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. 2004 Feb;72(2):766-73.
doi: 10.1128/IAI.72.2.766-773.2004.

Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade

Arnoud H M van Vliet  1 , Ernst J Kuipers, Jeroen Stoof, Sophie W Poppelaars, Johannes G Kusters
Affiliations

Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade

Arnoud H M van Vliet et al. Infect Immun. 2004 Feb.

Abstract

Although the adaptive mechanisms allowing the gastric pathogen Helicobacter pylori to survive acid shocks have been well documented, the mechanisms allowing growth at mildly acidic conditions (pH approximately 5.5) are still poorly understood. Here we demonstrate that H. pylori strain 26695 increases the transcription and activity of its urease, amidase, and formamidase enzymes four- to ninefold in response to growth at pH 5.5. Supplementation of growth medium with NiCl2 resulted in a similar induction of urease activity (at low NiCl2 concentration) and amidase activity (at > or = 500 micro M NiCl2) but did not affect formamidase activity. Mutation of the fur gene, which encodes an iron-responsive repressor of both amidases, resulted in a constitutively high level of amidase and formamidase activity at either pH but did not affect urease activity at pH 7.0 or pH 5.5. In contrast, mutation of the nikR gene, encoding the nickel-responsive activator of urease expression, resulted in a significant reduction of acid-responsive induction of amidase and formamidase activity. Finally, acid-responsive repression of fur transcription was absent in the H. pylori nikR mutant, whereas transcription of the nikR gene itself was increased at pH 5.5 in wild-type H. pylori. We hypothesize that H. pylori uses a repressor cascade to respond to low pH, with NikR initiating the response directly via the urease operon and indirectly via the members of the Fur regulon.

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Figures

FIG. 1.
FIG. 1.
Acid-responsive induction of urease (A), amidase (B), and formamidase (C) in H. pylori is mediated at the transcriptional level (D). Graphs A, B and C represent enzyme activities, measured in wild-type H. pylori 26695, grown in pH 7.0 and pH 5.5 BBC medium. The activity of urease, amidase, and formamidase was determined by measuring ammonia production from urea, acrylamide, and formamide, respectively. The results shown are the averages of three independent growth experiments. Bars: ▪, pH 7.0□, pH 5.5. Errors bars denote the standard deviations. Asterisks indicate a significant difference in enzyme activity at pH 5.5 compared to pH 7.0 (P < 0.05, Mann-Whitney U test). (D) Northern hybridization with probes specific for the ureA, amiE, and amiF genes by using RNA isolated from cultures grown at pH 7.0 and pH 5.5. Staining of transferred RNA by methylene blue was included for comparison of the RNA amounts. A Northern hybridization with the pH-independently transcribed hspA gene is included as a control. The probes used are indicated on the far left, whereas the position of the specific mRNAs is indicated on the right; relevant RNA marker sizes (in kilobases) are indicated on the left.
FIG. 2.
FIG. 2.
Nickel supplementation of H. pylori growth medium mimics acidification. Wild-type H. pylori 26695 was grown in unsupplemented BBC medium or in medium supplemented with NiCl2 to final concentrations of 1 to 1,000 μM (as indicated on the x axis). The activities of urease (A), amidase (B), and formamidase (C) were determined after ∼20 h of growth. The results shown are the averages of three independent growth experiments; errors bars denote the standard deviations. Asterisks indicate a significant change in enzyme activity of the respective enzymes upon NiCl2 supplementation compared to unsupplemented medium (P < 0.05; Mann-Whitney U test).
FIG. 3.
FIG. 3.
The H. pylori NikR and Fur regulators mediate acid-responsive induction of urease and both amidase enzymes. H. pylori 26695 wild-type and mutant derivatives were grown for 24 h in pH 7.0or pH 5.5 BBC medium, and the urease (A), amidase (B), and formamidase (C) enzyme activities were determined. The results shown are the averages of three independent growth experiments. Bars: ▪, pH 7.0; □, pH 5.5. Errors bars denote the standard deviations. Asterisks indicates a significant difference in enzyme activity at pH 5.5 compared to pH 7.0 (P < 0.05, Mann-Whitney U test), whereas the statistical evaluation of the differences between the wild-type and nikR mutant strains at pH 5.5 is given above the respective graphs. NS, not significant.
FIG. 4.
FIG. 4.
Acid-responsive regulation in H. pylori is mediated by a regulatory NikR-Fur cascade. (A) Putative transcriptional organization and predicted length of the nikR and fur mRNA species. (B) Transcription of the nikR gene is acid-induced, whereas fur transcription is acid repressed. Northern hybridization of H. pylori 26695 RNA isolated from cultures grown in BBC medium of pH 7.0 and pH 5.5, withprobes specific for the nikR and fur genes. (C) Transcription of the fur gene is derepressed in a H. pylori 26695 nikR mutant. Northern hybridization of RNA isolated from H. pylori 26695 (WT) and its isogenic nikR mutant (39) grown in BBC medium of pH 7.0 was carried out with a probe specific for the fur gene. Staining of transferred RNA by methylene blue is included for comparison of the RNA amounts. The probes used are indicated on the far left, whereas the position of the specific mRNAs is indicated on the right; relevant RNA marker sizes (in kilobases) are indicated on the left.
FIG. 5.
FIG. 5.
Model of the H. pylori regulatory cascade mediating the acid-responsive transcriptional induction of ammonia-producing enzymes as described in the present study. Acidification of the environment results in increased transcription of the nikR gene; the corresponding increase in NikR protein directly affects the urease system and indirectly affects expression of the amidase system via repression of transcription of the fur gene. The decrease in Fur repressor subsequently leads to increased activity of the amidase enzyme. Since the Fur protein does not directly regulate formamidase expression (40), one or more additional regulatory systems are putatively involved, and this is represented by the circled question mark.
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