SEARCH

SEARCH BY CITATION

Keywords:

  • ferric uptake regulator;
  • growth phase;
  • Helicobacter pylori;
  • proteome profile

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The ferric-uptake regulator (Fur) protein is an Fe2+-dependent transcriptional repressor. To clarify the global regulation of Helicobacter pylori proteins by Fur according to the growth phase, we compared the proteome profiles of H. pylori 26695 and its isogenic fur mutant, harvested during in vitro culture. Clustering analysis of the proteome profiles of the two strains revealed that the growth-phase-dependent protein regulation in the wild-type strain was largely altered in the fur mutant. Reverse transcriptase-PCR analysis of several H. pylori genes showed that a major switch in transcription occurred 12 h earlier than in the wild type, indicating that the fur mutation induced an earlier transcriptional switch from log to stationary phase. Several H. pylori proteins also showed changes in their patterns of protein post-translational modification (PTM). In particular, the HydB protein, which was detected as four spots on 2-dimensional electrophoresis gels, underwent two types of PTM, which were under different kinds of regulation. These data demonstrate that a fur mutation affects the growth-phase-dependent regulation of proteins and mRNAs, suggesting a role for Fur in controlling the global regulation of cellular processes in response to changing growth environments.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Ferric-uptake regulator (Fur) is a global transcriptional repressor present in many Gram-negative and some Gram-positive bacteria (Crosa, 1997). Dimeric Fur forms a complex with ferrous ion, and the complex binds to specific consensus sequences, called Fur-box, present in the target promoters of iron-regulated genes and consequently represses the transcription of the genes in an iron-rich environment (Baichoo & Helmann, 2002). In several Gram-negative pathogens, Fur represses the transcription of genes involved in iron transport but also in the detoxification of oxygen radicals, acid tolerance, the production of virulence factors, and metabolic processes (Hassett et al., 1996; Ochsner & Vasil, 1996; Horsburgh et al., 2001). However, in some cases, Fur also acts as a positive regulator rather than a negative regulator (Hall & Foster, 1996; Dubrac & Touati, 2000).

Helicobacter pylori is a microaerophilic Gram-negative human pathogen that is associated with chronic gastritis, peptic ulcers, and gastric cancer (Blaser & Atherton, 2004). Like other Gram-negative bacteria, H. pylori expresses the Fur protein, which regulates genes in iron-uptake systems in an iron-dependent manner (Delany et al., 2001; van Vliet et al., 2002). Helicobacter pylori Fur is also involved in acid tolerance, the detoxification of reactive oxygen species, and energy metabolism, suggesting a role for Fur as a global regulatory protein in H. pylori (Bijlsma et al., 2002; van Vliet et al., 2003; Alamuri et al., 2006). Transcriptome analysis of the growth-phase-dependent response of H. pylori to iron starvation and of an H. pylori fur mutant cultured in iron-depleted and replete media identified numerous genes regulated in an iron-dependent manner by Fur (Merrell et al., 2003; Ernst et al., 2005). In our previous study, a comparison of the proteome profiles of H. pylori wild type and its isogenic fur mutant cultured under iron-rich and iron-depleted conditions also revealed diverse groups of H. pylori genes under Fur-mediated control (Lee et al., 2004). This confirms a role for Fur as a global regulator of gene expression in H. pylori and suggests the presence of Fur-mediated positive regulation as well as negative regulation of genes in H. pylori. Danielli et al. (2006) identified Fur regulons using immunoprecipitation analysis of Fur-chromatin complexes, some of which included genes that are positively regulated by Fur.

In pathogenic bacteria, the expression of genes responsible for bacterial survival in the host and for virulence are often influenced by the growth phases of the bacteria (Bachman & Swanson, 2001; Gaynor et al., 2005). Thompson et al. (2003) analyzed the transcriptional profiles of H. pylori harvested during in vitro culture and showed that there is a transcriptional switch in the expression of the virulence gene between the late log and stationary phases during culture. Genes related to virulence were induced at this stage of culture, suggesting that the late log phase may correspond to the most virulent phase of growth in H. pylori. The expression of genes involved in iron homeostasis also changed dramatically at the switch, and bacteria in log and stationary phases responded differently to iron starvation, suggesting that H. pylori cells differ physiologically during various growth phases and respond differently to environmental changes (Merrell et al., 2003; Thompson et al., 2003).

