INTRODUCTION

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Salmonella typhimurium can propel itself in a liquid environment by the rotation of 6 to 12 individual flagellar structures that are located peritrichously on the cell surface. The signal transduction pathway of the chemosensory system allows for the biased movement of an individual bacterium across a chemical gradient, a process called chemotaxis (reviewed in references 2 and 43). The flagella can be signaled to rotate in a clockwise manner, which causes the bacterium to tumble and change direction, or if this signal is suppressed, they will rotate in a counterclockwise manner, which propels the bacterium in a forward direction. The flagella are biased toward counterclockwise rotation when approaching an attractant or moving away from a repellent and are biased toward clockwise rotation when moving away from an attractant or toward a repellent.

The flagellum is generally broken down into three main structural components: (i) the basal body, (ii) the hook, and (iii) the long external filament (for recent reviews of flagellar structure and assembly, see references 1 and 33). Several stages of flagellar assembly are diagrammed in Fig. 1. The basal body traverses from the cytoplasm to the outside of the cell. Assembly begins with formation of the C- and MS-rings at the cytoplasmic base of the basal body. The C-ring extends into the cytoplasm and includes a type III secretion machinery and proteins that control the direction of flagellar rotation in response to the chemotactic signal transduction system. The C-ring is attached to the MS-ring, which is embedded within the cytoplasmic membrane. The next structure to be assembled is the rod that extends from the MS-ring to the lipopolysaccharide (LPS) layer. This is followed by P- and L-ring assembly in the peptidoglycan and LPS layers, respectively, which must occur before an external hook structure can polymerize. Assembly of extracytoplasmic components of the hook-basal body (HBB) requires the type III secretion system, whereas P- and L-ring subunits are exported out of the cytoplasm via the signal peptide-dependent general secretory pathway. Initiation of hook assembly can occur prior to P- and L-ring assembly, but elongation is blocked by the peptidoglycan and LPS layers (21). The hook is thought to act as a universal joint between the rotating part of the basal body and the long external filament. Hook completion is determined by the fliK gene product, which somehow signals the type III export apparatus to stop the export of hook proteins and initiate export of the flagellin subunits and associated proteins (13, 25, 47). The flgK and flgL genes encode the hook-associated proteins, which form small subunit rings at the hook-filament junction (17). The fliD gene encodes a flagellar cap protein located at the tip of the flagellum (18). Flagellin is encoded by alternatively expressed fliC and fliB genes encoding antigenically distinct flagellin subunits (44). Flagellin subunits polymerize first between the cap and the FlgL ring and then between the cap and the tip of the growing filament.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Morphological pathway of flagellar assembly. MS-ring assembly in the inner membrane (IM) is followed by assembly of the switch complex and the flagellum-specific type III export apparatus. The rod components require the type III system to be exported across the inner membrane and into the periplasmic space, where they are assembled into a structure that penetrates the peptidoglycan layer (PG). The P- and L-rings (peptidoglycan and lipopolysaccharide, respectively) are assembled independently of hook initiation; however, hook elongation through the outer membrane (OM) requires the assembled P- and L-rings. After ring assembly, the hook is completed and the biosynthesis of the HBB intermediate structure is finished. At this point the class 3 flagellar proteins, including FlgM, are secreted. The hook-associated proteins and Cap are added to the end of the hook, followed by flagellin polymerization initially between the hook-associated proteins and Cap and then between the Cap and the elongating filament.

Regulation of the 50-plus genes in the flagellar and chemotaxis regulon occurs in coordination with flagellar assembly. The expression of these genes is organized into a regulatory hierarchy of three major classes: class 1, class 2, and class 3 (22). Class 1 genes, flhD and flhC, are cotranscribed and represent the top of the flagellar transcriptional hierarchy. They are transcribed from a ⁷⁰-dependent promoter, which is affected by a large number of global regulatory signals (28, 30, 31). The FlhD and FlhC proteins are required for expression of the rest of the genes in the regulon. These proteins form a heteromultimeric transcriptional activator complex which directs ⁷⁰-dependent transcription of promoters for class 2 genes (30). Class 2 genes encode proteins required for the structure and assembly of the HBB intermediate flagellar structure and the FlgM and ²⁸ regulatory proteins. Class 3 genes include ²⁸-dependent promoters and encode proteins required late in flagellar assembly, including the flgK, flgL, and fliD genes, and the filament genes fliC and fliB. In addition, genes required for chemotactic signal transduction are also class 3 genes.

This initial simplicity was complicated by the finding that multiple promoters transcribe most of the flagellar genes. The ²⁸ protein is an alternative transcription factor that interacts with RNA polymerase to direct transcription specifically from class 3 promoters but will also transcribe class 2 flagellar genes (23, 37). Several of the class 2 gene promoter regions have been shown to be transcribed by either ²⁸ holoenzyme or ⁷⁰ holoenzyme directed by the FlhDC complex (32). In vivo, all class 2 operons were shown to be dependent on either the flhDC operon or the ²⁸ structural gene, fliA, for expression (23). These results suggest that initiation of flagellar biosynthesis is completely dependent on FlhDC but that continued HBB gene expression results from FlhDC-dependent and/or ²⁸-dependent class 2 transcription. The class 3 structural genes are of two types; the flgK, flgL, and fliD genes are transcribed from both a class 2 promoter and a ²⁸-dependent class 3 promoter (26). The remaining class 3 structural genes are exclusively transcribed by ²⁸-dependent promoters (23).

