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Motility of Helicobacter priori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog
No full textσ54 is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of Helicobacter pylori genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified σ54-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the σ28-regulated flaA flagellin gene promoter, while changes in DNA topology repress transcription of the σ54-regulated flaB flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in H. priori shows similarities with that in enterobacteriaceae and Caulobacter
The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori
No full textIn the present study, we provide evidence that the groESL, hrcA-grpE-dnaK and cbpA-hspR-orf operons encoding the major chaperones of the human gastric pathogen Helicobacter pylori are transcribed by the vegetative sigma factor σ80 and are regulated negatively by the transcriptional repressor HspR. In vitro studies with purified recombinant HspR protein established that the protein represses transcription by binding to large DNA regions centred around the transcription initiation site in the case of the P(cbp) promoter, and around -85 and -120 in the case of the P(gro) and P(hrc) promoters respectively. All three binding sites contain DNA motifs with some similarity to the HAIR sequence identified as a consensus for the HspR protein of Streptomyces. In contrast to the situation in Streptomyces, in which transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible by thermal stimuli. Transcription of the groESL and cbpA-hspR-orf operons is induced by osmotic shock, while transcription of the hrcA-grpE-dnaK operon, although HspR dependent, is not affected by salt treatment. The possibility that HspR could constitute a global transcriptional regulator for diverse cellular functions with implications for pathogenesis is discussed
Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription
No full textWe have cloned the rpoD gene encoding the principal sigma (σ) factor of Helicobacter pylori. The deduced amino acid sequence reveals a predicted polypeptide of 676 residues that has amino acid homology with the principal σ factors of a number of divergent prokaryotes. We have designated this factor σ80. Amino acid sequence analysis suggests that region 1.1 is missing in σ80 and that a region with homology to a regulatory protein from Bacillus subtilis phage SPO1 is present. Genetic studies have indicated that σ80 is not compatible with the transcriptional machinery of Escherichia coil. However, in vitro σ80 could be assembled into the E. coli RNA polymerase and could bind to E. coli and H. pylori promoters, suggesting that the σ80-containing RNA polymerase has the same stoichiometry as the native complex. By exchanging protein domains between E. coil and H. pylori σ factors, we demonstrate that the σ80 domain inhibiting transcription from E. coli promoters is confined within the non- conserved spacer region, implying that the spacer region of prokaryotic primary σ factors plays an important role in the process of transcription. Consistent with its restricted niche and with the availability of a very restricted number of transcriptional regulators, H. pylori may have evolved a spacer region of the σ factor to modulate total transcription and to quickly respond to microenvironmental changes
Growth phase-dependent regulation of target gene promoters for binding of the essential orphan response regulator HP1043 of Helicobacter pylori
No full textHelicobacter pylori encodes three two-component systems and two orphan response regulators (RRs) that are predicted to be involved in transcriptional regulation. The HP1043 gene encodes an essential OmpR-like RR, 1043RR, for which no histidine kinase has been identified. Gel filtration and cross-linking experiments on the purified 1043RR protein reveals that this protein is a dimer and in vivo dimerization assays localize the dimerization to the N-terminal regulatory domain. DNA-binding studies have revealed two targets for specific binding of the 1043RR protein and moreover, phosphorylation of the protein was not needed for the activation of binding. Footprinting analysis demonstrated that the 1043RR protein binds to its own promoter, P1043, overlapping the -35 promoter element from positions -17 to -45, suggesting that this protein is autoregulatory. In addition, it binds at a similar location, spanning nucleotides from positions -22 to -51 at the promoter of the methyl-accepting chemotaxis tlpB gene, PtlpB. A possible inverted repeat was identified in the binding sites of both promoters. In an attempt to overexpress 1043RR in H. pylori, the 10-fold induction in transcription of a second copy of HP1043 with use of an inducible promoter failed to increase cellular levels of the RR protein, suggesting that 1043RR is tightly regulated at a posttranscriptional level. The P1043 and PtlpB promoters were demonstrated to be coordinately regulated in response to growth phase in H. pylori. The essential role of HP1043 in encoding a cell cycle regulator is discussed
Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation
No full textWe identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification. The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase. In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H. pylori. Knockout mutants showed that CheY is essential for bacterial motility and that CopA, but not CopP, relieves copper toxicity. Transcriptional analyses indicated that this locus is regulated by a single promoter and that a positive effect on transcription is exerted by the addition of copper to the medium and by temperature upshift from 37 to 45°C. The possible role of this locus in H. pylori virulence is discussed
