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

Florian D. Ernst1,2,3, Stefan Bereswill2,{dagger}, Barbara Waidner2, Jeroen Stoof1, Ulrike Mäder3,{ddagger}, Johannes G. Kusters1, Ernst J. Kuipers1, Manfred Kist2, Arnoud H. M. van Vliet1 and Georg Homuth3,{ddagger}

1 Department of Gastroenterology and Hepatology, Erasmus MC – University Medical Center Rotterdam, Dijkzigt L-455, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
2 Department of Microbiology and Hygiene, Institute of Medical Microbiology and Hygiene, University Hospital of Freiburg, D-79140 Freiburg, Germany
3 Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität Greifswald, D-17487 Greifswald, Germany

Correspondence
Arnoud H. M. van Vliet
a.h.m.vanvliet{at}erasmusmc.nl


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


The complete MIAME information for the identification of Fur- and iron-regulated gene expression in the Helicobacter pylori dataset is shown in Supplementary Table S1, available online as supplementary data with the online version of this paper at http://mic.sgmjournals.org.

{dagger}Present address: Institut für Mikrobiologie und Hygiene, Campus Charité Mitte, Berlin, Germany.

{ddagger}Present address: School of Cell and Molecular Biosciences, Newcastle University Medical School, Newcastle-upon-Tyne, UK.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection with the human pathogen Helicobacter pylori results in persistent gastritis which can develop into peptic ulcer disease and adenocarcinoma of the distal stomach (Blaser & Berg, 2001). Approximately half of the world's human population is colonized by H. pylori, leading to significant morbidity and mortality. For these reasons, the infection is considered an important public health problem with serious economic consequences. The only known habitat of H. pylori is the mucus layer overlaying the epithelial cells in the human stomach. Colonization of this acidic and variable environmental niche has necessitated the development of adaptive stress responses by H. pylori.

In the gastric environment, changes in iron availability represent one of the important environmental stimuli for H. pylori. Iron is an essential element for almost all living organisms, as it is a cofactor of many enzymes and acts as a catalyst in electron transport processes. However, in the presence of oxygen, iron potentiates the formation of toxic oxygen radicals. Therefore regulation of intracellular iron homeostasis, as mediated by the ferric uptake regulator (Fur) protein, is of critical importance (Andrews et al., 2003). Regulation of gene expression via Fur has been extensively investigated in several Gram-negative and Gram-positive bacteria, where it is involved in the regulation of many cellular processes, including iron metabolism, oxidative stress defence and central intermediary metabolism (Andrews et al., 2003; Hantke, 2001). However, while the absence of Fur affects many cellular processes, several of the regulatory phenomena described mostly in E. coli are only indirectly affected by Fur (Masse & Gottesman, 2002).

Fur is a transcriptional repressor protein, which displays iron-dependent binding to conserved DNA sequences (Fur boxes) located in the promoters of iron-regulated genes (Hantke, 2001). In most bacteria, including H. pylori, the iron-complexed form of Fur binds to promoters of iron-uptake genes, thus repressing iron uptake in iron-replete conditions (Delany et al., 2001b; van Vliet et al., 2002a). However, H. pylori Fur has acquired the thus far unique ability also to bind the pfr promoter in its iron-free form, thus repressing expression of iron-storage proteins in iron-restricted conditions (Bereswill et al., 2000; Delany et al., 2001a; Waidner et al., 2002).

The relative paucity of transcriptional regulators in H. pylori, combined with the necessity to respond to environmental stresses, may have resulted in H. pylori Fur being involved in the regulation of other adaptive responses. Other than regulation of iron metabolism, H. pylori Fur has also been implicated in the regulation of acid resistance (Bijlsma et al., 2002; Bury-Mone et al., 2004; van Vliet et al., 2004), nitrogen metabolism (van Vliet et al., 2001, 2003) and oxidative stress resistance (Barnard et al., 2004; Cooksley et al., 2003; Harris et al., 2002). Fur-mediated regulation is also required for gastric colonization by H. pylori, as demonstrated in a mouse model of infection (Bury-Mone et al., 2004).

