Helicobacter pylori flagellar hook-filament transition is controlled by a FliK functional homolog encoded by the gene HP0906

Abstract

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regulation pattern, and because of the properties of an ortholog in Campylobacter jejuni (N. Kamal and C. W. Penn, unpublished data).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 sigma(54) regulon during progression of the flagellar gene expression cascade.

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.

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.

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.

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.

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.

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.

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-β-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.

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.