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ABSTRACT: Urease is an essential component of gastric acid acclimation by H. pylori. The increased level of urease in gastric acidity is due, in part, to acid activation of the two-component system consisting of the membrane sensor HP0165 (ArsS) and its response regulator HP0166 (ArsR), which regulates transcription of the seven genes in two separate operons (ureAB, and ureIEFGH) of the urease gene cluster. Recently, we identified a novel cis-encoded antisense small RNA, 5' ureB-sRNA, targeted at the 5' end of ureB, which down regulates ureAB expression by truncation of the ureAB transcript at neutral pH. It is not known whether the truncated transcript is due to transcription termination or processing of the full-length mRNA by co-degradation of ureAB mRNA-sRNA hybrid complex. S1 nuclease mapping assays show that the truncated transcript is due to transcription termination. Further studies using an in vitro transcription assay found that 5' ureB-sRNA promotes premature termination of transcription of ureAB mRNA. These results suggest that the antisense small RNA, 5' ureB-sRNA, down-regulates ureAB expression by enhancing transcription termination at 5' of ureB. With this mechanism, a limited amount of 5' ureB-sRNA is sufficient to regulate the relatively high level of ureAB transcript.
Journal of bacteriology 10/2012; · 3.94 Impact Factor
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ABSTRACT: Helicobacter pylori survives and grows at low pHs via acid acclimation mechanisms that enable periplasmic pH homeostasis. Important components include a cytoplasmic urease; a pH-gated urea channel, UreI; and periplasmic α-carbonic anhydrase. To allow the rapid adjustment of periplasmic pH, acid acclimation components are recruited to the inner membrane in acid. The ArsRS two-component system, in an acid-responsive manner, controls the transcription of the urease gene cluster and α-carbonic anhydrase. The aim of this study is to determine the role of ArsS in protein trafficking as a component of acid acclimation. H. pylori wild-type and ΔarsS bacteria were incubated at acidic and neutral pHs. Intact bacteria, purified membranes, and total protein were analyzed by Western blotting and urease activity measurements. The total urease activity level was decreased in the ΔarsS strain, but the acid activation of UreI was unaffected. A 30-min acid exposure increased the level and activity of urease proteins at the membrane in the wild type but not in the ΔarsS strain. The urease levels and activity of the ΔarsS strain after a 90-min acid exposure were similar to those of the wild type. ArsS, in addition to its role in urease gene transcription, is also involved in the recruitment of urease proteins to the inner membrane to augment acid acclimation during acute acid exposure. Urease membrane recruitment following prolonged acid exposure in the absence of ArsS was similar to that of the wild type, suggesting a compensatory mechanism, possibly regulated by FlgS, underscoring the importance of urease membrane recruitment and activation in periplasmic pH homeostasis.
Journal of bacteriology 08/2012; 194(20):5545-51. · 3.94 Impact Factor
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ABSTRACT: Expression of urease is essential for gastric colonization by Helicobacter pylori. The increased level of urease in gastric acidity is due, in part, to acid activation of the two-component system (TCS) consisting of the membrane sensor HP0165 and its response regulator, HP0166, which regulates transcription of the seven genes of the urease gene cluster. We now find that there are two major ureAB transcripts: a 2.7-kb full-length ureAB transcript and a 1.4-kb truncated transcript lacking 3' ureB. Acidic pH (pH 4.5) results in a significant increase in transcription of ureAB, while neutral pH (pH 7.4) increases the truncated 1.4-kb transcript. Northern blot analysis with sense RNA and strand-specific oligonucleotide probes followed by 5' rapid amplification of cDNA ends detects an antisense small RNA (sRNA) encoded by the 5' ureB noncoding strand consisting of ∼290 nucleotides (5'ureB-sRNA). Deletion of HP0165 elevates the level of the truncated 1.4-kb transcript along with that of the 5'ureB-sRNA at both pH 7.4 and pH 4.5. Overexpression of 5'ureB-sRNA increases the 1.4-kb transcript, decreases the 2.7-kb transcript, and decreases urease activity. Electrophoretic mobility shift assay shows that unphosphorylated HP0166 binds specifically to the 5'ureB-sRNA promoter. The ability of the HP0165-HP0166 TCS to both increase and decrease ureB expression at low and high pHs, respectively, facilitates gastric habitation and colonization over the wide range of intragastric pHs experienced by the organism.
