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Journal of Bacteriology, August 2005, p. 5742-5750, Vol. 187, No. 16
0021-9193/05/$08.00+0     doi:10.1128/JB.187.16.5742-5750.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Helicobacter pylori Flagellar Hook-Filament Transition Is Controlled by a FliK Functional Homolog Encoded by the Gene HP0906

Kieran A. Ryan,¹ Najma Karim,² Mulugeta Worku,³ Charles W. Penn,⁴ and Paul W. O'Toole^1*

Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland,¹ Department of Microbiology, Imperial College School Medicine, St. Mary's Hospital, London, United Kingdom,² Department of Medicine, Imperial College School Medicine, St. Mary's Hospital, London, United Kingdom,³ School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom⁴

Received 22 April 2005/ Accepted 24 May 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Helicobacter pylori is a human gastric pathogen which is dependent on motility for infection. The H. pylori genome encodes a near-complete complement of flagellar proteins compared to model enteric bacteria. One of the few flagellar genes not annotated in H. pylori is that encoding FliK, a hook length control protein whose absence leads to a polyhook phenotype in Salmonella enterica. We investigated the role of the H. pylori gene HP0906 in flagellar biogenesis because of linkage to other flagellar genes, because of its transcriptional regulation pattern, and because of the properties of an ortholog in Campylobacter jejuni (N. Kamal and C. W. Penn, unpublished data). A nonpolar mutation of HP0906 in strain CCUG 17874 was generated by insertion of a chloramphenicol resistance marker. Cells of the mutant were almost completely nonmotile but produced sheathed, undulating polyhook structures at the cell pole. Expression of HP0906 in a Salmonella fliK mutant restored motility, confirming that HP0906 is the H. pylori fliK gene. Mutation of HP0906 caused a dramatic reduction in H. pylori flagellin protein production and a significant increase in production of the hook protein FlgE. The HP0906 mutant showed increased transcription of the flgE and flaB genes relative to the wild type, down-regulation of flaA transcription, and no significant change in transcription of the flagellar intermediate class genes flgM, fliD, and flhA. We conclude that the H. pylori HP0906 gene product is the hook length control protein FliK and that its function is required for turning off the ⁵⁴ regulon during progression of the flagellar gene expression cascade.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Helicobacter pylori is a causative agent for duodenal and peptic ulcers (17, 49), and infection with H. pylori is a risk factor for gastric adenocarcinoma (14) and for B-cell MALT lymphoma (37). H. pylori infection in Western countries occurs in up to 50% of the population (16) and may reach 90 to 100% in developing countries (15), where antibiotic resistance rates are also high (15). Detailed understanding of the metabolism and cell biology of H. pylori may facilitate development of new therapies for treating H. pylori infection.

Motility is an essential colonization factor for H. pylori in experimental infection models (10-12, 30). Selected flagellar genes have been shown to be transcribed in infection of humans and mice (23, 38). In addition to imparting motility, there is cross-talk between H. pylori flagellar biogenesis and adhesion mechanisms (8, 38), and the sialic acid-specific adhesin HpaA is enriched in the flagellar sheath (20). Because of the contribution of flagella to virulence, the genomic basis of H. pylori flagellum production is therefore of considerable interest. The genetics and genomics of flagellar biogenesis have been studied in detail in Salmonella enterica and Escherichia coli (28). Relative to these paradigms, the H. pylori genome (4, 47) contains homologs of most of the expected complement of flagellar genes (reviewed in reference 36). The flagellar filament is composed of a major flagellin, FlaA, and a minor flagellin, FlaB (43). The hook is composed of the FlgE protein (35). The gene for the anti-²⁸-factor FlgM, which was not originally annotated, was identified by direct experimental methods (9) and a bioinformatics approach (21). Expression of flagellar genes is controlled by at least three RNA polymerase sigma factors, ⁸⁰, ⁵⁴, and ²⁸ (1, 3, 47), and a two-component system for ⁵⁴-regulated genes (42). The FlhA protein is required for expression of three flagellar genes (40), and an FlhF homolog (HP1035) is also essential for flagellar gene regulation (34). Recent global transcript analysis of strains mutated in flagellar regulatory genes allowed establishment of a model for flagellar gene regulation in H. pylori (34). In this model, class 1 gene expression is controlled by the ⁸⁰ factor, as is intermediate class gene expression (34). Class 2 genes are controlled by the ⁵⁴ factor (RpoN), while class 3 genes are expressed by the ²⁸-FlgM-controlled system. Seven novel genes dependent on ⁵⁴ were identified, as were a number of hypothetical proteins showing differential expression in flagellar regulatory mutants compared to the wild type (34).

