scholarly journals Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 533-546 ◽  
Author(s):  
Florian D. Ernst ◽  
Stefan Bereswill ◽  
Barbara Waidner ◽  
Jeroen Stoof ◽  
Ulrike Mäder ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Iron Homeostasis ◽  
Transcriptional Profiling ◽  
Northern Hybridization ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Gastric Colonization ◽  
Genes Encoding ◽  
H Pylori ◽  
Regulated Gene Expression

Intracellular iron homeostasis is a necessity for almost all living organisms, since both iron restriction and iron overload can result in cell death. The ferric uptake regulator protein, Fur, controls iron homeostasis in most Gram-negative bacteria. In the human gastric pathogen Helicobacter pylori, Fur is thought to have acquired extra functions to compensate for the relative paucity of regulatory genes. To identify H. pylori genes regulated by iron and Fur, we used DNA array-based transcriptional profiling with RNA isolated from H. pylori 26695 wild-type and fur mutant cells grown in iron-restricted and iron-replete conditions. Sixteen genes encoding proteins involved in metal metabolism, nitrogen metabolism, motility, cell wall synthesis and cofactor synthesis displayed iron-dependent Fur-repressed expression. Conversely, 16 genes encoding proteins involved in iron storage, respiration, energy metabolism, chemotaxis, and oxygen scavenging displayed iron-induced Fur-dependent expression. Several Fur-regulated genes have been previously shown to be essential for acid resistance or gastric colonization in animal models, such as those encoding the hydrogenase and superoxide dismutase enzymes. Overall, there was a partial overlap between the sets of genes regulated by Fur and those previously identified as growth-phase, iron or acid regulated. Regulatory patterns were confirmed for five selected genes using Northern hybridization. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization. These findings further delineate the central role of Fur in regulating the unique capacity of H. pylori to colonize the human stomach.

scholarly journals Essential Role of Ferritin Pfr in Helicobacter pylori Iron Metabolism and Gastric Colonization

2002 ◽  
Vol 70 (7) ◽  
pp. 3923-3929 ◽  
Author(s):  
Barbara Waidner ◽  
Stefan Greiner ◽  
Stefan Odenbreit ◽  
Holger Kavermann ◽  
Jyoti Velayudhan ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Iron Metabolism ◽  
Essential Element ◽  
Iron Starvation ◽  
Iron Storage ◽  
Gastric Colonization ◽  
H Pylori ◽  
Iron Pool

ABSTRACT The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment.

scholarly journals Characterization of the ArsRS Regulon of Helicobacter pylori, Involved in Acid Adaptation

2006 ◽  
Vol 188 (10) ◽  
pp. 3449-3462 ◽  
Author(s):  
Michael Pflock ◽  
Nadja Finsterer ◽  
Biju Joseph ◽  
Hans Mollenkopf ◽  
Thomas F. Meyer ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Acid Resistance ◽  
Two Component System ◽  
Gene Encoding ◽  
Acid Adaptation ◽  
Genes Encoding ◽  
H Pylori ◽  
A Site

ABSTRACT The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR∼P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR∼P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the −10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR∼P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene.

scholarly journals Regulation of Ferritin-Mediated Cytoplasmic Iron Storage by the Ferric Uptake Regulator Homolog (Fur) of Helicobacter pylori

2000 ◽  
Vol 182 (21) ◽  
pp. 5948-5953 ◽  
Author(s):  
Stefan Bereswill ◽  
Stefan Greiner ◽  
Arnoud H. M. van Vliet ◽  
Barbara Waidner ◽  
Frank Fassbinder ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Iron Homeostasis ◽  
Mrna Level ◽  
Ferric Uptake Regulator ◽  
Iron Starvation ◽  
Iron Storage ◽  
Ferritin Synthesis ◽  
In The Wild ◽  
H Pylori ◽  
Medium Supplementation

ABSTRACT Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed thatH. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in theH. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

scholarly journals Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

2015 ◽  
Vol 197 (11) ◽  
pp. 1921-1930 ◽  
Author(s):  
Jennifer Tsang ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Basal Body ◽  
Protein Interactions ◽  
Transcript Levels ◽  
Content Type ◽  
Genes Encoding ◽  
H Pylori ◽  
Flagellar Biogenesis ◽  
Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

scholarly journals In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

2005 ◽  
Vol 187 (22) ◽  
pp. 7703-7715 ◽  
Author(s):  
Isabel Delany ◽  
Raffaele Ieva ◽  
Alice Soragni ◽  
Markus Hilleringmann ◽  
Rino Rappuoli ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Binding Site ◽  
Gene Networks ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Target Sequence ◽  
Gene Promoters ◽  
Direct Role ◽  
H Pylori

