Characterization of the HspR-Mediated Stress Response inHelicobacter pylori

Abstract

The 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 P cbp , P gro , and P hrc 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 P gro and P cbp promoters while no such response is detectable at the P hrc 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.The heat shock proteins of the gastric pathogen Helicobacter pylori have been studied in some detail both because of their general role in the protection of the bacteria from the hostile environment represented by the human stomach and because of their involvement in specific pathogenic processes. The H. pylori GroEL (Hsp60) homologue (12, 20) has been proposed to play a role in regulating the activity of the nickel-dependent urease enzyme, which generates ammonia ions from the hydrolysis of urea and therefore protects the bacteria from the low pH of the stomach lumen (4, 6). Its cochaperone GroES (Hsp10) is thought to contribute to this regulation by controlling the availability of nickel ions by means of its intrinsic metal-binding activity (10,20). GroEL as well as another major heat shock protein, DnaK (Hsp70), can furthermore be found in association with the outer membrane, and this surface localization has been suggested to modify the glycolipid binding specificity of H. pylori cells at a low pH (5, 9, 15).Because of their multiple functions in both the general stress response and in specific disease mechanisms, the H. pylori heat shock proteins are expected to be tightly regulated in their expression levels. It was recently demonstrated that transcription of the groESL, hrcA-grpE-dnaK, and cbpA-hspR-orf operons encoding the major chaperones of H. pylori is negatively regulated by HspR (17). HspR is a homologue of the repressor of the dnaK operon of Streptomyces coelicolor (2, 7). By RNA analyses and footprint experiments with purified protein preparations, we have shown that HspR represses transcription by binding to regions of 75 bp on the three chaperone genetranscribing promoters P gro , P hrc , and P cbp . In an attempt to identify the environmental stresses, which induce transcription from these promoters, we subjected H. pylori cultures to both osmotic shock and thermal shock at 45°C. While the P gro and P cbp promoters were found t...

Dual Control ofHelicobacter pyloriHeat Shock Gene Transcription by HspR and HrcA

“…The transcript quantification in Fig. 5B shows that the maximal induced level is ϳ5-fold higher than in nonstressed cells, thus confirming previous results obtained in our laboratory (36). In contrast, in G27(P gro ::cat) cells virtually no increase in transcription can be observed at the P gro promoter after temperature upshift, indicating that transcriptional control is lost in this mutant.…”

“…Total H. pylori RNA was isolated as described previously (36). Transcription from the P gro and P hrc promoters was assayed by primer extension analysis with oligonucleotides groS and hrcA as detailed previously (35,36).…”

“…Unfortunately, transient changes in oligomerization, as are typical of the heat shock response (28,34,36), cannot be measured with this assay.…”

Identification of the cis -Acting Site Involved in Activation of Promoters Regulated by Activity of the Type III Secretion Apparatus in Shigella flexneri

“…RNA preparations were performed essentially as described by Spohn et al (40). S. flexneri strains M90T (wild type) and SF622 (ipaD) were grown in 25 ml of LB broth at 37°C to mid-log phase and harvested.…”

Transcriptional Regulation of Stress Response and Motility Functions inHelicobacter pyloriIs Mediated by HspR and HrcA

“…Initial studies indicated that while transcription of the groESL and cbpA-hspR-orf operons was strongly inducible by treatment with 300 mM NaCl, no induction was observed when cultures were incubated at 45°C (31). Subsequently, Homouth and coworkers (15) showed that transcription of these operons is strongly inducible by a mild heat shock at 42°C, suggesting that HspR can indeed mediate the transcriptional response to a sudden temperature increase, characterized by fast and strong induction of transcription and a subsequent shutoff phase, whose onset is determined largely by the stability of the respective mRNAs (29). Until recently, the study of HrcAdependent regulation was hampered by difficulties in purifying a recombinant protein to map the HrcA binding sites on the promoters (25,31).…”