We sought to identify additional flagellar genes which were not identified in the original genome annotations (1, 4, 47). That such genes might have been "missed" because of sequence divergence is exemplified by the later discovery of FlgM (9, 21). One such "missing" H. pylori gene was that for FliK, the hook length control protein. The FliK protein of Salmonella enterica serovar Typhimurium is required for termination of hook assembly, and its ablation led to a "polyhook" phenotype in which flgE expression continues unchecked, with failure to pass the hook-filament checkpoint (19). The FliK proteins of Salmonella enterica serovar Typhimurium and E. coli are only 50% identical, which is low for flagellar proteins of these organisms (24), and suggests that FliK sequences may be significantly divergent between more-distant species. Residue identity between Salmonella and E. coli FliK proteins is highest in the carboxy-terminal domain. This region is rich in glutamine residues, while a central domain is proline rich (24). The organizational model for Salmonella enterica serovar Typhimurium FliK was recently refined (32). The amino-terminal domain contains an export signal, and a carboxy-terminal domain controls substrate-specificity switching. It is proposed that the highly elongated structure of the FliK molecule allows it to span the region from the export apparatus to the hook cap and to switch substrate specificity when the hook has reached its mature length (32).

A bioinformatics approach was recently employed by one of us (C.W.P.) to postulate that the Campylobacter jejuni gene Cj0041 encoded the FliK protein (N. Kamal and C. W. Penn, unpublished data). A mutant defective in Cj0041 expression had a polyhook phenotype and repressed production of flagellins. The Cj0041 gene product is only 8.7% identical to the FliK protein of S. enterica; the closest database matches were H. pylori gene HP0906 and hypothetical proteins of unknown function in H. hepaticus and Wolinella succinogenes. We thus investigated HP0906 as a possible H. pylori FliK ortholog, while a role in motility was also implied by multiple other lines of evidence. The HP0906 protein was previously identified in the secreted proteome of H. pylori (7), despite its lacking a signal peptide, which would be consistent with the exported nature of FliK (31). In common with some other flagellar genes, HP0906 is transcribed from a ⁵⁴-dependent promoter (42). Global transcript analysis showed that HP0906 was coregulated with 11 other class 2 flagellar genes (34). Expression of HP0906 was also significantly up-regulated upon exposure to cultured epithelial cells (25) and was repressed by iron-bound Fur (13), as was the expression of other ⁵⁴-dependent motility genes such as flaB and flgE (13). Finally, the genomic organization of Cj0041, HP0906, and presumptive orthologs in other epsilon proteobacterial genomes (Fig. 1), particularly linkage to flgD and flgE2, is suggestive of conservation and possible functional equivalence of Cj0041 and its orthologs. We now describe microscopic, biochemical, and transcriptional analysis of a mutant defective in HP0906 production and complementation of fliK in Salmonella spp. The data support the annotation of this gene as fliK in H. pylori and provide new insights into the functional organization of this divergent flagellar protein.

