Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori

Abstract

About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

FIG. 1.

Overproduction and purification of recombinant HP0166-His₆. SDS-PAGE analysis of crude extracts from E. coli BL21 Star(DE3) carrying pET100/D-TOPO-HP0166 without IPTG induction (lane 2) and with 2 h of IPTG induction (lane 3) and purified HP0166-His₆ protein after Ni-nitrilotriacetic acid affinity chromatography (lane 4). Molecular weight standards were loaded in lane 1.

FIG. 2.

Control experiments for EMSA with HP0166-His₆ purified from E. coli (HP0166-WT) (left parts), HP0166-His₆ subsequently phosphorylated in vitro by carbamylphosphate (HP0166-WT∼P) (center parts), and a mutated response regulator (HP0166-D52N) that is not able to be phosphorylated at the aspartate phosphorylation site (right parts). A previously identified HP0166 target promoter region for pHP1408-1412 (10) was used as a positive control (A). A 161-bp probe for pHP0294 (amiE) that did not bind to HP0166-His₆ protein under the same experimental conditions served as a negative (nonspecific) control (B). PCR products for promoters were labeled with [γ-³³P]ATP. Labeled probes were incubated in the absence (lanes 1) or presence of increasing amounts of response regulator protein (15, 30, 60, and 120 pmol, lanes 2 to 5, respectively) or in the presence of both the protein (120 pmol) and a 50-fold excess of unlabeled probe as a specific competitor (lanes 6). The open and line arrows indicate the shifted bands and free probes, respectively.

FIG. 3.

HP0166 target promoters in group A. HP0166 target promoters in group A may be HP0165 dependent and responsive to the periplasmic pH. (A) Enlargement of the gene cluster segment assigned to group A from the cluster analysis of gene expression patterns showing microarray experiments at a medium pH of 6.2 versus a medium pH of 7.4 with and without urea. The presence of urea on this group of genes up-regulated at a medium pH of 6.2 abolishes their up-regulation. The HP0166 target genes identified by EMSA are highlighted with rectangles. The medium pH for each individual microarray experiment is indicated. The color scale used to represent the expression ratios is shown at the bottom. (B) HP0166 target promoters in group A identified by EMSA. PCR products for promoters were labeled with [γ-³³P]ATP. Labeled probes were incubated in the absence (lane 1) or presence of increasing amounts of wild-type HP0166-His₆ (HP0166-WT) or mutated protein HP0166-D52N (15, 30, 60, and 120 pmol, lanes 2 to 5, respectively) or in the presence of both HP0166-His₆ (120 pmol) and a 50-fold excess of unlabeled probe as a specific competitor (lane 6). The open and line arrows indicate the shifted bands and free probes, respectively.

FIG. 4.

HP0166 target promoters in group B. HP0166 target promoters in group B may be HP0165 independent and responsive to a cytoplasmic pH lower than pH 6.5. (A) Enlargement of the gene cluster segment assigned to group B on the basis of the cluster analysis of gene expression patterns in microarray experiments at a medium pH of 5.5 versus a medium pH of 7.4 with and without urea. In this group, the presence of urea also abolished up-regulation of these genes at a fixed medium pH of 5.5. The HP0166 target genes identified by EMSA are highlighted with rectangles. The medium pH for each individual microarray experiment is indicated. The color scale used to represent the expression ratios is shown on the bottom. (B) HP0166 target promoters in group B identified by EMSA. PCR products for promoters were labeled with [γ-³³P]ATP. Labeled probes were incubated in the absence (lane 1) or presence of increasing amounts of wild-type HP0166-His₆ (HP0166-WT) or mutated protein HP0166-D52N (15, 30, 60, and 120 pmol, lanes 2 to 5, respectively) or in the presence of both HP0166-His₆ (120 pmol) and a 50-fold excess of unlabeled probe as a specific competitor (lane 6). The open and line arrows indicate the shifted bands and free probes, respectively.

FIG. 5.

HP0166 target promoters in group C. HP0166 target promoters in group C may be HP0165 independent and responsive to a cytoplasmic pH lower than pH 5.3. (A) Enlargement of the gene cluster assigned to group C from the cluster analysis of gene expression patterns showing microarray experiments at a medium pH of 4.5 versus a medium pH of 7.4 with and without urea. In this group also, the presence of urea abolished up-regulation at pH 4.5. The HP0166 target genes identified by EMSA are highlighted with rectangles. The fixed medium pH for each individual microarray experiment is indicated. The color scale used to represent the expression ratios is shown at the bottom. (B) HP0166 target promoters in group C identified by EMSA. PCR products for promoters were labeled with [γ-³³P]ATP. Labeled probes were incubated in the absence (lane 1) or presence of increasing amounts of wild-type HP0166-His₆ (HP0166-WT) or mutated protein HP0166-D52N (15, 30, 60, and 120 pmol, lanes 2 to 5, respectively) or in the presence of both HP0166-His₆ (120 pmol) and a 50-fold excess of unlabeled probe as a specific competitor (lane 6). The open and line arrows indicate the shifted bands and free probes, respectively.

FIG. 6.

HP0166 binding sites for the HP0871-0866 (hypA) and HP1186 (carbonic anhydrase) promoter regions. DNase I footprinting analysis of HP0166 binding to the HP0871-0866 (hypA) and HP1186 (carbonic anhydrase) promoter regions was carried out with purified HP0166-His₆. Each lane contains 0.5 pmol of the γ-³³P-end-labeled nontemplate strand of HP0871-0866 (−34, −351) (A) or the nontemplate strand of HP1186 (−47, −271) (B). Fragments were incubated with increasing amounts of purified HP0166-His₆: lane 1, no protein; lane 2, 15 pmol; lane 3, 30 pmol; lane 4, 60 pmol; lane 5, 120 pmol; lane 6, 240 pmol; lane 7, 480 pmol. Sequencing reaction mixtures of appropriate DNA fragments were loaded next to lane 7. Regions protected by HP0166 are indicated by vertical solid lines (at a lower HP0166 concentration) and dotted lines (at a higher HP0166 concentration). The double-stranded sequences of regions protected from DNase I digestion by the binding of HP0166 for HP0871-0866 (C) and HP1186 (D) are shown. (E) Alignment of the protected regions for the HP0871-0866 (hypA) and HP1186 (carbonic anhydrase) promoter regions. The conserved, AT-rich sequence (AATG/CATT) found between protected regions for HP0871-0866 and HP1186 is underlined. Positions relative to the translational start codon are indicated.