A single nucleotide change affects fur-dependent regulation of sodB in H. pylori.
ABSTRACT: Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) . Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter.
Project description:In Helicobacter pylori, the ferric uptake regulator (Fur) has evolved additional regulatory functions not seen in other bacteria; it can repress and activate different groups of genes in both its iron-bound and apo forms. Because little is understood about the process of apo-Fur repression and because only two apo-Fur-repressed genes (pfr and sodB) have previously been identified, we sought to expand our understanding of this type of regulation. Utilizing published genomic studies, we selected three potential new apo-Fur-regulated gene targets: serB, hydA, and the cytochrome c553 gene. Transcriptional analyses confirmed Fur-dependent repression of these genes in the absence of iron, as well as derepression in the absence of Fur. Binding studies showed that apo-Fur directly interacted with the suspected hydA and cytochrome c553 promoters but not that of serB, which was subsequently shown to be cotranscribed with pfr; apo-Fur-dependent regulation occurred at the pfr promoter. Alignments of apo-regulated promoter regions revealed a conserved, 6-bp consensus sequence (AAATGA). DNase I footprinting showed that this sequence lies within the protected regions of the pfr and hydA promoters. Moreover, mutation of the sequence in the pfr promoter abrogated Fur binding and DNase protection. Likewise, fluorescence anisotropy studies and binding studies with mutated consensus sequences showed that the sequence was important for apo-Fur binding to the pfr promoter. Together these studies expand the known apo-Fur regulon in H. pylori and characterize the first reported apo-Fur box sequence.
Project description:We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni2+-limiting conditions, as measured by reduced transcript levels and 63Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.
Project description:The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.
Project description:The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild-type (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.
Project description:Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and delta fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and delta fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the delta fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.
Project description:In Helicobacter pylori, iron balance is controlled by the Ferric uptake regulator (Fur), an iron-sensing repressor protein that typically regulates expression of genes implicated in iron transport and storage. Herein, we carried out extensive analysis of Fur-regulated promoters and identified a 7-1-7 motif with dyad symmetry (5'-TAATAATnATTATTA-3'), which functions as the Fur box core sequence of H. pylori. Addition of this sequence to the promoter region of a typically non-Fur regulated gene was sufficient to impose Fur-dependent regulation in vivo. Moreover, mutation of this sequence within Fur-controlled promoters negated regulation. Analysis of the H. pylori chromosome for the occurrence of the Fur box established the existence of well-conserved Fur boxes in the promoters of numerous known Fur-regulated genes, and revealed novel putative Fur targets. Transcriptional analysis of the new candidate genes demonstrated Fur-dependent repression of HPG27_51, HPG27_52, HPG27_199, HPG27_445, HPG27_825 and HPG27_1063, as well as Fur-mediated activation of the cytotoxin associated gene A, cagA (HPG27_507). Furthermore, electrophoretic mobility shift assays confirmed specific binding of Fur to the promoters of each of these genes. Future experiments will determine whether loss of Fur regulation of any of these particular genes contributes to the defects in colonization exhibited by the H. pylori fur mutant.
Project description:Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and causes peptic ulcers and gastric carcinoma. H. pylori strain 26695 has a small genome (1.67 Mb), which codes for few known transcriptional regulators that control bacterial metabolism and virulence. We analyzed by qRT-PCR the expression of 16 transcriptional regulators in H. pylori 26695, including the three sigma factors under different environmental conditions. When bacteria were exposed to acidic pH, urea, nickel, or iron, the sigma factors were differentially expressed with a particularly strong induction of fliA. The regulatory genes hrcA, hup, and crdR were highly induced in the presence of urea, nickel, and iron. In terms of biofilm formation fliA, flgR, hp1021, fur, nikR, and crdR were induced in sessile bacteria. Transcriptional expression levels of rpoD, flgR, hspR, hp1043, and cheY were increased in contact with AGS epithelial cells. Kanamycin, chloramphenicol, and tetracycline increased or decreased expression of regulatory genes, showing that these antibiotics affect the transcription of H. pylori. Our data indicate that environmental cues which may be present in the human stomach modulate H. pylori transcription.
Project description:Flagellin component D (FlgD) from Helicobacter pylori is involved in the assembly of the hook of flagella, helical tubular structures that provide motility in non-filamentous bacteria. Data provided in this article refer to HpFlgD from strains 26695 (HpFlgD_26695) and G27 (HpFlgD_G27). Within this article, information on the secondary structure content and different type of interfaces found in the two crystal forms of HpFlgD (monoclinic, HpFlgD_m and tetragonal, HpFlgD_t) are provided, as well as the list of the hydrogen bonds between monomers that are relevant for their assembly into a tetramer. Additionally, data involving investigation of the size of HpFlgD in the solution and the crystallized HpFlgD are presented, "Crystal structure of truncated FlgD from the human pathogen Helicobacter pylori" . The superposition of the different domains of HpFlgD (Fn-III and tudor domains) with the similar domains found in other species is shown, as well as the superposition of HpFlgD and modeled HpFlgE (flagellar hook protein).
Project description:We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porgammadeltaalphabeta), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations in rdxA. However, the mechanism of rdxA inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.
Project description:Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.