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FIG. 1. Comparison of the genomic regions of epsilon proteobacteria that include their respective HP0906 orthologs (shaded). Arrows indicate positions of named genes in respective genomes. Gene arrangements, locus numbers, and gene annotations are from the TIGR Comprehensive Microbial Resource (46). Hp, H. pylori; Cj, C. jejuni; Hh, H. hepaticus; Ws, W. succinogenes. Right-angled arrows over the H. pylori region indicate promoters with the indicated sigma factor specificity.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains, media, and growth conditions. H. pylori strain CCUG 17874 (Culture Collection University of Gothenburg, Gothenburg, Sweden) (equivalent to NCTC 11637, the type strain of H. pylori) was used in this study. Bacteria were cultured on chocolate blood agar plates (Columbia agar base [Merck, Darmstadt, Germany] with 10% [vol/vol] heat-inactivated whole horse blood [Charles River Laboratories, Wilmington, MA]) for 48 h at 37°C in an atmosphere containing 5% CO₂. H. pylori liquid cultures were grown in brain heart infusion broth (BHI broth; Oxoid Ltd, Basingstoke, Hampshire, United Kingdom) with 10% heat-inactivated fetal calf serum (HI-FCS; Sigma, St. Louis, MO) under agitation in microaerobic conditions generated by CampyGen sachets (Oxoid). E. coli strain TOP10 (Invitrogen, Carlsbad, CA) was used as the host strain for routine molecular biology and protein expression and was cultured in Luria broth or agar (39). Salmonella enterica serovar Typhimurium wild-type motile strain SJW1103 (51), and a polyhook fliK mutant SJW108 derived from it (50), were kind gifts from R. Macnab and T. Minamino, respectively. For motility testing, Salmonella strains were inoculated onto soft agar (Brucella broth [Oxoid] with 0.3% agar) and incubated at 37°C. Antibiotics were added to growth media as required, using the following levels for E. coli and Salmonella spp.: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; chloramphenicol, 10 µg/ml. H. pylori transformants were selected on chocolate blood agar plates containing chloramphenicol at 10 µg/ml and kanamycin at 10 µg/ml.

Microscopy. For transmission electron microscopy, samples were subjected to negative staining. Whole cells of H. pylori, recovered from a plate of BHI broth-1% HI-FCS agar incubated for 24 h, were gently suspended with a loop in 2% ammonium molybdate with 70 µg ml^–1 bacitracin as a wetting agent to just-visible turbidity. A drop was applied immediately to a copper grid covered with a carbon-coated Formvar film. The excess sample was withdrawn by touching the edge of the grid to a cut edge of Whatman No. 1 filter paper. The grids were examined in a JEOL JEM-1200EX transmission electron microscope operated at an accelerating voltage of 80 kV.

Analysis by computer image processing technology coupled to phase-contrast microscopy (Hobson BacTracker) was performed three times as previously described (22), using H. pylori cultures grown for 24 h in BHI broth-10% HI-FCS. Curvilinear velocity (CLV) and run length (RL) were measured, and statistical differences were calculated using Student's t test.

Molecular cloning and bioinformatics. Helicobacter DNA was isolated as previously described (35). Custom primers were purchased from MWG (Ebersberg, Germany). Standard procedures and plasmids were employed for plasmid cloning experiments using E. coli (39). The chloramphenicol acetyltransferase gene was amplified from the plasmid pRY109 (52). The plasmid pHEL3, for cloning in H. pylori, has been previously described (18). The vector pQE60 (QIAGEN, Crawley, United Kingdom) contains a phage T5 promoter regulated by a Lac repressor expressed in trans from the lacI^q gene expressed on the pREP4 plasmid (QIAGEN). Oligonucleotides HP0906res_for (5'-AGCAGCGGATCCGCAAGCGCCAACGCAAACGCT-3') and HP0906res_rev (5'-AGCAGCGAATTCGGATGTCTTTAAGGGTTTTTGGC 3') were designed for the amplification of a 538-bp intragenic fragment of the HP0906 gene that contained a unique BglII restriction site. Following cloning of the HP0906 fragment into pUC19, the resultant plasmid was cut with BglII and ligated with the chloramphenicol acetyltransferase gene. H. pylori cells were transformed with 1 µg of this plasmid for double cross-over gene disruption as previously described (35). PCRs were performed using 3 µM of each primer and 0.5 units per reaction of Vent polymerase (New England Biolabs) or 1.5 units per reaction of Taq polymerase (BioLine, London, United Kingdom). For complementation of a Salmonella fliK mutant, the HP0906 gene was amplified with primer pair HP0906-FP2 and 0906QE-R and primer pair HP0906-FP2 and 0906QE-R(stop) (see Table S1 in the supplemental material). The latter primer pair includes the stop codon of HP0906, preventing fusion to a C-terminal His tag (see below). The amplicons were digested with NcoI and BamHI and ligated to similarly restricted pQE60. Salmonella cells were transformed by electroporation using standard protocols (39). Electrocompetent Salmonella fliK mutant cells (strain SJW108) were first transformed with pREP4 plasmid, and transformants were selected on ampicillin (100 µg/ml). Salmonella pREP4 transformants were then made electrocompetent and transformed individually with the two pQE60-HP0906 constructs, and double transformants were selected on kanamycin (50 µg/ml) and ampicillin (100 µg/ml).