DNA array technology has proved to be a powerful technique for the study of global gene regulation in many organisms (Conway & Schoolnik, 2003), and has also been successfully applied to study alterations in H. pylori gene expression (Bury-Mone et al., 2004; Forsyth et al., 2002; Kim et al., 2004; Merrell et al., 2003a, 2003b; Thompson et al., 2003; Wen et al., 2003) and genetic variation between isolates (Israel et al., 2001a; Salama et al., 2000). In this study, we have applied DNA array technology to growth experiments with the well-characterized H. pylori reference strain 26695, both to define the H. pylori responses to variation in iron availability and to identify new members of the H. pylori Fur regulon.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and growth conditions.
The H. pylori strains used in this study were reference strain 26695 (Tomb et al., 1997) and its isogenic fur mutant (Bijlsma et al., 2002; van Vliet et al., 2002a). H. pylori strains were routinely cultured on Dent agar at 37 °C under microaerophilic conditions (10 % CO2, 5 % O2 and 85 % N2). Broth cultures were grown in brucella broth (Difco) supplemented with 3 % newborn calf serum (Gibco) (BBN). Broth cultures were continuously shaken at 40 r.p.m. under microaerophilic conditions. Iron restriction was achieved by supplementing BBN with desferal (deferoxamine mesylate, Sigma) to a final concentration of 20 µM. Iron-replete conditions were achieved by supplementing desferal-treated BBN with ferric chloride (Sigma) to a final concentration of 100 µM (van Vliet et al., 2002a).

Purification and analysis of RNA.
Total RNA was isolated using Trizol (Gibco), according to the manufacturer's instructions. The amount of RNA was determined spectrophotometrically. RNA electrophoresis, blotting, hybridization with DIG-labelled RNA probes, and detection of bound probe were carried out as described previously (Homuth et al., 1997). Directly after transfer, the membranes were stained with methylene blue to confirm the integrity of the RNA samples and to confirm loading of equal amounts of RNA based on the relative intensities of the 16S and 23S rRNA (van Vliet et al., 2001). Chemiluminescence was detected using a Lumi-Imager (Roche Diagnostics), and chemiluminographs were quantified using the Lumi-Analyse software package (Roche Diagnostics). The DIG-labelled specific RNA probes were synthesized by in vitro transcription using T7 RNA polymerase (Roche Diagnostics), and PCR products were amplified using the primers listed in Table 1.


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Table 1. Oligonucleotide primers used in this study

Primer sequences were derived from the H. pylori 26695 genome sequence (Tomb et al., 1997). Lower-case letters indicate a 5' extension with T7 promoter sequence for the creation of an antisense RNA probe.

 
Synthesis of labelled cDNA for transcriptome analysis.
H. pylori strain 26695 and its isogenic fur mutant were grown in iron-restricted and iron-replete conditions (van Vliet et al., 2002a), and total RNA was isolated from cells grown for 20 h and checked by Northern hybridization using an amiE-specific probe (Fig. 1). For annealing of the specific oligonucleotide primers complementary to the mRNAs specified by all H. pylori genes, 1 µg total RNA (concentration determined photometrically) was hybridized to 4 µl cDNA primer mix (0·05 pmol µl–1) (Eurogentec) in hybridization buffer (10 mM Tris/HCl, pH 7·9, 1 mM EDTA, 250 mM KCl) in a total volume of 30 µl (1 h, 42 °C). After annealing, 30 µl of reverse transcription premix [12 µl 5x First Strand Buffer (Gibco-BRL), 6 µl 0·1 mM DTT (Gibco-BRL), 2 µl 10 mM dATP, 2 µl 10 mM dGTP, 2 µl 10 mM dTTP, 4·5 µl [{alpha}-33P]dCTP (10 µCi µl–1, Amersham Pharmacia), 1·5 µl reverse transcriptase (Superscript II; Gibco-BRL)] was added, and reverse transcription was carried out for 1·5 h at 42 °C. Next, 2 µl 0·5 M EDTA was added to stop all reactions. Alkaline hydrolysis of the RNA was performed by addition of 6 µl 3·0 M NaOH and incubation of the solution for 30 min at 65 °C, followed by 15 min at room temperature. The solution was neutralized with 20 µl 1 M Tris/HCl, pH 8·0, and 6 µl 2N HCl. Finally, the cDNA was precipitated overnight at –20 °C after the addition of 10 µl 3 M sodium acetate, pH 5·2, and 400 µl ethanol. The cDNA was pelleted by centrifugation at 17 600 g for 15 min at 4 °C, washed with 70 % (v/v) ethanol, dried, and resuspended in 100 µl sterile water. Labelling efficiency was determined by liquid scintillation measurement.