In a previous study, to clarify the global regulation of H. pylori proteins in response to changing growth phases, we analyzed the proteome profiles of H. pylori cultured in vitro and observed a substantial discordance in the mRNA and protein patterns (Choi et al., 2008). In this study, we investigated whether Fur plays a role in the regulation of proteins in response to the growth phase in this human pathogen.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Bacterial strains and culture conditions

Helicobacter pylori strain 26695 was purchased from the American Type Culture Collection (Manassas, VA). An isogenic fur mutant strain of H. pylori 26695 was kindly provided by Dr A. van Vliet of Erasmus MC University, the Netherlands (Bijlsma et al., 2002). The bacteria were precultured for 24–36 h on Brucella broth agar plates containing 10% horse serum at 37 °C in an incubator under a 10% CO2 atmosphere. Cultured H. pylori were collected from the agar plates, and the suspensions were adjusted with Brucella broth to an OD600 nm of 1.0 and diluted to an OD600 nm of 0.05 with Brucella broth supplemented with 10% newborn bovine serum. Aliquots (20 mL) were distributed into six 100-mL flasks, which were filled with mixed gas (N2 : CO2 : O2=85 : 10 : 5, v/v/v). Bacterial cells were cultured at 37 °C with agitation at 200 r.p.m., and one flask each was taken for cell harvest at 6, 12, 24, 36, 48, and 60 h. Helicobacter pylori cells were harvested by centrifugation at 5000 g for 10 min at 4 °C, washed in ice-cold phosphate-buffered saline (PBS, pH 7.0), aliquoted, and kept frozen at −70 °C until use.

Proteome analysis

Preparation of bacterial cell lysates, isoelectric focusing, and second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed as described previously (Lee et al., 2004). Image analysis of 2-dimensional electrophoresis (2-DE) gels was performed using pdquest software, as described by the manufacturer (BioRad, Hercules, CA). The reference pattern used for the spot analysis was obtained from 2-DE gels of the wild-type culture harvested at 12 h, as described previously (Choi et al., 2008), and protein spots detected only in the fur mutant were added to the reference pattern. All spot files were matched to the reference pattern, and each matched spot on the gels was numbered. The quantity of protein in each spot was normalized to the total valid spot intensity. 2-DE analysis was carried out three times using cultures independently grown on different days. Protein spots that were detected at all time points in either the wild type or fur mutant were selected and analyzed further. Fold changes in the protein levels were determined by comparison with the 6 h culture of the wild type or with that of each corresponding strain, and the mean fold change from three gels was calculated. Self-organizing map (SOM) analysis was performed using the avadis program (Strand Genomics, Bangalore, India) to group and display protein spots with similar profiles. Protein spots showing altered intensity on 2-DE gels were identified by enzymatic digestion of spots with trypsin, Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis followed by database search as described previously (Choi et al., 2008).

Semiquantitative reverse transcriptase (RT)-PCR analysis

Total cellular RNA was isolated from the harvested bacteria using TRIzol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. The purified RNA was quantified spectrophotometrically and resolved on an ethidium bromide-containing agarose gel to check its integrity and correct quantification. To measure mRNA transcript levels, RT–PCR was performed using gene-specific primers, as described previously (Choi et al., 2008).

Measurement of total intracellular iron content using inductively coupled plasma (ICP)-atomic emission spectrometry

The levels of total intracellular iron accumulated in H. pylori strains were determined for bacterial cells grown in liquid media for 12 h. Bacterial lysate containing 500 μg of protein was dried in a glass beaker and, to release the iron from the storage proteins, acid hydrolyzed by boiling in concentrated HNO3 for 3 h. After evaporation of the acid, the iron content of the sample was analyzed using an Ultima 2C ICP-atomic emission spectrometer (Horiba Jobin Yvon, Longjumeau, France) at the Korea Basic Science Institute (Seoul, Korea).