Alignment of genomic regions was performed using The Institute for Genome Research (TIGR) Comprehensive Microbial Resource (46). Protein sequences were aligned with ClustalW (45) and shaded using GeneDoc (33).

Protein electrophoresis and blotting. H. pylori liquid cultures were centrifuged at 20,800 x g for 1 min. Residual medium was removed from the cell pellet after an additional spin of 1 min at 20,800 x g. Pellets were resuspended in sterile water, boiled for 10 min, and stored at –70°C. Standard conditions were employed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (39), in gels containing 10% acrylamide, and subsequent immunoblotting. Rabbit polyclonal antibodies against H. pylori flagellin and hook protein were prepared as described previously (35). An anti-hexahistidine mouse monoclonal antibody was obtained commercially (Sigma). Separated proteins were transferred from SDS-PAGE gels to nitrocellulose paper by the methanol Tris-glycine system described by Towbin et al. (48). Bound antibodies were detected using horseradish-peroxidase-coupled goat anti-rabbit immunoglobulin (DAKO, Glostrup, Denmark), with hydrogen peroxide and 4-chloro-1-naphthol (Sigma) as chromogenic reagents. Cell fractionation was carried out as previously described (35).

Transcription analysis. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was employed to determine relative transcript amounts of selected flagellar genes. RNA was extracted from H. pylori strain 17874 or HP0906 mutant cells grown in liquid using an Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. A minimum of 0.5 µg of RNA was reverse transcribed using 20 ng of random primer and the Improm-II reverse transcriptase enzyme (both from Promega, Madison, WI) per the manufacturer's recommended protocol. Real-time PCR primers for seven flagellar-associated genes and two housekeeping genes were designed using the Primer3 online software package (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer sequences are listed in Table S1 in the supplemental material. qRT-PCR was performed using an ABI7000 thermocycler (Applied Biosystems, Foster City, CA). Each 12.5-µl reaction mixture contained a 50 nM concentration of each primer, 6.25 µl of 2x Master Mix, and 1/60,000 SYBR green I (both from Biogene, Kimbolton, United Kingdom). Individual amplification reactions were first established and optimized for single-band specificity and verified by running pilot reaction products on gel and monitoring of dissociation curves of all subsequent test reactions. Reactions were performed in triplicate, and crossing threshold (ct) values were averaged. Fold change in expression was calculated according to the standard formula 2^{(En – Rn) – (Et – Rt)}, where En is the ct of the experimental gene in the normal (wild-type strain) sample, Rn is the ct of the reference gene in the normal sample, Et is the ct of the experimental gene in the treated (knockout strain) sample, and Rt is the ct of the reference gene in the knockout sample. qRT-PCRs were repeated on three different sets of cultures collected on separate days, and fold expression changes were averaged.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bioinformatic analysis of HP0906. The Campylobacter jejuni locus Cj0041 was recently proposed to encode a flagellar hook length control protein (Kamal et al., unpublished). The product of Cj0041 is 27% identical and 44% similar to HP0906, which was suggestive of shared function. The HP0906 protein of H. pylori strain 26695 is 90% identical to the homolog JHP0842 in the H. pylori strain J99 genome, with the majority of the 29 mismatches occurring in the amino-terminal half of the aligned sequences (not shown). HP0906 is 29.6% identical to HH0185 of H. hepaticus and 30.2% identical to WS1761 of W. succinogenes, both of which are annotated as hypothetical proteins of unknown function (2, 44). Multiple alignment of these protein sequences and the FliK proteins of Salmonella spp. and E. coli highlights the lack of sequence identity in the amino-terminal two-thirds of the molecules but also the significant conservation in the carboxy-terminal 100 amino acids (Fig. 2). The divergent amino terminus of FliK includes the export signal recently identified (32), while the conserved carboxy-terminal moiety is responsible for substrate-specificity switching and interaction with FlhB (32). Thus, the aligned proteins, though significantly divergent, display regional sequence constraints consistent with orthology with FliK proteins of Salmonella spp. and E. coli.

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FIG. 2. Multiple sequence alignments of H. pylori HP0906 and orthologs in C. jejuni (Cj0041), H. hepaticus (HH0185), W. succinogenes (WS1761), Salmonella enterica serovar Typhimurium LT2 (STM1974), and E. coli K12 (b1943). The shading of the alignment was created with the GENEDOC program; residues in gray are identical in at least half the aligned proteins, and residues in black shading are identical in all sequences.