The class 3 genes were originally defined by their dependence on a functional HBB structure for their expression (22). This ability to couple class 3 gene expression to HBB completion is accomplished by the action of the FlgM protein on ²⁸-dependent transcription (8, 9). FlgM is an anti-²⁸ factor (38). When the HBB structure is incomplete or defective, FlgM interacts directly with ²⁸ to inhibit its activity as a transcription factor. FlgM can sense that the HBB structure is complete and export competent for external class 3 flagellar proteins by itself being a class 3 exported protein (16, 24). When the HBB structure is defective, FlgM inhibits ²⁸-dependent transcription and is not found in the external growth medium. In a strain that makes the HBB structure, FlgM is found in the external growth medium and ²⁸-dependent transcription occurs.

Recently, a novel flagellar regulatory gene, flk, was described as a gene whose product can sense the completion of the flagellar P- and L-rings of the basal body, which occurs just prior to hook assembly (20). The flgA and flgI genes are required for P-ring assembly, where the flgI gene encodes the structural component, and the flgH gene encodes the structural component for the L-ring (33). Like all genes involved in HBB assembly, mutations in any of the flgA, flgH, and flgI genes result in FlgM-dependent inhibition of ²⁸ activity (8). Unlike the other HBB genes, however, the negative regulatory effect of mutations in the flgA, flgH, or flgI gene can be suppressed by recessive insertion mutations in either flgM or flk (20). Insertions in flgM, but not those in flk, will suppress the negative regulatory effect caused by the loss of the remaining HBB genes (8, 20). In this paper, we provide evidence that the flk gene plays a role in the translational control of flgM gene expression.

	MATERIALS AND METHODS

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Strains. The bacterial strains used in this study and their origins are listed in Table 1.

Media and standard genetic manipulations. Media, growth conditions, transductional methods, and motility assays were performed as described previously (7, 8).

Plasmid constructions. Plasmids p322KS and p322SK were constructed as described previously (20). The pGEM3Z vector was obtained from Promega Corp (Madison, Wis.). pJK282 served as a template for RNase T2 probes and was made as follows. A BspDI-HindIII 3,455-bp fragment from pMH71 (14) containing the flgAMN operon as well as the flgB and flgC genes was ligated into BspDI-HindIII-cut p322KS. pTX592 contained a 1.3-kbp insert obtained by PCR amplification with pJK286 (20) and primers 5'-GCCGGATCCTTCCCGTCCACGCA and 5'-TTAAAGCTTCGACGCGTTCCATATTAA. This insert contained the first 182 bp of the flgB coding region, the entire flgA gene, and the first 198 bp of the flgM coding region. The amplified PCR product was digested with BamHI and HindIII at sites that were engineered in the primers and were ligated to BamHI- and HindIII-cut pGEM3Z to give plasmid pTX592. Plasmids pTX593 and pTX594 were obtained by digesting the 1.3-kbp PCR products with BamHI and AvaI and HindIII and AvaI, respectively, and ligating the 777- and 511-bp fragments to the pGEM3Z vector cut with the same enzymes. pTX593 contained the first 182 bp of the flgB coding region, the intercistronic region of flgB and flgA, and the first 439 bp of the flgA coding region. pTX594 contained the last 221 bp of the flgA coding region, the intercistronic region of flgA and flgM, and the first 218 bp of the flgM coding region. pTX595 contained a 704-bp BamHI-to-HpaI fragment from pMS531 (42) cloned into the pGEM3Z vector. It contained 122 bp of the region upstream from fliA and the first 582 bp of the fliA coding region.

Tandem chromosomal duplications of flgM-lac operon fusions. Duplications used to measure transcription separately from the flgM class 2 and class 3 promoters were constructed as described previously (15). Separately, a P22 lysate, grown on either the flgA5211::MudA or the flgM5207::MudA strain, was mixed with an equal titer of P22 lysate grown on the purB1879::MudA strain. The mixed lysates were used to transduce strain LT2 to MudA-encoded ampicillin resistance. Because of the large size of the MudA transposon (38 kbp) relative to the size of DNA packaged by P22 (42 kbp), inheritance of MudA by homologous recombination requires that two transduced fragments, each carrying part of the Mud transposon, enter a recipient cell to inherit the Mud insertion. Inheritance of MudA by homologous recombination involves three recombinational exchanges; one exchange joins the donor MudA fragments and two exchanges occur between the composite fragment and the recipient chromosome (15). Mixed lysates from either TH2575 (flgA::MudA) or TH2877 (flgM::MudA) and TT10213 (purB::MudA) were used to transduce LT2 to MudA-encoded ampicillin resistance. Duplication recombinants were distinguished from the parental recombinant types by the fact that they maintained wild-type copies of the purB and flgA genes and when cultures were grown in the absence of ampicillin, such that selection for MudA at the duplication join point was removed, they gave off Ap^s Lac (white in the presence of X-Gal [5-bromo-4-chloro-3-indolyl--D-galactopyranoside]) segregants. Loss of MudA is indicative of recombination between the duplicated homologies when selection for the MudA insertion at the join point is removed (15). Duplications were constructed in flgA and flgG to -L genetic backgrounds by transduction, selecting for MudA-encoded ampicillin resistance and screening for nonmotile transductants that kept the flg allele(s) of the recipient strain.