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Fig. 1. Selection of the growth phase of cultures of H. pylori 26695 wild-type isogenic fur mutant for isolation of RNA and subsequent transcriptome analysis. (a) Growth curves of H. pylori 26695 wild-type (circles) and fur mutant (triangles) grown in iron-restricted (open symbols) and iron-replete conditions (closed symbols). Growth is expressed as OD600. (b) Verification of iron- and Fur-responsive regulation at 8, 20 and 30 h of growth in iron-restricted (–Fe) and iron-replete (+Fe) conditions using the iron- and Fur-repressed amiE gene. Northern hybridization of RNA isolated from H. pylori 26695 (WT) and its isogenic fur mutant (fur) with a probe specific for the amiE gene. The position of the two relevant RNA marker sizes (in kb) is indicated on the left.

 
Hybridization of labelled cDNA to DNA macroarrays.
H. pylori arrays (Eurogentec): nylon membranes carrying PCR products which represented 97 % of all H. pylori 26695 and J99 protein-encoding genes (n=1578), were incubated for 10 min in 50 ml saline sodium phosphate EDTA (SSPE) buffer (0·18 M NaCl, 10 mM sodium phosphate, pH 7·7, 1 mM EDTA). Prehybridization was carried out in 10 ml hybridization solution [5x Denhardt solution, 5x SSC, where 1x SSC is 0·15 M NaCl plus 0·015 M sodium citrate, 0·5 % SDS, 100 µg denatured salmon sperm DNA (Sigma) ml–1] for 2 h at 65 °C. Subsequently, hybridization was performed for 20 h at 65 °C in 5 ml hybridization solution containing the labelled cDNA probe which had been boiled for 5 min and rapidly cooled on ice before hybridization. Arrays were washed twice with 200 ml 2x SSC and 0·1 % SDS (5 min at room temperature and 20 min at 65 °C) and once with 200 ml 0·2x SSC and 0·1 % SDS. Finally, arrays were air-dried for 2 min, sealed in plastic bags, and exposed to PhosphorImager screens. The transcriptome analysis was carried out twice, using two independently isolated sets of RNA preparations and two different array batches. Exposed PhosphorImager screens were scanned with a Storm 860 PhosphorImager (Molecular Dynamics) at a resolution of 50 µm and a colour depth of 16 bit. To remove the labelled cDNA from the arrays prior to subsequent hybridizations, the membranes were incubated three times (2, 5 and 60 min) in 300 ml boiling stripping buffer (10 mM Tris/HCl, pH 8·0; 1 mM EDTA; 1 % SDS). Exposure of the arrays after stripping revealed that the complete activity was successfully removed from the membrane. Using this method, it was possible to use the macroarrays five times without significant loss in quality.

Analysis of transcriptome data.
For quantification of the hybridization signals and background values, the ArrayVision software (Imaging Research) was used (Eymann et al., 2002). Subsequently, a quality score was calculated for each spot reflecting the ratio between the signal intensity and the background intensity. This quality score was utilized to identify hybridization signals close to the detection limit. Data normalization and data anlysis were done with the GeneSpring software (Silicon Genetics). After background subtraction, normalized intensity values of the individual spots were calculated (median normalization). Only genes specifying signals which significantly exceeded the background signal level (determined by the quality scores) under at least one condition were included in further data analysis. The average of the normalized intensity values of the duplicate spots of each gene was used to calculate the expression level ratios. Induction or repression ratios >=2 in both experiments were considered as significant and used in subsequent analysis (Eymann et al., 2002).

Final evaluation of the macroarray data included the consideration of putative operon structures derived from the genome sequence as well as previously known operons. Genes exhibiting significant expression ratios were analysed for their transcriptional organization using the PyloriGene database (http://genolist.pasteur.fr/PyloriGene/) (Boneca et al., 2003). The complete dataset is shown in Supplementary Table S1, which is available online as supplementary data with the online version of this paper at http://mic.sgmjournals.org.

Furbox Analyses.
Sequences of Fur-regulated genes (Table 2) were obtained from the H. pylori 26695 genome sequence using the PyloriGene database (http://genolist.pasteur.fr/PyloriGene/). Sequences included the intergenic regions upstream of the regulated gene when applicable. Genes were designated as being located downstream of co-transcribed genes (fliP, murB, ispE, pdxJ and hp0241) when there was less than 10 bp between the stop codon of the preceding gene and the start codon of the following gene. All genes included in this analyses had putative ribosome-binding sites (RBS) located upstream of the translational start codon.