The amino acid composition of HP0906 is also consistent with the domain organization of FliK (24). The central region of the HP0906 molecule, from residues 201 to 385, is 10.75 mol% proline, compared to 8.16 mol% for the rest of the molecule. The carboxy-terminal domain (residues 386 to 527, by reference to the domain structure of FliK proposed by Kawagishi and colleagues) (24) is 11.27 mol% glutamine, compared to 4.95 mol% glutamine for the rest of the molecule. Thus, HP0906 has both the potential domain organization and the domain amino acid composition expected for FliK.

HP0906 is required for flagellum production. The closest FliK homolog to HP0906 is Cj0041, which is only 27% identical. To investigate HP0906 function, an intragenic fragment of 538 bp was amplified by PCR from the genome of H. pylori strain 17874 and cloned in the E. coli vector pUC19. The cat gene of pRY109 was then cloned as a BamHI restriction fragment into the unique BglII restriction site of the cloned HP0906 fragment. The resulting plasmid was used for marker exchange mutagenesis of HP0906 in H. pylori strain 17874, which is flagellated and highly motile. Chloramphenicol-resistant transformants were verified for the expected insertional mutation in HP0906 by PCR, using HP0906-specific and HP0906-cat-specific primer pairs (not shown).

The H. pylori HP0906 mutant was nonmotile, as judged by phase-contrast light microscopy. To corroborate this, the mutant was examined by BacTracker, a digitized bacterial motility tracking system which produces objective quantitative measurements of bacterial motility (22) (Fig. 3). Lack of motility is indicated when the measured value for curvilinear velocity (CLV; the length of the track traveled by a bacterium divided by the time taken) is 5.5 µm/s or below, which is baseline movement due to Brownian motion (22). The HP0906 mutant displayed significantly impaired motility, as measured by CLV (mean 6.4 µm/s) and run length (RL; the length traveled by a bacterium between two stops). However, the CLV value for the mutant did indicate residual motility, as it was reproducibly greater than that previously determined for strains totally lacking motility (22). The wild-type strain 17874 grown and examined in parallel showed normal spiraling, tumbling, and straight runs (Fig. 3). Thus, HP0906 was required for normal-level production or assembly of flagellum components or energization of rotation of the flagellar filament.

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FIG. 3. A HP0906 mutant displayed impaired motility as measured by CLV (A) and RL (B) compared to the wild-type (WT) strain. Statistical differences between the wild-type and HP0906 mutant are indicated. *, overall P value of 0.05, as determined with Student's t test; KO, knockout.

Disruption of HP0906 could have polar effects; as shown in Fig. 1, it is linked to HP0907 (flgD) and HP0908 (flgE2). However, in contrast to HP0906 results, transcription of these genes is not RpoN dependent (34), and a ⁸⁰-dependent promoter was tentatively identified. To rule out polar effects, we performed RT-PCR with primers for HP0907. This showed that HP0907 expression was unchanged in the HP0906 mutant compared to the wild type (Fig. 4), meaning that phenotypic changes in the mutant could be attributed to lack of expression of HP0906 alone. We attempted to complement the HP0906 mutation and successfully cloned the HP0906 gene into pHEL3 in E. coli (data not shown). However, transformation of this plasmid into H. pylori was repeatedly unsuccessful. Colonies from the transformation grew poorly on double-drug selection and could not be subcultured. This may be because of protein toxicity due to gene dosage effects or genetic instability due to recombination between the cloned gene and homologous sequences in the disrupted gene in the chromosome. Successful complementation in Salmonella spp. (see below) makes the latter explanation more likely, and together with evidence for lack of polarity, these findings argue strongly that the phenotypic changes in the HP0906 mutant were due to lack of this gene product alone.

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FIG. 4. Transcription of HP0907 is unaffected in the HP0906 mutant. Primer pairs specific for HP0907 or the era reference gene were employed to amplify respective target sequences from reverse-transcribed RNA of wild-type and HP0906 mutant strains, as indicated, for 30 cycles.