Tandem chromosomal duplications of flgM-lacZ gene fusions. Duplications used to measure translation of the flgM5208-lacZ gene fusion separately from either the flgM class 2 or class 3 promoters were constructed by first transducing strains TH3590, TH3591, TH3592, TH3593, TH3594, and TH3595 with a P22 transducing lysate grown on strain TH3907 (flgA5210::Tn10dTc flgM5223 flgM5208::MudK) selecting for Tc^r. Recombination between the lac operon regions and the chromosomal regions clockwise to the site of the flgA5210::Tn10dTc insertion in both the donor and recipient DNA segments yielded Tc^r, Ap^r, and Km^s transductants in which the flgA5211::MudA duplication join point was converted to an flgM5208::MudB join point to yield strains TH4023, TH4024, TH4025, TH4026, TH4027, and TH4028, respectively. All of these constructs carry the polar flgA5210::Tn10dTc near the join point and the flgM5223 (L66S) allele in the FlgM-LacZ fusions. This means that the FlgM-LacZ fusion containing the flgM5223 (L66S) allele is transcribed and translated only from the class 3 flgMN promoter. Ampicillin was added to the growth medium to select for the duplication. When grown in the absence of ampicillin, the duplication strains segregated, yielding Ap^s, Lac, and Tc^s segregants, as expected.

-Galactosidase assays. -Galactosidase assays were performed in triplicate on mid-log-phase cells as previously described (34). -Galactosidase activities are expressed as nanomoles per minute per optical density unit at 650 nm.

Quantitative immunoblots. Intracellular levels of FlgM protein were determined by quantitative immunoblotting (3) with the following modifications. Cell lysates were prepared from mid-log-phase cells. Cells were pelleted from 3 ml of culture, resuspended in 50 µl of 1× sample buffer (29), and heated for 3 min. Protein concentrations from dilutions of the prepared lysates were determined with a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, Calif.). A total of 50 µg of each cell lysate was separated on 16.5% Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (40) and electrotransferred to Westran polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, N.H.) in CAPS (3-[cyclohexylamino]-1-propane sulfonic acid) buffer (36). The blots were probed with anti-FlgM rabbit polyclonal antibodies and were immunostained by an alkaline phosphate-based reaction as described previously (16). Densitometric measurements of stained bands were accomplished by scanning the blots on a Agfa Arcus II Flatbed scanner by using Adobe Photoshop support software and then quantifying the image intensities with ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.). Standard protein concentration curves with purified FlgM protein were performed and showed the linearity intensity of immunostaining versus the protein concentration to be between 1 and 25 ng.

Pulse-chase experiments. Bacterial cells were grown in 1× E minimal medium supplemented with 0.2% glycerol and amino acid pools 6 to 11 without methionine (7) at 37°C to 40 Klett units. The cells were labeled for 1 min with [³⁵S]methionine-cysteine protein labeling mix (New England Nuclear, Boston, Mass.) at a final concentration of 50 µCi/ml. Cold methionine was added at a final concentration of 0.5%, and duplicate 1-ml samples were taken at various time points. Trichloroacetic acid (TCA) was added to a final concentration of 5%, and the mixture was incubated on ice for 20 min. The TCA precipitate was centrifuged in an Eppendorf microcentrifuge at 16,000 × g for 5 min at 4°C and was then washed two times in 80% cold acetone and dried. The pellet was resuspended in 50 µl of 1× SDS-sample buffer (29) and boiled for 3 min. Equal counts per minute from the time course samples were added to 900 µl of radioimmunoprecipitation buffer (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) (12) as well as a radiolabeled lysate from TH3746. Lysate from TH3746 has an N-terminal flgM fusion protein with glutathione S-transferase (39 kDa versus 10 kDa for native FlgM protein) and served as an internal control for the immunoprecipitation reaction. Anti-FlgM antibody (16) was added, and the mixture was incubated on ice for 1 h. A total of 50 µl of IgGSORB prepared as per the manufacturer's instructions (The Enzyme Center, Malden, Mass.) was added, and the mixture was incubated on ice for 30 min. The immunoprecipitated material was washed three times with cold RIPA buffer and then resuspended in 25 µl of 1× SDS-sample buffer. The samples were electrophoresed on 10% Tricine SDS-PAGE gels (40), and the gels were fixed in 10% methanol-10% acetic acid and dried. The gels were analyzed with a PhosphorImager (Molecular Dynamics), and the band intensities were quantified (phosphorimager units [PU]) by using ImageQuant software (Molecular Dynamics). Relative levels of FlgM were calculated as (PU of FlgM band/PU of GST-FlgM band) × 100.

FlgM export assay. Bacterial cells were grown in 25 ml of 1XE minimal medium supplemented with 0.4% glycerol and amino acid pools 6 to 11 (7) to 100 Klett units. The cells were pelleted at 17,000 × g for 30 min at 4°C, and the supernatant was saved and filtered through a 0.45-µm-pore-size cellulose acetate filter to remove any remaining bacterial cells. A 10-ml sample of filtered supernatant was filtered onto a prewetted BA85 0.2-µm-pore-size nitrocellulose filter (Schleicher & Schuell). The proteins were eluted by the addition of 50 µl of 1× SDS-sample buffer (29) and were heated at 65°C for 30 min. Samples were then analyzed by immunoblotting using anti-FlgM antibodies as described above.

