Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation

Abstract

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR approximately P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR approximately P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR approximately P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene.

FIG. 1.

Analysis of transcription of selected ArsR∼P target genes by slot blot Northern hybridization (A) and primer extension (B). Equal amounts of RNA extracted from H. pylori G27 (lane 1) and G27/HP165::km (lane 2) exposed to pH 5.0 for 1 h were used in the respective experiments performed with DNA probes or radioactively labeled oligonucleotides specific for the indicated ORFs. RNA slot blot analysis with a labeled probe specific for 16S RNA was performed as a control (not shown). +, presence of gene; −, absence of gene.

FIG. 2.

Analysis of transcription of the amidase genes in H. pylori G27 and the isogenic ArsS-deficient mutant G27/HP165::km grown at neutral pH and exposed to pH 5.0. (A) Primer extension experiments using the radiolabeled oligonucleotide amiE-PE were performed on equal amounts of RNAs extracted from H. pylori G27 (lanes 1 and 2) and G27/HP165::km (lanes 3 and 4) that were grown at neutral pH (lanes 1 and 3) or incubated at pH 5.0 for 1 h (lanes 2 and 4). In addition, RNA extracted from strain G27/ΔarsSΔnikR exposed to pH 5.0 (lane 5) was analyzed. (B) Primer extension analysis using the radiolabeled oligonucleotide amiF-PE was performed on RNAs prepared from strains G27 (lanes 1 and 2) and G27/HP165::km (lanes 3 and 4) grown at neutral pH (lanes 1 and 3) or exposed to pH 5.0 (lanes 2 and 4). The respective cDNAs are indicated by an arrow on the right. The sequences of the −10 element of the P_amiE and P_amiF promoter are given on the left. The sequencing ladders (lanes T, A, G, C) were obtained by annealing primers amiE-PE and amiF-PE to plasmids pSL-P_amiE and pSL-P_amiF, respectively. +, presence of gene; −, absence of gene.

FIG. 3.

Binding of ArsR∼P to the P_amiE promoter. (A) DNase I footprint experiments were performed on a 328-bp EcoRI-BamHI fragment containing the P_amiE promoter derived from plasmid pSL-P_amiE, which was end labeled at the EcoRI terminus by adding increasing amounts of His₆-ArsR phosphorylated in vitro by acetylphosphate. In lanes 2 to 6, His₆-ArsR is present in concentrations of 0 (lane 2), 0.37 (lane 3), 0.75 (lane 4), 3.0 (lane 5), and 4.5 μM (lane 6). The numbers on the left indicate nucleotide positions with respect to the transcriptional start site, which is marked by an arrow. The solid bar on the right indicates the region of maximum DNase I protection. The arrow on the right indicates a band of hypersensitivity to DNase I digestion. Lane 1 contained a G+A sequence reaction mixture with the DNA probe used as a size marker (19). (B) Schematic representation of the P_amiE promoter. The −10 promoter element is highlighted by black shading, and the transcriptional start site is indicated by an arrow above the double-stranded sequence. The gray box indicates the region with maximum protection from DNase I digestion by ArsR∼P binding to the P_amiE promoter probe labeled at the EcoRI-terminus. The black bar below the sequence indicates the minimum size of the ArsR∼P binding site protected from DNaseI digestion. Numbers above the sequence indicate the nucleotide position with respect to the transcriptional start site (+1, not shown). The translational start codon of the amiE gene is shown in italics.

FIG. 4.

Binding of ArsR∼P to the P_amiF promoter. (A) DNase I footprint experiments were performed on a 318-bp EcoRI-BamHI fragment containing the P_amiF promoter, which was end labeled at the EcoRI terminus by adding increasing amounts of His₆-ArsR phosphorylated in vitro by acetylphosphate. In lanes 2 to 7, His₆-ArsR is present in concentrations of 0 (lane 2), 0.37 (lane 3), 0.75 (lane 4), 1.5 (lane 5), 3.0 (lane 6), and 4.5 μM (lane 7). The numbers on the left indicate nucleotide positions with respect to the transcriptional start site, which is marked by an arrow. The solid bar on the right indicates the maximum region of DNase I protection. Lane 1 contained a G+A sequence reaction mixture with the DNA probe used as a size marker (19). (B) Schematic representation of the P_amiF promoter. The −10 promoter element is highlighted by black shading, and the transcriptional start site is indicated by an arrow above the double-stranded sequence. The gray box indicates the region with maximum protection from DNase I digestion by response regulator binding to the P_amiF promoter probe labeled at the EcoRI terminus. The black bar below the sequence indicates the minimum size of the ArsR∼P binding site protected from DNase I digestion. Numbers above the sequence indicate the nucleotide position with respect to the transcriptional start site (+1, not shown). The translational start codon of the amiF gene is given in italics.

FIG. 5.

Analysis of transcription of rocF and ORF HP0682 in H. pylori G27 and G27/HP165::km. (A) Primer extension experiments using the radiolabeled oligonucleotide rocF-PE were performed on RNAs extracted from the wild-type strain G27 grown at pH 7.0 and pH 5.0 (lanes 1 and 2) and the ArsS-deficient mutant G27/HP165::km grown at neutral pH and pH 5.0 (lanes 3 and 4). The elongated primer products are indicated by an arrow on the right. The sequences of the −10 element of the P_rocF promoter is given on the left. The sequencing ladder (lanes T, A, G, C) was obtained by annealing primer rocF-PE to plasmid pSL-P_rocF. (B) Primer extension experiments using the radiolabeled oligonucleotide 682-PE were performed on RNAs extracted from strains G27 (lanes 1 and 2) and G27/HP165::km (lanes 3 and 4) grown at pH 7.0 (lanes 1 and 3) and exposed to pH 5.0 for 1 h (lanes 2 and 4). The cDNAs specific for ORF hp682 are indicated by an arrow on the right. +, presence of gene; −, absence of gene.

FIG. 6.

Binding of ArsR∼P to the P_rocF promoter. (A) DNase I footprint experiments were carried out on the P_rocF promoter fragment labeled at the BamHI terminus. In lanes 2 to 9, His₆-ArsR was added in concentrations of 0 (lane 2), 0.09 (lane 3), 0.18 (lane 4), 0.37 (lane 5), 0.75 (lane 6), 1.5 (lane 7), 3.0 (lane 8) and 4.5 μM (lane 9). The numbers on the left indicate nucleotide positions with respect to the transcriptional start site, which is marked by an arrow. The solid bar on the right indicates the region of DNase I protection. Lane 1 contained a G+A sequence reaction mixture with the DNA probe used as a size marker (19). (B) Schematic representation of the P_rocF promoter. The −10 promoter element is highlighted by black shading, and the transcriptional start site is indicated by an arrow above the double-stranded sequence. The gray box indicates the region protected from DNase I digestion by response regulator binding to the P_rocF promoter probe. The black bar below the sequence highlights the complete ArsR∼P binding site, which covers the −10 promoter element. Numbers above the sequence indicate the nucleotide position with respect to the transcriptional start site (+1, not shown). The translational start codon of the rocF gene is given in italics.