Negatively stained cells of wild-type and HP0906 mutant were further examined by electron microscopy (Fig. 5). In contrast to the wild type, which produced normal flagella, cells of the HP0906 mutant lacked typical flagellar filaments. Cells of the mutant instead produced sheathed, undulating structures approximately 100 to 2,000 nm in length and similar in appearance to classical polyhooks of Salmonella spp. (19). To our knowledge, this is the first report of production of sheathed polyhook structures in a bacterial fliK mutant. The "wavelength" of these undulations was approximately 100 nm, similar to that of the individual wild-type hook structure visible in Fig. 5, and the radius of curvature of the undulations was also similar to that of the wild-type hook structure. The polyhook structures were approximately 22 nm in diameter, in comparison to about 20 nm for the wild-type flagellar filament, and were generally "straight" corkscrews for stretches of 500 nm or more, interrupted by kinks or bends. This straightness may have been due in part to constraint by the sheath structure, which, when empty as in the distal portion in Fig. 5C, generally showed a very straight form. The polyhooks were located at the normal unipolar position in the cell (Fig. 5). Since the cells of the HP0906 mutant retained residual levels of motility, it may be concluded that the polyhook structures are still capable of rotation. This is also consistent with the fact that HP0906 was not identified as an essential colonization factor by signature-tagged mutagenesis, while other motility-related genes were (23).

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FIG. 5. Electron micrographs of negatively stained cells or flagellar structures of H. pylori strain 17874 wild-type (A and B) or HP0906 mutant (C and D) cells. Bars, 1,000 nm (A and C), 100 nm (B and D). The white line in panel B indicates the hook structure in the wild-type flagellar structure.

We predicted that the dramatically different flagellar morphology in the HP0906 mutant would be reflected in flagellar protein production. This was examined by Western immunoblotting of whole cell lysates with anti-FlgE (hook protein) antiserum and anti-flagellin antiserum. Both antisera significantly cross-react, due to either cross-reactivity with shared amino-terminal sequences or minor flagellin contamination of the FlgE used for immunization (35). Compared to the wild type, the HP0906 mutant showed a significant increase in FlgE production and a significant decrease in flagellin production (Fig. 6). The flagellin protein that was produced by the HP0906 mutant was divided almost equally between cytoplasm and the cell envelope fraction, suggesting that the polyhook structures may incorporate the residual flagellin protein. The overproduced FlgE protein was present in significant amounts in the cytoplasm and was abundant in the cell envelope compared to the wild type, as expected. This phenotype is consistent with a hook length control function for FliK/HP0906 and is similar to the properties of fliK mutants of Salmonella spp. or C. jejuni (27; Kamal et al., unpublished). It was difficult to discern differences between the two flagellin proteins due to the similarity in molecular masses (FlaA, 53 kDa; FlaB, 54 kDa) and also the fact that FlaA is so much more abundant (26). Comparison of the total protein profiles of the wild-type and HP0906 mutant strains revealed no discernible differences (Fig. 6D), demonstrating that no pleiotropic mutations had occurred and that the HP0906 mutation did not affect general cell physiology.

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FIG. 6. Mutation of HP0906 causes altered flagellin and hook protein production. (A and B) Cell lysates of the strains indicated above the figure were subjected to Western immunoblotting with antiflagellin (A) or antihook protein (B). The hook protein FlgE and flagellin Fla (composed of FlaA and FlaB) are arrowed. (C) Cell fractions of the respective strains were tested by immunoblotting with antisera recognizing FlgE and flagellins. (D) Total protein profiles in cell lysates of wild-type and HP0906 mutant strains were compared by SDS-PAGE and Coomassie staining. WT, wild-type H. pylori 17874; HP0906, cat insertion mutant of HP0906 gene.

