Regulatory protein that inhibits both synthesis and use of the target protein controls flagellar phase variation in Salmonella enterica

Abstract

Flagellin is a major surface antigen for many bacterial species. The pathogen Salmonella enterica switches between two alternative, antigenic forms of its flagellin filament protein, either type B or C. This switching (flagellar phase variation) is achieved by stochastic inversion of a promoter that produces both type B flagellin (FljB) and an inhibitor (FljA) of type C flagellin formation. When the fljB-fljA operon is expressed, only type B flagella are produced; when the operon is not transcribed, the gene for type C flagellin (fliC) is released from inhibition and forms type C flagella. Long thought to be a transcription repressor, the FljA inhibitor is shown here to block both translation and use of the FliC protein by binding to an mRNA region upstream from the translation start codon. Bypass mutants resistant to this inhibition alter this mRNA region, and some prevent FljA-RNA binding. Other bypass mutations are duplications within the leader mRNA that make FljA essential for FliC assembly. Certain bypass mutations allow FljA to block FliC-dependent motility without blocking production of the FliC protein, per se. Other mutations in the FliC mRNA leader block expression of the unlinked fljB gene. Results suggest that mRNAs for types B and C flagellin compete for occupancy of a site that directs the product toward assembly and that FljA influences this competition. This mechanism may serve to prevent assembly of flagella with a mixture of subunit types, especially during periods of switching from one type to the other.

Fig. 1.

Flagellar phase variation in Salmonella. The reversible, Hin-mediated inversion of a 996-bp segment of the S. typhimurium chromosome results in the inversion of a promoter driving the expression of the fljB flagellin gene and fljA, which encodes an inhibitor of the alternative flagellin gene fliC. The Hin recombinase in conjunction with the Fis protein catalyzes a site-specific recombination reaction between the hixL and hixR recombination sites. (Upper) fljBA is transcribed to produce FljB flagellin, whereas fliC expression is inhibited by the action of FljA. (Lower) The promoter for the fljBA transcript is removed, and only fliC is expressed.

Fig. 2.

The 5′ UTR region of the fliC gene. (A) The RNA sequence of the 5′ UTR region of fliC from the +1 site of transcription through 12 bases after the translation-initiating AUG codon was subjected to the mfold RNA-folding program of Zucker (www.bioinfo.rpi.edu/applications/mfold). Five potential SL structures (SL1–SL5) were identified, corresponding to the two lowest-energy folds. SL3 and SL4 are competing SL structures and are depicted in Upper and Lower, respectively. The translation-initiating AUG codon is boxed. (B) Mutational changes in the fliC 5′ UTR that allow fliC-lacZ expression (Lac⁺) in cells expressing the FljA protein. Mutations in green were isolated in this study, and mutations in the fliC 5′ UTR that allow fliC⁺ expression (motility) in cells expressing the FljA protein (10) are depicted in black. The regions of the two duplications are highlighted in red [DUP-(−15 to −19)] and orange [DUP-(−13 to +15)].

Fig. 3.

Gel-shift assays of purified FljA binding to DNA and RNA. (A) Assay of purified His₆-FljA binding to a DNA fragment containing the fliC promoter, 5′ UTR, and N-terminal coding sequences from −160 to +100 relative to the ATG start codon. (B) Assay of purified His₆-FljA binding to an RNA sequence including the 5′ UTR of fliC and the first 15 bases of the coding sequence. B, bound; U, unbound. (C) Competition assays of purified His₆-FljA binding to the RNA sequence in B with either unlabeled RNA of the same sequence (Left) or tRNA (Right) as the chase. All reactions contained 264 nM His₆-FljA; excess unlabeled RNA was added at 20-, 200-, 2,000-, and 20,000-fold molar excess with respect to the concentration of the added fliC transcript (see Materials and Methods).

Fig. 4.

Filter-binding assays of purified FljA binding to 5′ UTR duplication mutants and single-base mutants. (A) Assay of purified His₆-FljA binding to in vitro-transcribed RNA from the following fliC 5′ UTR mutants: −62 to +15 bp from AUG of fliC for WT and the fliC duplication mutants DUP-(−15 to −19) and DUP-(−13 to +15). Because DUP-(−13 to +15) is a longer transcript, a second WT transcript, WT −62 to +26, was used as a control. (B) Assays of purified His₆-FljA binding to in vitro-transcribed RNA from WT, −24G → C, −4G → U, and Δ(−13U).

Fig. 5.

Toeprint analysis of FljA binding to the fliC mRNA 5′ UTR. Equal amounts of synthetic RNA transcripts and ³²P-labeled oligonucleotide primer were incubated with FljA bound to Ni-NTA–agarose (+FljA; lanes 1, 4, and 7) or Ni-NTA–agarose alone (−FljA; lanes 2, 5, and 8), and the associated RNA was toeprinted. The RNAs analyzed contained WT fliC 5′ UTR (TH437; lanes 1–3), fliC5751[FljA-BP Δ(−13U) from AUG] (TH5802; lanes 4–6), fliC5755[FljA-BP DUP-(−15 to −19) from AUG] (TH5806; lanes 7–9), and fliC6323[DUP-(−13 to +15) relative to AUG] (TH8980; lanes 10–12). A reaction lacking RNA but containing radiolabeled oligonucleotide and FljA bound to Ni-NTA–agarose (−RNA, lane 13) showed that all signals in lanes 1–9 were RNA-specific. Primer-extension analysis of RNA in the presence of Ni-NTA–agarose (R; lanes 3, 6, 9, and 12) was used as a control to identify protein-specific signals. The PCR templates used to produce RNA were sequenced with the oligonucleotide used for primer extension so that toeprint sites could be precisely mapped. The nucleotide complementary to the dideoxynucleotide added to each sequencing reaction is indicated above the corresponding lane (C, T, A, and G) so that the sequence of the template can be directly deduced; the 5′–3′ sequence reads from top to bottom. The positions of an identically placed FljA-dependent signal observed with TH437, TH5806, and TH8930 are indicated with filled blue circles; the corresponding position in the TH5802 experiments is indicated with an open blue circle; FliC start codon positions are indicated with blue lines. The duplicated region in TH8980 includes the start codon (red line) and the FljA toeprint site (red filled circle). Blue arrowheads indicate the primer-extension products corresponding to mRNA 5′ ends; filled and open green arrowheads indicate toeprint sites at SL1 and SL2, respectively.

Fig. 6.

Motility of FljA-bypass mutants in the fliC operator. (A) Nine FljA-bypass mutants were placed in strains containing either Δhin-5717::FRT (fljBA^OFF) or Δhin-5718::FRT (fljBA^ON) and assayed for the ability to swim in motility agar. (B) Effect of the 5-bp duplication DUP-(−15 to −19) in a fliC-lacZ gene fusion on expression of the unlinked fljB flagellin gene.