1,721,010 research outputs found
Expression of factor H binding protein of meningococcus responds to oxygen limitation through a dedicated FNR-regulated promoter
No full textFactor H binding protein (fHBP) is a surface-exposed lipoprotein in Neisseria meningitidis, which is a component of several investigational vaccines against serogroup B meningococcus (MenB)currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene, fhbp. We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, P(fhbp). Transcription from the promoter P(fhbp) responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a P(fhbp) probe in vitro. Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood
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
In the NadR regulon, adhesins and diverse meningococcal functions are regulated in response to signals in human saliva.
No full textThe Neisseria meningitidis regulator NadR was shown to repress expression of the NadA adhesin and play a major role in NadA phase-variable expression. In this study, we identified through microarray analysis over 30 genes coregulated with nadA in the NadR mutant and defined members of the NadR regulon through in vitro DNA-binding assays. Two distinct types of promoter architectures (I and II) were identified for NadR targets, differing in both the number and position of NadR-binding sites. All NadR-regulated genes investigated were found to respond to 4 hydroxyphenylacetic acid (4HPA), a small molecule secreted in human saliva, which was previously demonstrated to induce nadA expression by alleviating NadR-dependent repression. Interestingly, two types of NadR 4HPA responsive activities were found on different NadR targets corresponding to the two types of genes identified by different promoter architectures: while NadA and the majority of NadR targets (type I) are induced, only the MafA adhesins (type II) are corepressed in response to the same 4HPA signal. This alternate behavior of NadR was confirmed in a panel of strains in response to 4HPA and after incubation in saliva. The in vitro NadR binding activity at type I and type II promoter regions is differentially affected by 4HPA, suggesting that the nature of the NadR binding sites may define the regulation to which they will be subjected. We conclude that NadR coordinates a broad transcriptional response to signals present in human saliva, mimicked in vitro by 4HPA, enabling the meningococcus to adapt to the relevant host niche
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
The iron-responsive regulator Fur is transcriptionally autoregulated and not essential in Neisseria meningitidis
No full textFur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. The fur gene in Neisseria meningitidis has been described as an essential gene that may regulate a broad array of genes. We succeeded in obtaining an N. meningitidis mutant with the fur gene knocked out and used it to undertake studies of fur-mediated iron regulation. We show that expression of both Fur and the transferrin binding protein Tbp2 is iron regulated and demonstrate that this regulation is Fur mediated for the Tbp2 protein. Footprinting analysis revealed that Fur binds to two distinct sites upstream of its coding region with different affinities and that these binding sites overlap two promoters that differentially control transcription of the fur gene in response to iron. The presence of two independently regulated fur promoters may allow meningococcus to fine-tune expression of this regulator controlling iron homeostasis, possibly during infection
An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori
No full textThe Fur protein acts as a regulator of iron-dependent gene transcription in bacteria. In Helicobacter pylori, Fur regulates iron-activated and iron-repressed promoters. It also acts as an autoregulatory rheostat of transcription to fine-tune its own expression in response to iron by binding to three operators at its own promoter Pfur. Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at -110) is essential for iron regulation of Pfur and functions as an anti-repression site that is bound by the iron-free form of Fur to induce transcription. Furthermore, operator I (centred at -50) may have a dual role both as a high-affinity binding site for Fur and as an UP element. We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level. Our data supports a novel promoter architecture and mechanism of regulation by Fur
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