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Table 2. Fur-regulated genes of Helicobacter pylori strain 26695

The Gene number shown is from the complete genome sequences of H. pylori strain 26695 (Tomb et al., 1997) and strain J99 (Alm et al., 1999). The Predicted function column shows the functions and functional categories as defined on the PyloriGene database (Boneca et al., 2003). Values in the Ratio columns show the ratio of expression levels in H. pylori wild-type (WT) or fur mutant (fur) strain, in iron-restricted (–Fe) or iron-replete (+Fe) conditions. The value shown is the average ratio of two independent array experiments. Values in italic type indicate significant down-regulation of expression; values in bold type indicate significant upregulation of expression. Significant regulation was defined as at least twofold changes in the mRNA levels in both independent array experiments. ND, not detectable: the signal on the array was below the detection threshold in both array experiments.

 
To find putative binding sequences for iron-cofactored Fur, promoter sequences were analysed for the presence of consecutive NAT triplets (van Vliet et al., 2002b) using the GeneRunner program (http://www.generunner.com). Putative binding sequences for apo-Fur were identified by aligning the promoter sequences with the Pfr boxes I and II (Delany et al., 2001a) using the Clone Manager 7 Suite (Scientific and Educational Software).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of iron- and Fur-regulated H. pylori genes by transcriptional profiling
For characterizing Fur- and iron-regulated gene expression, H. pylori strain 26695 was selected, as this allowed direct comparison with the available genome sequence (Tomb et al., 1997). In addition, H. pylori 26695 has been extensively characterized for the role of Fur and iron in the regulation of genes putatively involved in iron transport (van Vliet et al., 2002a). The H. pylori 26695 isogenic fur mutant used in this study contains the Campylobacter coli chloramphenicol resistance cassette in the unique BclI restriction inside the fur coding region, and was characterized previously with regard to acid resistance (Bijlsma et al., 2002), iron uptake (van Vliet et al., 2002a) and iron- and acid-responsive regulation (Bury-Mone et al., 2004; van Vliet et al., 2004).

We selected a single time-point (20 h) to compare gene expression, as this is when H. pylori 26695 reaches the late exponential phase (Fig. 1a). To confirm that the 20 h time-point was representative for identification of iron- and Fur-regulated genes, RNA samples isolated at 8, 20 and 30 h post-inoculation were hybridized with a probe specific for the amiE gene (Fig. 1b). The amiE gene was previously demonstrated to be iron- and Fur-repressed (van Vliet et al., 2003), and this regulation is apparent at each of the three time-points (Fig. 1b). The amiE mRNA, with a size of ~1 kb, was detected in the wild-type strain only in iron-restricted conditions, but was constitutively expressed in the fur mutant (Fig. 1b). Although the amiE gene has also been reported to be growth-phase regulated (Merrell et al., 2003b; Thompson et al., 2003), this was not apparent in the conditions used in this study.

RNA for array testing was isolated from two independent cultures of H. pylori 26695 and its isogenic fur mutant, grown in iron-restricted and iron-replete conditions. Subsequently the RNA samples were used for transcriptional profiling using the Eurogentec H. pylori DNA array, which contains 97 % of all ORFs of H. pylori strain 26695. To exclude potential artefacts, only genes with a signal to noise ratio >3 were included in the subsequent data analysis. In total, 1248 out of 1551 genes (80·5 %) fullfilled these criteria for at least one of the conditions in both array experiments, and this percentage of genes exhibiting significant expression signals is relatively high compared to that reported in previous studies (~50 %) (Ang et al., 2001; Merrell et al., 2003a; Thompson et al., 2003). In total, data for iron regulation were available for 1241 genes in the wild-type strain, and for 964 genes in the fur mutant. Data for Fur regulation were available for 994 genes in iron-restricted conditions, and 909 genes in iron-replete conditions.

For each of the 1248 genes, iron regulation was assessed by calculating the ratio between expression levels in iron-restricted conditions (–Fe) and the expression levels in iron-replete conditions (+Fe). To assess Fur regulation, the ratio between expression levels in the wild-type strain (WT) was compared with the expression levels in the fur mutant strain (fur/WT ratio). Since H. pylori Fur affects transcription in both iron-replete and iron-restricted conditions, the fur/WT ratio was calculated for both –Fe and +Fe conditions. Genes were considered to be regulated by either iron or Fur when the repression or induction ratio was >2 in both independent RNA preparations. Genes regulated by Fur, together with the different ratios, are presented in Table 2.

In total, 61 genes (4·9 %) displayed iron-repressed expression in the wild-type strain, whereas 36 genes (2·9 %) displayed iron-induced expression. Of these 97 iron-regulated genes, only 10 still displayed iron-dependent regulation in the fur mutant, with data for 22 genes not being available in the fur mutant. This underlines the central role of Fur in iron-regulated gene expression in H. pylori. Sixteen genes displayed derepressed expression in the fur mutant in iron-replete conditions, and thus these genes probably are regulated by the iron-complexed form of Fur. Conversely, 16 genes displayed derepressed expression in iron-restricted conditions, possibly representing repression by the iron-free form of Fur.