RNA isolation and RNase T2 assays of chromosomal transcripts. RNA was purified from cells grown exponentially (50 to 65 Klett units at 660 nm) at 37°C by adding portions of bacterial cultures directly to lysis solutions without intervening steps as described previously (46). RNase T2 protection assays of transcripts from the bacterial chromosomes were completed as described previously (46). RNA probes P1 to P4 covering parts of the flgB, flgA, and flgM regions are shown in Fig. 6 and were synthesized by using the following phage RNA polymerase and linearized plasmid templates: probe P1, SP6 and pTX592 with EcoRI; probe P2, SP6 and pTX594 with EcoRI; probe P3, T7 and pTX592 with HindIII; and probe P4, SP6 and pTX593 with EcoRI. An RNA probe for the detection of fliA transcript was synthesized by SP6 polymerase and pTX595 linearized with BamHI. A series of labeled RNA molecules of known lengths were used as size standards to determine the lengths of the protected probes (46). Each hybridization reaction mixture contained 40 µg of total RNA or tRNA as a negative control. The opposite strand of probe P2 and the fliA probe were also synthesized and used to test for DNA contamination or antisense transcription. No bands were detected with these probes. The radioactivity in the bands on the gels was measured with the InstantImager (Packard, Meriden, Conn.).

Determination of mRNA stability. The stability of the flgAMN and flgMN mRNA was determined by previously described methods (11, 45). Rifampin was added to exponentially grown cells to a final concentration of 500 mg/ml, and the RNA was purified at different time points following addition of rifampin. Transcripts expressed from the flgAMN and flgMN promoters were analyzed as described above except that the radioactivity in the bands was measured with a PhosphorImager (Molecular Dynamics). The flgAMN and flgMN mRNA half-lives were calculated as described previously (45).

	RESULTS

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Mutations in the HBB structure result in an increase in the intracellular levels of FlgM, and loss of Flk restores wild-type levels of FlgM in ring mutant strains. Mutations in genes required for HBB formation result in the loss of class 3 gene expression (8). Because the negative regulator FlgM is believed to be exported in response to completion of the HBB structure, it was expected that a defective structure would lead to increased levels of FlgM protein in the cell. This model predicts that FlgM protein might accumulate in strains defective in HBB formation and result in the inhibition of ²⁸-dependent class 3 gene expression. In order to test if a defective HBB structure leads to an increase in the intracellular levels of FlgM, quantitative immunoblot assays were used to measure the intracellular levels of FlgM in a variety of flagellar mutant strains.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Effects of HBB mutations and Flk on intracellular levels of FlgM protein. A quantitative immunoblot was performed with anti-FlgM antibody on cell extracts prepared from exponentially growing cells of isogenic wild-type and flagellar mutant strains. WT, TH2592; flk, TH3282; flgB, TH2157; flgB flk, TH3303; flgA, TH3441; flgA flk, TH3451; flgH, TH3442; flgH flk, TH3452; flgI, TH3444; flgI flk, TH3454.

FlgM turnover is unaffected by mutations in flk. The reduction in the intracellular levels of FlgM protein by loss of Flk in the ring mutant strains would account for the increase in class 3 gene transcription. There are several mechanisms by which the loss of Flk could lead to reduced FlgM levels. Flk could affect FlgM stability, FlgM export, transcription of the flgM gene, flgM mRNA translation, flgM mRNA stability, FlgM anti-²⁸ activity, or some other mechanism that affects FlgM protein levels.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Turnover of intracellular FlgM protein as measured by pulse-chase experiments with [35S]Met-Cys on exponentially growing cells of isogenic strains. (A) Intracellular turnover of labeled FlgM in a wild-type (wt) strain (half-life = 7.3 min) (TH2592) and an isogenic flk mutant strain (half-life = 11 min) (TH3282). (B) Intracellular turnover of labeled FlgM protein in isogenic flgB (rod-defective, TH2157) and flgB flk (TH3303) mutant strains. (C) Intracellular turnover of labeled FlgM protein in isogenic flgHI (ring-defective, TH3445) and flgHI flk (TH3455) mutant strains.

FlgM export is unaffected by mutations in flk. The possibility that the reduction of intracellular FlgM levels by inactivation of flk in ring mutant strains was due to export of FlgM was assayed by measuring levels of exported FlgM in the external medium in a variety of flagellar mutant backgrounds. The results presented in Fig. 4 show that FlgM is present in the spent growth medium of a wild-type flagellar strain but not in that of isogenic ring mutant strains. Introduction of an insertion mutation in flk had no effect on FlgM export. In a wild-type Fla⁺ strain, FlgM is present in the external growth medium with or without a mutation in flk. In ring mutant strains no FlgM was detected in the spent growth medium even in the absence of a functional flk gene. These results do not support a model in which loss of Flk allows FlgM export in ring mutant strains.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Effect of Flk on the secretion of FlgM into the external growth medium. Immunoblot analysis was performed with anti-FlgM antibody on protein found in the spent growth medium of exponentially growing isogenic strains. WT, TH2592; flgA, TH3441; flgH, TH3442; flgI [left], TH3443; flgI [right], TH3444; flgHI, TH3445; WT flk, TH3282; flgA flk, TH3451; flgH flk, TH3452; flgI flk [left], TH3453; flgI flk [right], TH3454; flgHI flk, TH3455.