Statistical analysis

One-way anova was applied to the proteomic data using the avadis program to identify spots differentially regulated in response to the growth phase in the fur mutant (95% confidence interval). Two-way anova was used to identify spots differentially regulated in the fur mutant in response to the growth phase. A two-tailed Student's t-test was used to determine statistical differences in intracellular iron level between two strains, and P values <0.05 were considered significant.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Comparison of the proteome profiles of H. pylori wild type and fur mutant in various growth phases

To investigate the role of H. pylori Fur protein in protein regulation in response to the growth phase, we analyzed the proteome profiles of an H. pylori 26695 fur mutant strain harvested at various time points, using 2-DE, and compared them with those of the isogenic wild-type strain. The H. pylori 26695 fur mutant was cultured in liquid medium and harvested after 6, 12, 24, 36, 48, and 60 h. The growth curve obtained from the fur mutant showed a pattern similar to the parent strain till 48 h but declined thereafter (see Supporting Information, Fig. S1). Gram staining revealed that coccoid forms appeared with considerably high frequency in the fur mutant at 48 h while no coccoid forms were observed in the wild type till 60 h. Whole-cell lysates prepared from cultures harvested at each time point were subjected to 2-DE analysis, and their proteome profiles were obtained (see Fig. S2). A reference pattern for spot analysis was obtained from a gel of a 12-h culture of the H. pylori wild type from a previous study (Choi et al., 2008), and spots that were detected only in the fur mutant in this study were also added to the reference pattern. Each spot on the 2-DE gels was matched to the reference pattern and the spot intensities of each gel were determined. To obtain reliable data, we repeated the 2-DE analysis twice, with three sets of independent cultures grown on different days. In total, 1368 protein spots were detected on 2-DE gels, with an average of 488 spots on each gel. No significant differences in the spot numbers were observed between the two strains or among the cultures in various growth phases. A total of 323 protein spots were present on all 18 gel images of either the wild type or fur mutant strain, and were used for further analysis. Among the 323 protein spots, spots identified by MALDI-TOF analysis are listed with their levels in Table S1.

To determine the effects of H. pylori Fur on protein regulation as a function of the growth phase, the ratio of spot intensity at each time point relative to that of the 6 h culture of the wild type was calculated and used for SOM clustering analysis. The results revealed prominent changes in the overall protein profiles of the fur mutant throughout the culture period when compared with the wild type (Fig. 1a). About 20% of the spots were negatively regulated and 55% were positively regulated by Fur. A substantial reduction in intensity at 6 h was observed for many protein spots in the fur mutant, compared with the wild type. The SOM cluster of the 157 Fur-regulated spots clearly showed that the fur mutation tends to further intensify the growth-phase-dependent upregulation or downregulation of proteins (Fig. 1b). We used two-way anova to select those spots whose regulation was modulated by the fur mutation and/or the growth phase (Fig. 1c). The regulation of 157 spots was significantly influenced by the fur mutation and 101 spots by the growth phase. Among these, 60 spots were under both Fur- and growth-phase-mediated regulation, and 15 spots were regulated by the interaction between the two parameters, i.e. Fur and the growth phase affected the levels of the spots in opposite ways. Taken together, these data demonstrate that the fur mutation caused major changes in protein regulation in response to the growth phase, suggesting a great disturbance of the intracellular homeostasis in this mutant strain.

image

Figure 1.  Comparison of the protein spots of the Helicobacter pylori 26695 wild type and its isogenic fur mutant during in vitro culture. The cluster diagrams show SOMs of the profiles of 323 protein spots detected on all three sets of gels for either the wild type or fur mutant (a), and the profiles of 157 protein spots with significantly altered intensities between the two strains (b). The values shown are the relative intensities of the spots compared with those of the wild-type 6-h culture, and represent the means of three spots from different gels. In the clustering diagram, red color of increasing intensity indicates increasing protein levels, and green color indicates decreasing protein levels. Note that the bar denotes log10 of the fold-change in spot intensity. The clustering diagram for the wild-type strain has been described in a previous report and is shown for comparison (Choi et al., 2008). (c) A Venn diagram of the protein spots whose regulation was significantly modulated by either growth phase or the fur mutation, or by an interaction between the two factors, as determined by two-way anova (95% confidence interval).