Fur-repressed genes
In most Gram-negative bacteria, Fur binds its target promoters in an iron-dependent fashion, in other words, only in iron-replete conditions. Uniquely, the iron-free form of H. pylori Fur is also capable of repressing transcription, thus allowing differential expression of genes depending on iron availability in the cytoplasm (Delany et al., 2001a). Surprisingly, not all Fur-regulated genes identified in this study displayed iron-responsive expression (Table 2).

(i) Genes repressed by the iron-complexed form of Fur (Fe-Fur).
Of the 16 genes demonstrating derepressed expression under iron-replete conditions in the fur mutant, 10 also demonstrated iron-repressed expression in the wild-type strain (Table 2), with four displaying iron-independent expression; for two genes, data were not available. As predicted in previous studies (Delany et al., 2001a, 2001b; Fassbinder et al., 2000; van Vliet et al., 2002a), several of these genes (fecA1, fecA2, frpB1 and exbB2) encode homologues of iron transport and binding proteins, and probably play a role in the uptake and transport of iron to the cytoplasm. In addition, the hp1432 gene, encoding a nickel-binding histidine- and glutamine-rich protein (Gilbert et al., 1995), is also regulated by Fur, and this regulation was confirmed by Northern hybridization (Fig. 2) and RNA slotblot hybridization (data not shown). This gene has also been classified as nickel- and NikR-activated (Contreras et al., 2003), and acid-induced (Merrell et al., 2003a).



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Fig. 2. Confirmation of Fur- and iron-responsive regulation of a subset of genes selected from Table 2. (a) Staining of transferred RNA by methylene blue to allow for comparison of RNA amounts. (b) Northern hybridization with probes specific for five genes using RNA purified from H. pylori wild-type (WT) and fur mutant (fur) cells grown in iron-restricted (–Fe) and iron-replete (+Fe) conditions. Probes used are indicated on the left; the specific mRNA is indicated on the right.

 
The fur mutation also influenced several other classes of genes (Table 2). These included genes putatively involved in: i) biosynthesis of cofactors and prosthetic groups, including biotin (bioB), isoprenoid (ispE) and pyridoxal phosphate (pdxA); ii) production of cell envelope and surface structures, such as the murB peptidoglycan synthesis gene and the flaB and fliP flagellar biosynthesis genes (Josenhans et al., 2000); iii) energy metabolism, with both the paralogous amidases amiE and amiF (Skouloubris et al., 2001; van Vliet et al., 2003); iv) protein synthesis, in which the 16S rRNA dimethyltransferase gene ksgA is putatively involved. Finally, expression of the hypothetical protein HP906 was repressed by Fe-Fur. For two of the iron-repressed Fur-regulated genes (amiE and hp1432), the transcriptional pattern was confirmed using Northern hybridization (Fig. 2).

(ii) Genes repressed by the iron-free form of Fur.
Sixteen genes demonstrated increased expression in the fur mutant, when compared to the wild-type strain grown in iron-restricted conditions (Table 2). This unique form of regulation has so far only been reported for the pfr gene (Bereswill et al., 2000; Delany et al., 2001a; Waidner et al., 2002), but is probably more widespread in H. pylori. Of the 16 genes demonstrating derepressed expression under iron-restricted conditions in the fur mutant, nine genes also demonstrated iron-induced expression in the wild-type strain (Table 3), with five genes displaying iron-independent expression; for two genes, data were not available.


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Table 3. Iron-regulated (Fur-independent) genes of Helicobacter pylori strain 26695

The gene number shown is from the complete genome sequences of H. pylori strain 26695 (Tomb et al., 1997) and strain J99 (Alm et al., 1999). The Predicted function column shows the function and functional category as defined on the PyloriGene database (Boneca et al., 2003). Values in the Ratio columns show the ratio of expression levels in H. pylori wild-type (WT) or fur mutant (fur) strain, in iron-restricted (–Fe) or iron-replete (+Fe) conditions. The value shown is the average ratio of two independent array experiments. Values in italic type indicate significant down-regulation of expression; values in bold type indicate significant upregulation of expression. Significant regulation was defined as at least twofold changes in the mRNA levels in both independent array experiments. ND, not detectable: the signal on the array was below the detection threshold in both array experiments.