Effects of mutations in flk on expression of lac operon fusions to the flgM class 2 and class 3 promoters in wild-type and flagellar mutant strains. To measure the effect of Flk on expression from the flgAMN class 2 promoter and the flgMN class 3 promoter in response to flagellar structural mutations, tandem chromosomal duplications were constructed between MudA insertions in the purB and either the flgA or flgM gene by a previously described method (15). DNA sequence analysis revealed that the flgA5211::MudA and the flgM5207::MudA insertions used in these assays resulted from Mud transposition into the flgA gene just after codon 180 and into the flgM gene at codon 86 (CTC leu, 5'CT-Mud; however, insertion recreates a Leu codon at the fusion join point [10]). The duplication between MudA insertions in flgA and purB resulted in the lac operon being expressed from the class 2 promoter of the flgAMN operon (Fig. 5A). The duplication between MudA insertions in the flgM and purB genes resulted in the lac operon being expressed from the class 3 promoter of the flgMN operon (Fig. 5B). In this purB-flgM duplication, transcription from the upstream class 2 promoter is prevented by a polar flgA::Tn10dTc insertion mutation characterized previously (10).

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Effects of Flk on flgM gene expression from both the class 2 flgA promoter and class 3 flgM promoter and the effect of Flk on expression of the fliA gene. (A) Expression of an flgA-lac operon fusion from the class 2 flgA promoter in isogenic wild-type and flagellar mutant strains. WT, TH3590, a duplication strain carrying an flgA-lac fusion and an intact flgAMN operon; WT flk, TH3593 (TH3590 flk-5206::Tn10dCm), the identical strain defective in flk; flgG-L flk+, TH3592 (same as TH3590 except both duplicated regions carry the flgG-L2157 allele), a deletion of basal-body genes; flgG-L flk, TH3595 (TH3592 flk-5206::Tn10dCm); flgA flk+, TH3591 (same as TH3590 except the flgAMN operon carries a nonpolar internal deletion of the flgA gene); flgA flk, TH3594 (TH3591 flk-5206::Tn10dCm). (B) Expression of an flgM-lac operon fusion from the class 3 flgM promoter in isogenic wild-type and flagellar mutant strains. For the results from all the strains presented in this panel, expression of the intact flgMN operon from the flgA promoter is prevented by the introduction of a polar upstream flgA::Tn10dTc insertion. WT, TH3596, a duplication strain carrying an flgM-lac fusion and an intact flgMN operon; WT flk, TH3599 (TH3596 flk-5206::Tn10dCm), the identical strain defective in flk; flgG-L flk+, TH3598 (same as TH3596 except both duplicated regions carry the flgG-L2157 allele), a deletion of basal-body genes; flgG-L flk, TH3601 (TH3598 flk-5206::Tn10dCm); flgA flk+, TH3597 (same as TH3590 except the flgAMN operon carries a nonpolar internal deletion of the flgA gene); flgA flk, TH3600 (TH3597 flk-5206::Tn10dCm). (C) Expression of the lac operon from an isolated fliA promoter which includes DNA from 588 to +19 relative to the transcriptional start site from the fliA gene in the isogenic wild-type, the flagellar rod (flgB), and the different flagellar ring (flgA, flgH, and flgI) mutant strains with and without a mutation in the flk locus. WT, TH3558; WT flk, TH3565; flgB flk+, TH3559; flgB flk, TH3566; flgA flk+, TH3560; flgA flk, TH3567; flgH flk+, TH3561; flgH flk, TH3568; flgI flk+, TH3562; flgI flk, TH3569; flgHI flk+, TH3564; flgHI flk, TH3571.

Effect of mutations in flk on expression of lac operon fusions to the fliA promoter region in wild-type and flagellar mutant strains. We examined the effect of Flk on expression of the fliA promoter in a rod mutant and the different ring mutant strains (Fig. 5C). It was possible that the increased expression from class 3 promoters in ring mutant strains by introduction of an flk null allele could be due to a reduction of FlgM levels or an increase in FliA (²⁸) levels by increasing transcription from the fliA promoter. Introduction of the fliA gene on a multicopy plasmid overcomes FlgM inhibition of class 3 gene expression (16). A defective bacteriophage P22 vector carrying the lac operon transcribed from the fliA promoter region from 588 to +19 relative to the translational start site was constructed (5a). This phage was lysogenized into a variety of flagellar mutant strains with and without a functional flk gene, and the effect of Flk on fliA transcription was determined. Expression of lac from the fliA promoter was unaffected by a mutation in the rod structural gene, flgB, or any of the genes needed for P- or L-ring formation. Introduction of an insertion in the flk gene had no effect on expression of the fliA promoter in any of the strains tested (Fig. 5C). Thus, the increased expression from class 3 promoters in ring mutant strains by introduction of a flk null allele does not coincide with a reduction in transcription of the flgM gene or an increase in transcription of the fliA gene.

Effects of Flk on flgM mRNA synthesis. The effects of the flk mutation on class 2 and class 3 transcription of flgM were also examined at the level of mRNA synthesis by using RNase T2 protection assays. Levels of mRNA transcripts from the class 2 and class 3 flgM promoters were measured directly in wild-type, rod (flgB), and ring (flgA) mutant strains with and without a functional flk⁺ gene (Table 2; Fig. 6). The ring mutant used in these experiments was the flgA allele (flgA1529) previously shown to have no effect on transcription or translation of the downstream flgM gene (10). Protected transcripts were detected with RNA probes P1 to P4 shown in Fig. 6A. As controls, the levels of class 2 transcripts from the flgB to -L operon and the fliAZY operon (probe not shown) were also measured.

