FliT acts as an anti-FlhD2C2 factor in the transcriptional control of the flagellar regulon in Salmonella enterica serovar typhimurium

Abstract

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(2)C(2) heterotetramer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. Class 2 expression is known to be enhanced by a disruption mutation in a flagellar gene, fliT. In this study, we purified FliT protein in a His-tagged form and showed that the protein prevented binding of FlhD(2)C(2) to the class 2 promoter and inhibited FlhD(2)C(2)-dependent transcription. Pull-down and far-Western blotting analyses revealed that the FliT protein was capable of binding to FlhD(2)C(2) and FlhC and not to FlhD alone. We conclude that FliT acts as an anti-FlhD(2)C(2) factor, which binds to FlhD(2)C(2) through interaction with the FlhC subunit and inhibits its binding to the class 2 promoter.

FIG. 1.

Effect of FliT on FlhD₂C₂-dependent transcription in vitro. Plasmid pSIIA100 carrying the fliAZ promoter was transcribed in vitro with E. coli RNA polymerase holoenzyme with or without 57 nM FlhD₂/His-FlhC₂ in the presence of [α-³²P]UTP. His-FliT was added to the reaction mixture at the final concentration shown above each lane. Synthesized RNAs were separated on a 6% polyacrylamide gel containing 6 M urea and detected by autoradiography. nt, nucleotides.

FIG. 2.

Effect of FliT on binding of FlhD₂C₂ to the class 2 promoter. A 433-bp DNA fragment containing the FlhD₂C₂ box for the fliDST promoter was 5′-end labeled with ³²P. Each reaction mixture contained 0.05 nM labeled DNA. After incubation with FlhD₂/His-FlhC₂ and/or FliT at the final concentrations indicated above the lanes, the DNA-protein mixtures were separated on a native 5% polyacrylamide gel and labeled DNA was detected by autoradiography. Positions of free and bound DNAs are indicated by arrows to the left.

FIG. 3.

Pull-down and far-Western blotting analyses of the interaction between FliT and FlhD₂C₂. (A) Detection of FlhD and FlhC proteins in the cell lysate used in the pull-down assay. The FlhD and FlhC proteins were detected by Western blotting with anti-FlhD (α-FlhD) and anti-FlhC (α-FlhC) antibodies, respectively. The following strains were used: lanes 1 and 3, JM109 harboring pTrc99A; lanes 2 and 4, JM109 harboring pTrc99A-hDC. Positions of FlhD and FlhC are indicated by arrows. Positions of molecular mass markers are shown to the left. (B) Interaction between His-FliT and FlhD₂C₂. Cell lysates described above were treated with Ni⁺-NTA agarose containing His-FliT (lanes 2, 3, 5, and 7 to 9) or no protein (lanes 1, 4, and 6). Proteins eluted with 250 mM imidazole were separated by SDS-PAGE and detected by Coomassie brilliant blue staining (lanes 1 to 3) or Western blotting with antibodies against FlhD (lanes 4 and 5), FlhC (lanes 6 and 7), and FliT (lane 8). Several nonspecific bands were seen in lanes 1 and 2. For far-Western blotting analysis, the same blotted membrane was treated with His-FliT, which was then detected with anti-FliT antibody (lane 9). The following cell lysate were subjected to pull-down assay: lanes 1, 2, and 4 to 9, JM109 harboring pTrc99A-hDC; lane 3, no cell lysate.

FIG. 4.

Model for molecular mechanism of FliT-mediated negative regulation of the flagellar class 2 operons. FliT acts as an anti-FlhD₂C₂ factor, which binds to FlhD₂C₂ through interaction with the FlhC subunit and inhibits its binding to class 2 promoters. FliD acts as an anti-anti-FlhD₂C₂ factor, which binds to FliT to prevent its interaction with FlhD₂C₂. After hook-basal body completion, FliD is excreted outside the cell through the hook-basal body complex with the aid of the flagellum-specific type III export apparatus. The horizontal line and broken arrow indicate DNA and mRNA, respectively. Abbreviations used: C, FlhC; D, FlhD; iD, FliD; T, FliT; RPase, σ⁷⁰-RNA polymerase holoenzyme; −10, Pribnow box; HBB, hook-basal body.