 
Several genes associated with energy and oxygen metabolism displayed Fur-mediated repression of transcription, including the nickel/iron-cofactored hydrogenase subunit genes (hydABC) (Olson et al., 2001), a putative cytochrome c553, and the sodB gene encoding the iron-cofactored superoxide dismutase (Pesci & Pickett, 1994; Seyler et al., 2001; Spiegelhalder et al., 1993). Further genes regulated by the iron-free form of Fur included the chemotaxis gene cheV2, the hp0922 gene encoding a toxin-like outer-membrane protein, the serB gene, which is cotranscribed with pfr, the tryptophanyl-tRNA synthetase gene trpS, and five genes encoding hypothetical proteins (Table 2). For three of the iron-induced Fur-regulated genes (pfr, serB and hp0388), the transcriptional pattern was confirmed using Northern hybridization (Fig. 2).

(iii) Fur-induced genes.
Sixteen genes displayed decreased expression in the fur mutant when compared to the wild-type strain (Table 2). This inverse regulation is atypical for a repressor like Fur, and is likely to represent indirect regulation. This cluster of genes included the oipA gene encoding an outer-membrane protein, the murE gene involved in peptidoglycan synthesis, a DNA methyltransferase (mod), the ribonuclease HII-encoding rnhB gene, the periplasmic binding protein of the glutamine ABC transporter, two genes involved in protein synthesis, and eight genes encoding hypothetical proteins.

Identification of putative binding sequences for Fe-Fur and apo-Fur
All Fur-repressed genes listed in Table 2 were further investigated for the presence of putative Fur boxes in their respective promoters (Fig. 3). Firstly, the putative promoter region was identified for each gene using the PyloriGene database (see Methods for details). Putative binding sites for Fe-Fur were identified as up to six consecutive nAT triplets, with n representing any nucleotide (Delany et al., 2001b; van Vliet et al., 2002b). Each of the promoters included in this analysis contained a putative Fur box, with identities to the (nAT)6 sequence ranging from 6 to 11 per 12 residues (Fig. 3a).



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Fig. 3. Identification of putative binding sequences for Fe-Fur and for apo-Fur. (a) Putative Fe-Fur-repressed promoter sequences were searched for the presence of consecutive nAT triples, indicative of binding sequences for Fur (van Vliet et al., 2002b). Residues with black background are identical to the (NAT)6 Furbox, whereas residues with grey background represent A/T and T/A substitutions. (b) Putative apo-Fur-regulated promoters were aligned with the two high-affinity apo-Fur binding sequences identified in the H. pylori pfr promoter (Pfr box I and Pfr box II) (Delany et al., 2001a). Residues with black background are identical to the respective Pfr box, whereas residues with grey background represent A/T and T/A substitutions. For all aligned promoters, the position respective to the underlined ribosome-binding site (RBS) and underlined translational start codon (Start) are given. Designations above the alignments: Gene, gene designation given in Table 2; Prom, putative promoter of regulated gene. An asterisk indicates that the regulated gene is likely to be transcribed as a member of an operon, and the putative promoter of the gene at the beginning of the operon was analysed for the presence of a binding sequence for Fur-Fe or apo-Fur.

 
The identification of binding sites of apo-Fur is currently hampered by the absence of a consensus sequence, since only the pfr promoter has been analysed to date (Delany et al., 2001a). In this promoter there are two high-affinity sites for apo-Fur, designated Pfr box I and Pfr box II (Delany et al., 2001a). Therefore these boxes were aligned with the promoters of the genes putatively regulated by apo-Fur (Fig. 3b). All promoters contained sequences similar to each of the Pfr boxes, although this identity ranged from 17 to 24 per 41 residues (Pfr box I) and 15 to 22 per 37 residues (Pfr box II), respectively (Fig. 3b).

Iron-responsive regulation independent of Fur
While only approximately half of the Fur-repressed genes displayed iron-responsive expression, several other genes displayed iron-responsive expression which was not significantly altered in the fur mutant (Table 3). Forty-five genes displayed iron-repressed expression in the wild-type strain, i.e. higher mRNA levels in iron-restricted conditions, whereas twenty-five genes displayed iron-induced expression. As with the Fur-repressed genes, genes belonging to several functional classes were affected by iron restriction when compared to iron-replete conditions.