View this table: [in this window] [in a new window]   TABLE 2.   Effects of flk mutation on the amounts of transcripts from class 2 and class 3 promoters in parent, flgB2164, and flgA1529 strainsa

View larger version (43K): [in this window] [in a new window]   FIG. 6.   RNase T2 assay showing the effects of flk mutation on the amounts of transcripts in the flgB, flgA and flgM regions. (A) Structure and transcription of the flgB, flgA, and flgM regions in S. typhimurium. The figure, which is drawn to scale, is based on sequences in the GenBank database (accession no. D00498 for flgA and flgB and accession no. M74222 for flgM). The start sites of the promoters PflgB and PflgA were based on consensus sequences for promoters in class 2 flagellar operons (22). The start site of PflgM was mapped previously (9). Also indicated are the RNA probes P1 to P4 used in the RNase T2 protection assay. (B) Protected transcripts detected with RNA probes P1 to P4. Total RNA isolated from exponentially growing cells was hybridized with RNA probes P1 to P4 as described in Materials and Methods. The bands indicated correspond to transcription initiation from PflgA and PflgM, the flgA-flgM cotranscript, and transcripts in the flgB region. Lane 1, RNA probe P1; lane 2, protected transcripts obtained from hybridization of probe P1 with total RNA from TH3505 (wild type [wt]); lane 3, RNA probe P2; lanes 4 to 9, protected transcripts obtained from hybridization of probe P2 with total RNA obtained from TH3505 (wt), TH3506 (flk), TH3504 (flgB2164), TH3507 (flgB2164 flk), TH3508 (flgA1529), and TH3515 (flgA1529 flk), respectively; lanes 10 and 11, molecular weight standards [STD]; lane 12, RNA probe P3; lane 13, protected transcripts obtained from hybridization of probe P3 with total RNA from TH3505 (wt); lane 14, RNA probe P4; lanes 15 to 20, protected transcripts obtained from hybridization of probe P4 with total RNA obtained from TH3505 (wt), TH3506 (flk), TH3504 (flgB2164), TH3507 (flgB2164 flk), TH3508 (flgA1529), and TH3513 (flgA1529 flk), respectively. A total of 40 µg of total RNA was used in each hybridization. tRNA was used as a negative control (lane 21).

Effects of Flk on flgM mRNA stability. Another mechanism by which Flk could reduce FlgM levels in P- and L-ring mutant strains could be through an effect on flgM mRNA stability. Given that the class 3 flgMN mRNA transcript increases 10-fold in an flk ring mutant compared to that in the ring mutant strain alone, any reduction in flgM mRNA stability might be expected to occur on this transcript, although it is possible that both the flgAMN and the flgMN transcripts are affected. The stabilities of the class 2 flgAMN and class 3 flgMN transcripts were determined in wild-type (Fla⁺), HBB deletion (flgG to -L), and ring mutant backgrounds in the presence and absence of an flk null allele (Fig. 7 and Table 3). There was a slight increase in the mRNA stability (50 to 60%) of the class 2 transcript in the HBB deletion (flgG to -L) strain and no effect of the ring mutation on the stability of the class 2 transcript. There were 1.5- and 2-fold increases in the half-lives of the class 3 transcript in the HBB deletion mutant and ring mutant backgrounds, respectively, compared to that of the Fla⁺ background. Finally, there was no effect of flk on the class 2 transcript in the different backgrounds and a less than twofold increase in stability in the ring mutant background. There was a further slight increase (less than twofold) in the stability of the class 3 transcript in the ring mutant background when Flk was removed, but removal of Flk did not significantly affect the stability of the class 3 transcript in the HBB deletion background. Thus, there is no evidence suggesting that the reduction of intracellular FlgM levels in ring mutant backgrounds is due to a decrease in either class 2 or class 3 flgM mRNA stability. If anything, there was a three- to fourfold increase in the stability of the class 3 transcript in the ring flk double mutant strain compared to that of the Fla⁺ (flk^+/) strains.

View larger version (37K): [in this window] [in a new window]   FIG. 7.   Stability of flgM mRNA transcripts. Class 2 flgAMN and class 3 flgMN mRNA stability assays were performed on flagellar wild-type (WT: flk+, LT2; flk, TH2413), HBB deletion (flgG-L flk+, TH3586; flgG-L flk, TH3886), and ring mutant (flgHI flk+, TH3852; flgHI flk, TH3888) strains in the presence and absence of a functional flk gene. The results of RNAase T2 protection assays on RNA samples, taken at the different time points as indicated following addition of rifampin, are shown (see Materials and Methods). The probe used in these assays was the P2 probe depicted in Fig. 6A, which hybridizes to both class 2 flgAMN and class 3 flgMN transcripts.

View this table: [in this window] [in a new window]   TABLE 3.   Effects of flk mutation on the half-lives of mRNA transcripts from class 2 flgAMN and class 3 flgMN promoters in wild-type, ring mutant (flgHI), and HBB deletion (flgG to -L) strains

Characterization of a LacZ translational fusion to FlgM. We have shown that the loss of flk results in a reduction in intracellular FlgM levels in P- and L-ring mutant strains (Fig. 2). Reduction in FlgM levels leads to increased transcription of the class 3 promoter, including the class 3 promoter for the flgM gene itself (20) (Table 2 and Fig. 5 and 6). FlgM turnover, FlgM secretion, flgM transcription, and flgM mRNA stability were not found to account for the observed reduction in FlgM levels (Fig. 3, 4, and 7 and Table 3). Another possibility is that translation of the class 2 and/or the class 3 flgM mRNA transcripts might be reduced by loss of flk in the ring mutant strains. We tested this possibility with a lacZ translational fusion to flgM and used -galactosidase assays to examine the effects of flk on flgM translation. We previously reported the isolation of a MudK insertion in the flgM gene, flgM5208::MudK, that resulted in a translational fusion of lacZ to flgM (10). DNA sequence analysis revealed that the Mud transposed into the flgM gene at codon 86 (CTC Leu, 5'CT-MudK) (10). This is the exact same site of insertion for the flgM5207::MudA insertion used to characterize the effect of flk on flgM transcription from the class 3 promoter presented above (Fig. 5).