HP0906 can complement fliK-defective Salmonella mutants. To test whether HP0906 could complement a Salmonella enterica serovar Typhimurium mutant, we cloned the HP0906 coding sequence into pQE60. This vector has been previously used for complementation of Salmonella flagellar genes (6) and allows for gene fusion at an NcoI site, so there is no added sequence at the amino terminus of the protein. We constructed two plasmid derivatives, pQE60-HP0906 and pQE60-HP0906-His, with or without the HP0906 stop codon. The latter resulted in a hexahistidine tag at the carboxy terminus of the protein, allowing detection by anti-hexahistidine antiserum. Expression from both plasmids was verified in E. coli by SDS-PAGE (Fig. 7) and immunoblotting, and both procedures produced a protein of the expected size that, in the case of the His-tagged protein, reacted with the anti-hexahistidine antiserum (Fig. 7). The two constructs were introduced into the Salmonella fliK mutant strain SKW108, and protein expression was again verified (Fig. 7). On motility agar, the wild-type strain SJW1103 was visibly motile after 8 h or less (Fig. 7). In SJW108, neither the empty pQE-60 vector, nor the plasmid expressing the C-terminally His-tagged HP0906 protein, complemented the fliK mutation. In contrast to these controls, SJW108 cells with the plasmid expressing the untagged HP0906 protein had significant motility after 16 h (Fig. 7). Complementation clearly occurred but did not produce wild-type-level motility, as it required longer incubation time. This suggests that HP0906 can productively interact with the Salmonella flagellar export apparatus, including FliD and FlhB, but with lower efficiency than the native FliK protein. This reduced efficiency is perhaps not surprising, considering the low level of sequence identity between the proteins (Fig. 2), especially in the amino-terminal domain. Salmonella deletion studies have shown that the N-terminal domain of FliK is necessary for export of FliK but not of other proteins (31). This sequence divergence is sufficiently tolerated for export of the HP0906 gene product, as well as hook-cap protein interaction, to occur in Salmonella spp. The failure of the C-terminally His-tagged HP0906 protein to complement indicates that the tag interfered with interaction of the C-terminal domain with FlhB.

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FIG. 7. HP0906 complements a flik mutation in Salmonella. (A) Expression of HP0906 in E. coli. Western immunoblot, using anti-hexahistidine antibody, of E. coli (pQE60-HP0906-His) cell lysates uninduced (lane 2), induced with 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) (lane 3), or induced with 0.25 mM IPTG (lane 4). Size markers are in kilodaltons. (B) Expression of HP0906 in Salmonella cells. Western immunoblotting of Salmonella SJW108 lysates of cells harboring pQE60-HP0906-His (lane 1) or pQE60-HP0906 (lane 2). Size markers are in kilodaltons. (C) Motility agar tests of the indicated strains. SJW1103 was photographed after 8 h; all other strains were photographed after 16 h. All are shown at the same scale and are representative of three independent experiments.

Mutation of HP0906 affects transcription of other flagellar genes. FliK, in addition to controlling hook length, is also responsible for the switch in export specificity from hook to filament proteins (31). HP0906 in H. pylori has been designated a class 2 gene on the basis of a global transcription study (34), so we predicted that mutation of HP0906 would lead to incomplete assembly of the hook-basal body, lack of substrate-specificity switching, and failure to secrete FlgM, thus repressing flaA transcription. We therefore examined the transcription of selected flagellar genes by quantitative RT-PCR at intervals following dilution from an overnight broth culture. The abundance of flagellar gene transcripts was determined for each strain relative to that seen with two constitutively expressed housekeeping genes: glnA (glutamine synthetase) (29) and era (a posttranscriptionally regulated GTPase used for streptococcal qRT-PCR) (41). The genes examined (with the regulon classification of Niehus and colleagues [34] shown in parentheses) were flhA (class 1), rpoN (class 1), flaB (class 2), flgE1 (class 2), flaA (class 3), fliD (intermediate class), and flgM (intermediate class). The data shown in Fig. 8 are relative to the era gene; severalfold differences for gene transcription between wild-type and mutant relative to glnA were not significantly different (not shown).

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FIG. 8. Quantitative real-time PCR measurement of selected flagellar gene transcript abundance in the HP0906 mutant relative to the wild type. Values graphed are the means of three independent biological replicates; error bars represent standard errors of the means. Each transcript abundance measurement is relative to the era gene, as described in Materials and Methods.

Expression of the two class 1 genes investigated—the master regulator flhA and the alternative ⁵⁴ factor encoded by rpoN—was not significantly altered, showing lack of feedback from HP0906 to this level of the regulatory hierarchy. Transcription of the flaA gene was dramatically reduced at each culture time point in the HP0906 mutant, ranging from 0.03-fold to 0.25-fold of the wild-type level. This is consistent with the reduced flagellin production observed and with the expected effect of a class 2 gene (HP0906) on a class 3 gene (flaA). This effect may be partly rationalized in terms of failure to export FlgM from the cell, resulting in failure to relieve the anti-²⁸ inhibition by FlgM of flaA transcription. Suerbaum and colleagues note that the H. pylori sheath may impede FlgM secretion and that another mechanism for inactivating FlgM may operate (34). Disruption of ⁵⁴-dependent genes might also cause feedback inhibition through ⁵⁴ control of fliA (²⁸) expression, leading to reduction of flaA expression.