(i) Iron-repressed genes.
This group of iron-repressed genes includes five motility-associated genes, the fliD, fliI, fliM, fliY and flgI genes, encoding components of the flagellum of H. pylori (O'Toole et al., 2000). Their regulation by iron may explain the effect of iron restriction and acid exposure on the motility of H. pylori (Merrell et al., 2003b). In addition to motility-associated genes, iron repressed the expression of genes involved with the cell envelope and surface structures (lpp20), cell division (ftsH), peptidoglycan synthesis (murZ), LPS (kpsF, rfaC) and phospholipid synthesis (plsX). Other membrane-associated structures repressed by iron included a putative phosphate permease and one of the ferric citrate outer membrane receptors (fecA3). In addition to these genes, genes involved in protein synthesis, stress response, nucleotide metabolism and modification, and cofactor biosynthesis were also induced by iron restriction, as were several genes encoding hypothetical proteins (Table 3).

(ii) Iron-induced genes.
Many genes subject to Fur-independent, iron-induced transcriptional regulation encode major virulence factors of H. pylori. These include the VacA vacuolating cytotoxin, the CagA cytotoxin, and the HopA and HP0492 outer-membrane proteins. Also included in this category are genes encoding proteins functioning in stress response, such as the KatA catalase and the chaperones DnaK and ClpB (Table 3). Chemotaxis may also be iron responsive via the tlpB gene, which encodes a methyl-accepting chemotaxis protein. Finally, two genes involved in nucleotide metabolism/modification and eight genes encoding hypothetical proteins displayed Fur-independent, iron-induced expression.

(iii) Abberantly regulated genes.
Seven genes displayed iron-regulated expression in the fur mutant only, but were transcribed in an iron-independent manner in the wild-type strain. This cluster includes the HP0004 gene encoding carbonic anhydrase, the HP1458 gene encoding a putative thioredoxin, which is transcribed at higher levels in iron-replete conditions in the fur mutant, the HP0220 nifS gene, which is involved in the formation of Fe-S clusters (Olson et al., 2000), and three copies of the IS605 transposase (tnpB), whose expression is decreased in iron-replete conditions (Table 3).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In many bacteria, the Fur repressor is the central regulator of iron homeostasis (Andrews et al., 2003; Hantke, 2001). Fur mediates its iron homeostasis function via careful regulation of iron-acquisition and iron-storage systems: in iron-restricted conditions, iron-uptake systems are expressed and iron storage is repressed, but conversely in iron-replete conditions, iron-storage systems are expressed and iron uptake is abolished (Bereswill et al., 2000; Delany et al., 2001a; van Vliet et al., 2002a). The switch between repression and induction of iron uptake is coupled to iron availability in the cytoplasm: when iron is available, a Fur dimer forms a complex with ferrous iron and binds to Fur-binding sequences (Fur boxes) in the promoters of iron-uptake genes (Hantke, 2001). This situation is, however, not as clear for the switch in the repression and induction of ferritin-mediated iron storage: while iron induction of ferritin expression is found in several bacteria, the role of Fur in this process is not universal.

In this study, transcriptional profiling was used to identify H. pylori genes that are regulated by Fur and iron at the transcriptional level. Recent studies focusing on the effects of iron restriction, growth phase and acidic pH on gene expression in H. pylori have indicated that many genes classified in different functional categories are affected by these conditions (Allan et al., 2001; Ang et al., 2001; Kim et al., 2004; Merrell et al., 2003a, 2003b; Thompson et al., 2003; Wen et al., 2003). For 1248 genes, data were obtained on their regulation by iron or by Fur. In our study using the wild-type H. pylori strain 26695, 97 genes displayed iron-responsive regulation and 43 genes displayed Fur-dependent regulation.

Genes regulated by Fe-Fur and apo-Fur are classified in several functional categories (Table 2), indicative of the role of Fur as global regulator in H. pylori. This is consistent with the phenotypes reported for the fur mutant thus far, which displays increased iron uptake (van Vliet et al., 2002a), decreased acid resistance (Bijlsma et al., 2002), and attenuation in a mouse model of H. pylori infection (Bury-Mone et al., 2004). Rather surprisingly, while mutation of fur affects many cellular processes, the fur mutant is not significantly affected in growth under in vitro conditions (Fig. 1a).

