Dataset Information

The HP0165-HP0166 two-component system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in Helicobacter pylori by activating the pH-dependent promoter.

ABSTRACT: The periplasmic alpha-carbonic anhydrase of Helicobacter pylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 alpha-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechanism that regulates the expression of HP1186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acid-inducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type H. pylori strains (26695 and 43504) and mutants with HP0165 histidine kinase (ArsS) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HP1186 alpha-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of H. pylori membrane proteins confirmed the regulatory role of ArsS in HP1186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points (TSP1 and TSP2) located 43 and 11 bp 5' of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HP1186 gene. Quantitative primer extension analysis showed that the promoter from TSP1 (43 bp 5' of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from TSP2 may be responsible for control of the basal transcription of HP1186 alpha-carbonic anhydrase.

SUBMITTER: Wen Y

PROVIDER: S-EPMC1899393 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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The HP0165-HP0166 two-component system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in Helicobacter pylori by activating the pH-dependent promoter.

Wen Yi Y Feng Jing J Scott David R DR Marcus Elizabeth A EA Sachs George G

Journal of bacteriology 20070112 6

The periplasmic alpha-carbonic anhydrase of Helicobacter pylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 alpha-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechani ...[more]

PMID: 17220228

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Project description:One elusive area in the Helicobacter pylori field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of H. pylori was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the H. pylori lifecycle. Thus, herein we investigated homB expression, regulation, and contribution to biofilm formation. We found that in H. pylori strain G27, homB was expressed at a relatively low level until stationary phase. Furthermore, homB expression was suppressed at low pH in an ArsRS-dependent manner; mutation of arsRS resulted in increased homB transcript at all tested time-points. ArsRS regulation of homB appeared to be direct as purified ArsR was able to specifically bind to the homB promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that homB contributes to biofilm formation by H. pylori. Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of homB from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of homB in trans from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of homB had been deleted. Thus, homB is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner.

Project description: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 non-essential 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~P- dependent regulon consisting of 110 genes including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF and the rocF gene encoding arginase. Transcriptome analyses were performed using a whole-genome microarray containing 1649 PCR products generated with specific primer pairs derived from the genome sequences of H. pylori 26695 (Tomb et al., 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547) and J99 (Alm et al., 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180) which were spotted in duplicate. Microarrays were produced as described by Gressmann et al. (Gressmann et al., 2005. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet 1(4):e43). To determine genes which are differentially expressed in the ArsS-deficient mutant G27/HP165::km at pH 5.0, cDNA was prepared from RNA extracted from H. pylori G27 and G27/HP165::km after exposing the bacteria for one hour to acidic pH. A total of eight RNA samples from two independent RNA preparations from strain G27 and G27/HP165::km, respectively, was used for cDNA labelling und hybridisation. Dye reversal colour swaps were performed as follows: One cDNA sample was generated using Cy3-dCTP and the other using Cy5-dCTP resulting in four labelled cDNAs per colour swap. Cy5-dCTP and Cy3-dCTP labelled cDNAs were combined and hybridized to the H. pylori microarray. The slides were scanned using ScanArray HT and analysed by using the ScanArray express software (Perkin Elmer). Spots were flagged and eliminated from analysis when the signal to background ratio was less then three or in obvious instances of high background or stray fluorescent signals. Median intensities of spots were background corrected and differences in dye bias were normalized by using the LOWESS algorithm (Yang et al., 2002. Normalization for cDNA microarray data: a robuste composite method addressing single and multiple slide systematic variation. Nucleic Acid Res. 30:e15). The signal ratios as measure of differential expression between the red and green channels were obtained from processed signal intensities. Ratios were further analysed with Microsoft Excel (Microsoft) and SAM software for statistic significance (Tusher et al., 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98:5116-5121). To determine the significance of differential expression RNA was isolated from the H. pylori G27 wild-type grown in BHI broth (pH 5.0), and 20 Âµg of this RNA were labelled either with Cy3-dCTP or with Cy5-dCTP. The two cDNA probes generated were hybridized onto the same slide, and the data were analysed as mentioned above. Signal ratios < 0.5 and > 2.0 were analyzed further.

Dataset Information

The HP0165-HP0166 two-component system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in Helicobacter pylori by activating the pH-dependent promoter.

Publications

The HP0165-HP0166 two-component system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in Helicobacter pylori by activating the pH-dependent promoter.

Similar Datasets

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