Journal of bacteriology 10/2010; 193(1):40-51. · 3.94 Impact Factor
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ABSTRACT: Helicobacter pylori colonizes the normal human stomach by maintaining both periplasmic and cytoplasmic pH close to neutral in the presence of gastric acidity. Urease activity, urea flux through the pH-gated urea channel, UreI, and periplasmic alpha-carbonic anhydrase are essential for colonization. Exposure to pH 4.5 for up to 180 min activates total bacterial urease threefold. Within 30 min at pH 4.5, the urease structural subunits, UreA and UreB, and the Ni(2+) insertion protein, UreE, are recruited to UreI at the inner membrane. Formation of this complex and urease activation depend on expression of the cytoplasmic sensor histidine kinase, HP0244. Its deletion abolishes urease activation and assembly, impairs cytoplasmic and periplasmic pH homeostasis, and depolarizes the cells, with an approximately 7-log loss of survival at pH 2.5, even in 10 mM urea. Associated with this assembly, UreI is able to transport NH(3), NH(4)(+), and CO(2), as shown by changes in cytoplasmic pH following exposure to NH(4)Cl or CO(2). To be able to colonize cells in the presence of the highly variable pH of the stomach, the organism expresses two pH-sensor histidine kinases, one, HP0165, responding to a moderate fall in periplasmic pH and the other, HP0244, responding to cytoplasmic acidification at a more acidic medium pH. Assembly of a pH-regulatory complex of active urease with UreI provides an advantage for periplasmic buffering.
Journal of bacteriology 10/2009; 192(1):94-103. · 3.94 Impact Factor
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ABSTRACT: Helicobacter pylori colonizes the acidic gastric environment, in contrast to all other neutralophiles, whose acid resistance and tolerance responses allow only gastric transit. This acid adaptation is dependent on regulation of gene expression in response to pH changes in the periplasm and cytoplasm. The cytoplasmic histidine kinase, HP0244, which until now was thought only to regulate flagellar gene expression via its cognate response regulator, HP0703, was found to generate a response to declining medium pH. Although not required for survival at pH 4.5, HP0244 is required for survival at pH 2.5 with 10 mM urea after 30 min. Transcriptional profiling of a HP0244 deletion mutant grown at pH 7.4 confirmed the contribution of HP0244 to sigma(54) activation via HP0703 to coordinate flagellar biosynthesis by a pH-independent regulon that includes 14 flagellar genes. Microarray analysis of cells grown at pH 4.5 without urea revealed an additional 22 genes, including 4 acid acclimation genes (ureA, ureB, ureI, and amiE) that are positively regulated by HP0244. Additionally, 86 differentially expressed genes, including 3 acid acclimation genes (ureF, rocF [arginase], and ansB [asparaginase]), were found in cells grown at pH 2.5 with 30 mM urea. Hence, HP0244 has, in addition to the pH-independent flagellar regulon, a pH-dependent regulon, which allows adaptation to a wider range of environmental acid conditions. An acid survival study using an HP0703 mutant and an electrophoretic mobility shift assay with in vitro-phosphorylated HP0703 showed that HP0703 does not contribute to acid survival and does not bind to the promoter regions of several genes in the HP0244 pH-dependent regulon, suggesting that there is a pathway outside the HP0703 regulon which transduces the acid-responsive signal sensed by HP0244.
Journal of bacteriology 11/2008; 191(2):449-60. · 3.94 Impact Factor
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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.
Journal of Bacteriology 04/2007; 189(6):2426-34. · 3.83 Impact Factor
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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).
Journal of Bacteriology 04/2006; 188(5):1750-61. · 3.83 Impact Factor