Other than the genes functioning in metal metabolism, many of the genes regulated by Fe-Fur and apo-Fur have not been investigated previously and require experimental confirmation of their predicted function. However, based on homology, several of the proteins encoded by Fur-regulated genes are predicted to be iron-cofactored, like the biotin synthetase BioB (Sanyal et al., 1994). The E. coli BioB protein also requires pyridoxal phosphate (Ollagnier-De-Choudens et al., 2002), as synthesized by the PdxA protein, and in H. pylori this gene displays similar regulation to the bioB gene (Table 2). Furthermore, in E. coli, the ksgA gene is cotranscribed with the pdxA gene, and both are growth-phase regulated (Pease et al., 2002), while in H. pylori both genes are subjected to regulation by Fe-Fur (Table 2). Other Fe-Fur-regulated genes include the flaB and flgE genes, and taken together with the iron-responsive regulation of several fli genes (Table 2), this may explain the effect of iron on the motility of H. pylori (Merrell et al., 2003b). Finally, genes regulated by apo-Fur encode iron-cofactored enzymes like hydrogenase and superoxide dismutase (Table 2), and this form of regulation may ensure that these enzymes are only expressed when iron is available. Comparison with Fur and iron regulons in other bacteria is hampered by the lack of operon structure in the H. pylori genome sequence. The most closely related bacterium is Campylobacter jejuni, and recently its Fur and iron regulons were determined (Palyada et al., 2004). Interestingly, both in H. pylori and C. jejuni, motility-associated genes were affected by iron and the mutation of fur, suggesting a common mechanism behind the iron-responsive regulation of motility.

Iron-responsive genes were also recently identified in the mouse-adapted H. pylori strain SS1 (Merrell et al., 2003b; Thompson et al., 2003), and show partial overlap with the iron-responsive genes in our study. Unfortunately, a direct comparison with the two related studies is hampered by the use of different strains of H. pylori and differences in the experimental set-up. An important difference may be that in the previously published studies (Merrell et al., 2003b; Thompson et al., 2003), iron restriction was achieved via the use of 2,2-dipyridyl, which has a high affinity for ferrous iron and is membrane permeable, whereas in our study we used desferal, which is a siderophore-based iron chelator that removes ferric iron from the medium and makes it unavailable for H. pylori (van Vliet et al., 2002a). Comparison of the datasets is further complicated by the difference in H. pylori strains used. The complete genome sequence of H. pylori 26695, the strain used in this study, is available (Tomb et al., 1997), whereas the other two studies were based on H. pylori strain SS1 (Merrell et al., 2003b; Thompson et al., 2003), whose genome sequence is not yet known. Thus the gene order, promoter sequences and regulatory responses of H. pylori SS1 are unknown and may differ significantly from those in H. pylori 26695 (Alm et al., 1999; Israel et al., 2001a, 2001b; Salama et al., 2000; Tomb et al., 1997). However, the majority of Fur-regulated genes identified in our study display iron-responsive regulation (Table 2) and cluster mostly in the group of stationary-phase induced genes (Merrell et al., 2003b). This is consistent with our experimental set-up, since we used late-exponential-phase cells to isolate RNA for the transcriptome studies (Fig. 1a).

Rather surprising was the relative lack of operon structure in the transcriptome data. While several of the genes identified in this study are predicted to be transcribed as part of a multicistronic mRNA, this was not apparent from the array data. An example of this is the pdxA gene, which is predicted to constitute an operon with the upstream pdxJ gene. However, expression of the pdxJ gene seems not to be affected by the fur mutation or by iron restriction (see Supplementary Table S1). This may be partially due to differential mRNA degradation, as was described for the urease operon (Akada et al., 2000), and it is interesting to see that a ribonuclease gene (rnhB) is included in the list of iron-regulated genes (Table 3).

Despite its small genome, H. pylori is a highly successful colonizer of the human gastric mucosa, and is present for life unless eradicated by antibiotic treatment (Blaser & Berg, 2001). Its potential to adapt to hostile environmental niches with changing conditions is apparent, despite the relative paucity of transcriptional regulators. One of the possibilities explaining such adaptive capacity with relatively few regulators is that these regulators have broadened their regulatory potential, and this seems to be the case for H. pylori Fur. This protein, well known for its central role in iron homeostasis in bacteria, controls the expression of different pathways involved in normal metabolism, stress resistance, motility and virulence. This central role in these important pathways makes it a prime candidate for further study on the role of bacterial adaptation in long-term colonization of hostile environmental niches.


   ACKNOWLEDGEMENTS
 
We are indebted to Professor Michael Hecker, in whose laboratories part of this work was carried out. We thank Dr D. Scott Merrell for communicating results prior to publication. This study was financially supported by grants 901-14-206 and DN93-340 from the Nederlandse Organizatie voor Wetenschappelijk Onderzoek to A. H. M. v. V. and J. G. K., respectively, and grant Ki201/9-1 of the Deutsche Forschungsgemeinschaft to M. K.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 15 June 2004; revised 25 October 2004; accepted 2 November 2004.