Post-Transcriptional and Post-Translational Control of the Flagellar Regulon Rescues Motility of a Salmonella Enterica Type III Export Flio Mutant

The bacterial flagellum is made using a type III secretion pathway. For Salmonella enterica, this requires 6 transmembrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR. FliO is not absolutely required, because bypass mutations in the fliP gene can improve motility for a fliO mutant. In this study, an extended screen was made for randomly selected mutations that improved motility of cells bearing a ΔfliO null mutation. Using whole genome sequencing two novel mutations were identified, a “silent” mutation localized to the fliA gene, which encoded a U36C substitution in the fliAZ mRNA. A missense mutation was found in the clpP gene, and encoded a V20F substitution in the ClpXP protease. Transcriptional and translational fusions of the lacZ gene to the fliA promoter demonstrated that the silent mutation improved translation of the highly structured fliAZ mRNA. Real-time quantitative RT-PCR revealed that late flagellar gene expression was blocked in a ΔfliO mutant, but the clpP(V20F) or fliA(mRNA U36C) mutations improved expression. Examination of flagellar biogenesis by immunoblotting and transmission electron microscopy found that the fliO mutant synthesized rare flagella, but introducing the fliA(mRNA U36C) mutation together with previously identified mutations in fliP cooperatively restored flagellar biogenesis to near wild-type levels.