Transcriptional Regulation of Stress Response and Motility Functions in Helicobacter pylori is Mediated by HspR and HrcA
No full textThe hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were still to be identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA-binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system co-represses transcription together with HrcA. Comparative transcriptome analysis of the wild type strain versus hspR and hrcA single and double deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses on two representative genes, binding of either regulator to the respective promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 out of the 29 genes, which were found to be positively regulated by HspR or HrcA, code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed
Characterization of the HspR-mediated stress response in Helicobacter pylori
No full textThe major heat shock genes of Helicobacter pylori are regulated by the HspR repressor. In the present study we characterize the transcriptional response of the three known HspR-dependent promoters Pcbp, Pgro, and Phrc to different environmental stresses. A temperature shift from 37 to 42°C causes a typical heat shock response at all three promoters characterized by an immediate and strong induction phase of transcription and a subsequent adaptation phase, which is specific for each promoter and whose onset is determined partially by the half-lives of the respective mRNAs. Exposure to high osmolarity induces a similar response on the Pgro and Pcbp promoters while no such response is detectable at the Phrc promoter. Puromycin treatment induces transcription from all three HspR-dependent promoters, indicating that different environmental stresses are intracellularly sensed by the regulatory machinery through the accumulation of nonnative proteins. The implications of these data for the regulatory network controlling the heat shock response in H. pylori are discussed
In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori
No full textThe major chaperone genes of Helicobacter pylori are negatively regulated by HspR, a homologue of the repressor of the dnaK operon of Streptomyces coelicolor. Using an in vitro selection and amplification approach we identified two new chromosomal binding sites of the HspR protein. Both binding sites were characterized by footprinting analysis with purified HspR protein. Intriguingly, these HspR binding sites are located at the 3′ ends of two genes coding for predicted proteins with functions unrelated to those of chaperones. This suggests that H. pylori HspR may regulate the expression of genes encoding proteins with diverse functions. Nucleotide sequence alignment of HspR-binding sites highlights conserved nucleotides extending outside the previously proposed consensus binding sequence with structural features predicting geometry of HspR binding as an oligomer. © 2002 Elsevier Science B.V. All rights reserved
The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori
No full textThe ferric uptake regulator (Fur) protein is known to act as a Fe 2+-dependent transcriptional repressor of bacterial promoters. Here, we show that, in Helicobacter pylori, Fur can mediate the regulation of iron-activated genes in contrast to classical Fur regulation, in which iron acts as a co-repressor. Inactivation of the fur gene in the chromosome of H. pylori resulted in the derepression of a 19kDa protein that was identified by N-terminal sequencing as the non-haem-containing ferritin (Pfr). Growth of the wild-type H. pylori strain on media treated with increasing concentrations of FeSO4 resulted in induction of transcription from the Ppfr promoter and, conversely, depletion of iron resulted in repression of Ppfr, indicating that this promoter is iron activated. In the fur mutant, the Ppfr promoter is constitutively highly expressed and no longer responds to iron, indicating that the Fur protein mediates this type of iron regulation. Footprinting analysis revealed that Fur binds to the Ppfr promoter region and that Fe2+ decreases the efficiency of binding. In contrast, Fe2+ increased the affinity of Fur for a classical Fur-regulated promoter, the iron-repressed frpB gene promoter. To our knowledge, this is the first evidence of direct interaction between the Fur protein and the promoter of an iron-activated (-derepressed) gene. Our results support a model in which the iron status of the Fur protein differentially alters its affinity for operators in either iron-repressed or iron-activated genes
Regulation of transcription in Helicobacter pylori: Simple systems or complex circuits?
No full textA common strategy used by both Gram-negative and Gram-positive bacterial pathogens is based on the synchronisation of virulence gene expression using a variety of regulatory systems and networks to overcome host defence. During the last decade an exponentially growing number of studies on Helicobacter pylori, a human pathogen associated with diverse stomach diseases, have mainly focussed on the elucidation of mechanisms and functions of virulence factors. A subset of these studies were focussed on the molecular mechanisms regulating gene transcription in H. pylori with the aim of understanding the profound physiological changes that this pathogen, as well as other bacteria, undergoes during infection. Despite the limited number of putative regulatory proteins, as deduced from genome sequence analyses, evidence is accumulating for the existence of new and complex circuits regulating gene transcription and virulence of this bacterium. Here we will focus on the molecular mechanisms used by H. pylori to control gene transcription
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