Transcription levels of both flgE1 and flaB were increased, ranging 2- to 4.5-fold for flgE1 and from 3.5- to 7-fold for flaB. Both these genes are ⁵⁴-dependent flagellar genes, but their up-regulation is unlikely to be due to the minor changes in rpoN expression. It appears that, in its absence, the failure of FliK to either signal hook completion and/or affect substrate-specificity switching results in failure to turn off FlgS/FlgR-controlled expression. A similar generalized upregulation of ⁵⁴-dependent genes was observed in a fliK mutant of C. jejuni (Kamal et al., unpublished). The massive (60-fold) flgE transcription increase in the Cj0041 mutant of C. jejuni contrasts with the more modest upregulation we report here for the HP0906 mutant, but the latter is more in line with the concomitant level of increase of the respective FlgE protein. It is unclear why the severalfold increase in transcription of flaB in the HP0906 mutant, which is higher than that of flgE, is not reflected in production of flagellin proteins, which are globally reduced. This may be due to the low relative amount of FlaB production in the wild type.

Transcription of fliD was not altered in the HP0906 mutant. Expression of another so-called intermediate class gene, flgM, was also unaltered. It has been proposed (34) that the transcriptional behavior of the so-called intermediate class genes may be due to their being governed by both RpoN (⁵⁴) and FliA (²⁸). The potential ⁵⁴ promoter suggested for the flgM gene HP1122 (TTGGTA-N6-TGCAA) (21) is one nucleotide shorter in the spacer region than the rigid configuration (TTGG-N10-GC) inferred on the basis of primer extension for five ⁵⁴-dependent flagellar genes (42), and transcription of flgM was not significantly altered in an rpoN mutant (34). We therefore suggest that expression of the H. pylori flgM gene is not RpoN dependent. Transcription of two class 1 genes, flhA and rpoN, was also not significantly changed in the HP0906 mutant, confirming the expected lack of feedback of this class 2 gene to the hierarchical level above. The lack of significant change in rpoN expression shows that changes in the ⁵⁴-dependent regulon, represented by flaB and flgE, are mediated by components other than the sigma factor itself. Candidates include FlgS/FlgR (5, 42).

Identification of the H. pylori fliK gene adds another member, of known critical function, to the ⁵⁴-dependent flagellar regulon. We do not have data to explain the failure to turn off FlgS/FlgR-controlled expression but can now focus on HP0906-dependent components to identify such a mechanism. The polyhook structure produced by the HP0906 mutant is the result of reduced flagellin transcription and increased flgE transcription. However, it appears unlikely that increased flgE transcription alone can account for the more than 20-fold increase in hook protein apparently produced in the polyhook structures. The failure to detect significantly increased FlaB production in the HP0906 mutant may also imply posttranscriptional regulation. Further experiments will be required to compare global transcription levels and translation levels of genes affected by the HP0906 mutation in H. pylori and to determine whether all ⁵⁴-dependent flagellar genes are affected similarly in the HP0906 mutant. Notwithstanding the successful complementation of Salmonella fliK by HO0906, the conservation of the carboxy-terminal region in these and other FliK proteins, compared to the relative divergence of their amino-terminal domains, suggests lineage-specific divergence of export signals. There is clearly greater selection for retention of a conserved substrate-specificity switching domain. Experiments are in progress to confirm the interaction partners for these FliK domains in HP0906 of H. pylori.

 
Work in P.W.O.'s laboratory is supported by the Irish Research Council for Science, Engineering and Technology and by Science Foundation Ireland and in C.W.P.'s laboratory by the Biotechnology and Biological Sciences Research Council and the Darwin Trust of Edinburgh.

We acknowledge T. Minamino and the late R. Macnab for providing strains.

 
* Corresponding author. Mailing address: Department of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland. Phone: 353 21 490 3997. Fax: 353 21 490 3101. E-mail: pwotoole{at}ucc.ie.

Supplemental material for this article may be found at http://jb.asm.org/.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Journal of Bacteriology, August 2005, p. 5742-5750, Vol. 187, No. 16
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