Effects of mutations in flk on expression of flgM-lacZ gene fusions expressed from the flgM class 2 and class 3 promoters in wild-type and flagellar mutant strains. We decided to compare expression of -galactosidase with the flgM-lacZ gene fusion (where -galactosidase levels are dependent on both transcription and translation of flgM) to expression of -galactosidase with the flgM-lac operon fusion (where -galactosidase levels are dependent only on transcription of flgM). This is feasible only if the putative translational regulatory signals are 5' to the site of insertion of the Mud transposon at amino acid 86 of FlgM. To measure the effect of Flk on translation of FlgM-LacZ expressed from the class 2 and class 3 promoters in response to flagellar structural mutations, tandem chromosomal duplications were constructed as described in Materials and Methods. The duplications resulted in two tandem chromosomal copies of the flgM region. One copy contains a wild-type flgM gene. The second copy of the flgM chromosomal region contains the flgM-lacZ gene fusion expressed from either the class 2 promoter of the flgAMN operon or the class 3 promoter of the flgMN operon. In all cases the fusion protein carried the L66S substitution mutation. This is a base substitution mutation at codon 66 in FlgM that results in substitution of leucine by serine at this position (6). The L66S mutation renders FlgM defective in binding to ²⁸ (6). This mutation was used in these assays so that changes in the level of the FlgM-LacZ protein fusion would not have the capacity to feedback-regulate its own expression at the class 3 promoter. In the cases where the FlgM-LacZ fusion was expressed from the class 2 promoter, an internal, nonpolar deletion of flgA, flgA1529, was included which when duplicated for the flgA1529 allele renders the cell defective in P-ring assembly.

Effects of mutations in flk on expression of lac operon fusions to the flgM class 2 and class 3 promoters in wild-type and flagellar mutant strains. Expression of flgM-lacZ from the flgAMN class 2 promoter increased less than twofold in the presence of an HBB deletion and increased about 2.5-fold in the ring mutant background compared to the level in the isogenic wild-type strain (Fig. 8A). Introduction of an insertion mutation in the flk locus showed only a slight reduction of about 20% in the expression of the FlgM-LacZ fusion from the flgAMN class 2 promoter in all of the three backgrounds tested (Fig. 8A).

View larger version (27K): [in this window] [in a new window]   FIG. 8.   Effects of Flk on flgM-lacZ expression from the class 2 flgAMN and class 3 flgMN promoters. (A) Expression of the flgM5208-lacZ gene fusion from the class 2 flgA promoter in isogenic wild-type and flagellar mutant strains. All strains harbor tandem, chromosomal duplications of the flgM through purB genes with the flgM5208-lacZ gene fusion located at the join point of the duplicated region. WT, TH4045; WT flk, TH4048; HBB flgG-L flk+, TH4047 (same as TH4045 except both duplicated regions carry the flgG-L2157 allele), HBB flgG-L flk, TH4050 (TH4047 flk-5206::Tn10dCm); RING flgA flk+, TH4046 (same as TH4045 except both the flgAM-lacZYA and flgAMN operons carry a nonpolar internal deletion of the flgA gene); RING flgA flk, TH4049 (TH4046 flk-5206::Tn10dCm). All six strains carry the fliA5059::Tn10dTc insertion, which prevents expression of flgM and flgM-lacZ from the class 3 flgMN promoter (10). (B) Expression of the flgM5208-lac operon fusion from the class 3 flgM promoter in isogenic wild-type and flagellar mutant strains. All strains harbor tandem, chromosomal duplications of the flgM through purB genes with the flgM5208-lacZ gene fusion located at the join point of the duplicated region. WT, TH4023; WT flk, TH4026; HBB flgG-L flk+, TH4025 (same as TH4023 except both duplicated regions carry the flgG-L2157 allele); HBB flgG-L flk, TH4028 (TH4025 flk-5206::Tn10dCm); RING flgA flk+, TH4024 (same as TH4023 except that the flgAMN operon carries a nonpolar internal deletion of the flgA gene); RING flgA flk, TH4027 (TH4024 flk-5206::Tn10dCm). All six strains carry the flgA5210::Tn10dTc insertion, which prevents expression of flgM-lacZ from the class 2 flgAMN promoter (10).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Loss of Flk reduces FlgM levels in ring mutant strains. The flagellar regulon is a regulatory network of over 50 genes in which expression of these genes is tightly coupled to the assembly of the flagellar organelle. The FlgM negative regulator is responsible for keeping expression of ²⁸-dependent promoters off until the HBB structure is complete. A novel genetic locus, flk, was discovered which allowed class 3 gene expression in ring mutant strains. Results presented in this report demonstrate that restoration of class 3 gene expression in ring mutant strains by loss of Flk is the result of reduced FlgM levels in the cell. This work examined many possible mechanisms in which loss of Flk could result in a reduction in FlgM levels in the cell. This work demonstrates that the Flk effect on class 3 transcription in ring mutant strains is not due to any of the following: (i) a reduction in transcription of the flgM gene, (ii) a reduction in flgM mRNA levels, (iii) a reduction in flgM mRNA stability, (iv) a decrease in FlgM protein stability, or (v) the ability to export FlgM through a "ringless" HBB structure. Transcription of flgM from the class 3 promoter increased about 15-fold when flk was mutated, suggesting that the effect of Flk on FlgM levels is posttranscriptional. Indeed flgM translation was reduced when a null mutation in flk was introduced into a ring mutant background (discussed below).