ABSTRACT Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

scholarly journals Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

2020 ◽  
Vol 295 (46) ◽  
pp. 15454-15463 ◽  
Author(s):  
Chelsey R. Fontenot ◽  
Homyra Tasnim ◽  
Kathryn A. Valdes ◽  
Codrina V. Popescu ◽  
Huangen Ding
Keyword(s):  
Escherichia Coli ◽  
Iron Content ◽  
Iron Homeostasis ◽  
Ferric Uptake Regulator ◽  
Intracellular Iron ◽  
Free Iron ◽  
E Coli ◽  
Genes Encoding ◽  
Fur Protein ◽  
Mutant Cells

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron–sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

scholarly journals The Action of Bismuth against Helicobacter pylori Mimics but Is Not Caused by Intracellular Iron Deprivation

2004 ◽  
Vol 48 (6) ◽  
pp. 1983-1988 ◽  
Author(s):  
Michael V. Bland ◽  
Salim Ismail ◽  
Jack A. Heinemann ◽  
Jacqueline I. Keenan
Keyword(s):  
Helicobacter Pylori ◽  
Antimicrobial Effect ◽  
Iron Limitation ◽  
Intracellular Iron ◽  
Colloidal Bismuth Subcitrate ◽  
Iron Deprivation ◽  
Atp Levels ◽  
Bismuth Subcitrate ◽  
Membrane Changes ◽  
H Pylori

ABSTRACT Helicobacter pylori is highly susceptible to bismuth, a heavy metal with antimicrobial activity linked to its effect on bacterial iron uptake. Three strains of H. pylori were analyzed for indicators of iron limitation following exposure to the MIC of colloidal bismuth subcitrate (MICCBS). Similar morphologic and outer membrane changes were observed following growth in iron-limiting medium and at the MICCBS that inhibited the growth of all three strains. These changes, which were also observed for iron-limited bacteria, were alleviated by the addition of iron to the cultures. H. pylori ATP levels, reduced in iron-limiting medium, were below the limits of detection in two of the three strains following exposure to bismuth. The addition of iron partially restored bacterial ATP levels in these two strains, although not to normal concentrations. In contrast, exposure of the same strains to the MICCBS failed to deplete intracellular levels of iron, which were significantly reduced by culturing in iron-limiting medium. Thus, the antimicrobial effect of bismuth and of iron limitation on H. pylori may be similar. However, the respective mechanisms of intracellular action would appear to be mediated by different pathways within the cell.

scholarly journals Antisense RNA Modulation of Alkyl Hydroperoxide Reductase Levels in Helicobacter pylori Correlates with Organic Peroxide Toxicity but Not Infectivity

2007 ◽  
Vol 189 (9) ◽  
pp. 3359-3368 ◽  
Author(s):  
Matthew A. Croxen ◽  
Peter B. Ernst ◽  
Paul S. Hoffman
Keyword(s):  
Helicobacter Pylori ◽  
Rna Interference ◽  
Gene Function ◽  
Antisense Rna ◽  
In Vitro Growth ◽  
Protein Levels ◽  
Alkyl Hydroperoxide ◽  
Gastric Colonization ◽  
H Pylori

ABSTRACT Much of the gene content of the human gastric pathogen Helicobacter pylori (∼1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an ∼72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

scholarly journals The Orphan Response Regulator HP1021 of Helicobacter pylori Regulates Transcription of a Gene Cluster Presumably Involved in Acetone Metabolism

2007 ◽  
Vol 189 (6) ◽  
pp. 2339-2349 ◽  
Author(s):  
Michael Pflock ◽  
Melanie Bathon ◽  
Jennifer Schär ◽  
Stefanie Müller ◽  
Hans Mollenkopf ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Gene Cluster ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Acid Resistance ◽  
Housekeeping Genes ◽  
Open Reading Frames ◽  
Considerable Impact ◽  
Two Component Systems ◽  
H Pylori

ABSTRACT Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with the tight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021-regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region.

scholarly journals Identification and Characterization of Helicobacter pylori Genes Essential for Gastric Colonization

2003 ◽  
Vol 197 (7) ◽  
pp. 813-822 ◽  
Author(s):  
Holger Kavermann ◽  
Brendan P. Burns ◽  
Katrin Angermüller ◽  
Stefan Odenbreit ◽  
Wolfgang Fischer ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Infections ◽  
Meriones Unguiculatus ◽  
Type Iv ◽  
Gastric Colonization ◽  
Colonization Factors ◽  
Essential Function ◽  
H Pylori ◽  
Transformation Competence

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

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