effect on bypassing the loss of fliO function. This suggests that the function of FliO is closely associated with regulation of FliP during assembly of the flagellum.