FlgM accumulates in export-deficient cells despite a reduction in flgM transcription. The hypothesis that FlgM could sense the completion of the HBB structure by being a substrate for secretion through the completed structure was supported by the finding that intracellular levels of FlgM increase by about 2-fold in HBB mutant strains despite a 10-fold decrease in FlgM transcription from the class 3 promoter. We had shown previously that about 80% of flgM transcription during exponential growth comes from the class 3 promoter. We have determined that in HBB mutant strains, FlgM was stable and its turnover was dependent on the presence of a functional HBB structure, suggesting that turnover of FlgM in wild-type strains during exponential growth is dependent on a gene product produced or activated following HBB completion or that FlgM turnover occurs only on extracellular FlgM.

Flk couples flgM translation to ring assembly. The observation that the large amount of flgM-containing transcript from the class 3 promoter in a ring flk double mutant strain does not result in an increase in intracellular FlgM protein levels (Fig. 2 and 3) suggested that FlgM translation was reduced when flk was defective.

View larger version (23K): [in this window] [in a new window]   FIG. 9.   Model diagramming the role of the Flk protein in the regulation of flgM mRNA translation. The ringless basal body is depicted transversing the inner membrane (IM) and peptidoglycan (PG) layers but is unable to penetrate the outer membrane-LPS (OM) layer. Flk is depicted to interact with the ringless basal body through its C-terminal hydrophobic 18-amino-acid tail in the inner membrane. Prior to P- and L-ring assembly, the ringless basal-body structure either directly or indirectly inhibits flgM mRNA translation. This effect requires a functional hook gene (flgE), so either the ringless basal body contains a nascent hook structure or hook subunits act separately in the cytoplasm. A reduction in flgM mRNA translation and subsequent FlgM protein levels in the cell would relieve inhibition of 28 and allow class 3 flagellar gene expression. The negative regulatory effect of the ringless basal-body structure on flgM mRNA translation is inhibited either by the incorporation of the P- and L-rings into the basal body structure or by the action of the Flk protein. The genes are named above the chromosomal open reading frames (thickest arrows). mRNA transcripts are depicted below the genes, and the arrows indicate the direction of transcription. The arrow below each transcript indicates translation to the indicated protein product.

Homologies between Flk and RNA binding proteins of E. coli. An enhanced search of the protein database revealed homology between the so-called "S1 domain" of RNA binding proteins of E. coli (4) and a region of the deduced Flk protein sequence (amino acids 190 through 212 of S. typhimurium Flk and amino acids 192 through 214 of E. coli Flk). This could imply a direct interaction between Flk and the flgM mRNA.

FlgM, whose native structure is unfolded, is protected from intracellular degradation. While it is not unusual for proteins to be stable to turnover during exponential growth, the stability of intracellular FlgM seemed surprising given the recent discovery that the FlgM protein exists as an unfolded polypeptide (6). How does an unfolded protein stay resistant to protein degradation within the cell? One possible answer to this question is that intracellular FlgM stays bound by other proteins. The C-terminal half of FlgM contains the anti-²⁸ domain (6, 19), while the N-terminal portion is essential for FlgM export through the HBB structure (19). If binding of other proteins is necessary for FlgM stability, then two predictions can be made. First, in the absence of FlgM export, something may bind the N terminus of FlgM in the cell to prevent its degradation, while the ²⁸ protein binds the C terminus. If the level of FlgM exceeds that of ²⁸, something may bind the C terminus of FlgM to protect it from degradation. Furthermore, only insertions in the flgM gene relieve class 3 gene expression in strains defective in HBB genes, except in the case of ring mutants, which can be suppressed by insertions in flk as well as in flgM. This suggests that if binding proteins essential for intracellular FlgM stability exist, they may be either essential to cell viability or essential for class 3 gene expression. Otherwise, selections that yielded flgM mutants would have yielded mutations in other genes as well. A second prediction is that the ²⁸ protein may be required for FlgM stability unless the anti-²⁸ domain of FlgM binds other proteins as well as ²⁸.

The ringless basal body is necessary for translational inhibition of flgM expression. What is the mechanism by which ring completion is coupled to translation of flgM, and what is the role of Flk in this mechanism? All of the HBB genes except the ring genes, flgA, flgH, and flgI, are required to be functional for the Flk effect on class 3 gene expression to be seen. This suggests that it is the ringless HBB structure that is directly or indirectly responsible for reduction in FlgM protein levels in the absence of Flk (Fig. 9). If any one of the other 23 known HBB genes is mutated, then loss of Flk does not exhibit the large effect on class 3 gene expression. We propose that Flk is involved in detecting completion of the L- and P-rings for the purpose of initiating some class 3 gene derepression prior to completion of the L- and P-rings for the purpose of initiating some class 3 gene dererpression prior to completion of the HBB structure. Otherwise, class 3 gene derepression must wait until the hook is complete