Download figure to PowerPoint

Among 101 spots regulated according to the growth phase, 48 spots had been identified in our previous studies, and 53 of the 157 spots regulated by Fur had been identified (Lee et al., 2004; Choi et al., 2008). Some of the spots displaying differential regulation are shown with their types of regulation in Fig. 2a. Among them, hydantoin utilization protein A (HyuA) was detected as two spots, whose regulation pattern is shown in Fig. 2b. Both spots were under positive Fur regulation (two-way anova, P<0.0001), but only 7712R was under growth-phase-dependent regulation (P<0.05). Considering that hyuA mRNA levels were largely similar in the wild type and fur mutant strains (see Fig. 3, below), it is thought that in the fur mutant both HyuA protein spots were rapidly degraded by a protease(s) to basal levels regardless of the type of post-translational modification (PTM). This may have resulted in a greater reduction in protein level of 7712R, which showed higher spot intensity than 7705R at 12 h and thus yielded a growth-phase-dependent regulation pattern with 7712R.

image

Figure 2.  Identification of Helicobacter pylori protein spots differentially regulated by growth phase and/or Fur during in vitro culture. Different types of protein regulation are shown (a), and the types of regulation are marked on the spot numbers: agrowth-phase-dependent regulation, bFur-dependent regulation, and cregulation by an interaction between Fur and growth phase. The levels of the two spots for HyuA in the wild type and fur mutant are shown as means±SD of the values obtained from 2-DE gels in three independent experiments (b).

Download figure to PowerPoint

image

Figure 3.  Regulation of mRNA transcripts in the Helicobacter pylori fur mutant during in vitro culture. Three independent sets of cultures were analyzed and a representative set is shown. The mRNA transcript levels in the wild-type strain have been presented in a previous report and are shown here for comparison (Choi et al., 2008).

Download figure to PowerPoint

Modulation of the growth-phase-dependent regulation of H. pylori genes by a fur mutation

To determine whether the proteins whose levels changed according to the growth phase are regulated at the transcriptional level, we measured the mRNA transcript levels of 25 genes by semiquantitative RT-PCR analysis, including the 23S rRNA gene and fur gene as controls, and compared the expression patterns of these 23 genes of the fur mutant with those of the wild type. The results indicate that the genes can be classified into six groups according to their mRNA expression patterns as a function of growth phase and Fur (Fig. 3). Group I exhibited high transcript levels from 6 to 12 h in the fur mutant but underwent a marked reduction thereafter, indicating that a major switch in transcription occurs between 12 and 24 h in the fur mutant, which is 12 h earlier than that in the wild type. This result suggests that the fur mutation induces an earlier transcriptional switch from log to stationary phase. The expression of genes in Group II correlated inversely with the growth phase of the culture in the wild type, with an increase at 60 h (Choi et al., 2008), which was either shifted to 48 h or abolished in the fur mutant. This is consistent with the 12 h shift in the growth phase caused by the fur mutation. HP0697 represented another group of genes, whose mRNA transcripts remained constant throughout the culture but gradually decreased over time in the fur mutant. The genes in group IV are those whose expression was not responsive to the growth phase in the wild type but was induced in the fur mutant and came under growth-phase-dependent regulation. In contrast, the frdB gene in group V showed higher expression in the fur mutant but was not responsive to the growth phase in either strain. The expression of three genes in group VI did not show either growth phase or Fur dependence in either strain. Taken together with the proteomic data, these findings demonstrate that the growth-phase-responsive regulation of H. pylori genes is influenced by Fur, resulting in aberrant responses to growth phases in the fur mutant.

In the study using DNA microarrays, Ernst et al. (2005) compared the transcriptional profiles of H. pylori wild type and fur mutant strains and identified numerous genes regulated by Fur and iron. We compared the data obtained from our proteomic analysis with those from the study by Ernst and colleagues. There were only a few genes overlapped between the two studies, including pfr, amiE and hydB, all of which were negatively regulated by Fur. In this study as well as in our previous study, we observed a great discrepancy between the mRNA and protein levels of several genes obtained during the in vitro culture (Choi et al., 2008), which, we believe, is the reason for the lack of conformity between the studies.

Changes in the relative intensity of H. pylori protein spots caused by the fur mutation

Proteins often exist in several forms and appear as more than one spot on 2-DE gels. This has also been observed in H. pylori proteome studies (Jungblut et al., 2000; Cho et al., 2002). In a previous study, we assessed the relative intensities of multiple spots for each protein during the course of in vitro culture of the H. pylori wild type and observed that several H. pylori proteins underwent modifications, which were regulated as a function of the growth phase (Choi et al., 2008). In this study, we examined whether the fur mutation influences the relative ratios of H. pylori proteins (Fig. 4). The Pfr protein was present as five spots on 2-DE gels of the fur mutant, as seen in the wild type (Choi et al., 2008), and the relative ratios of the Pfr spots did not vary greatly between the two strains throughout the culture period. For SerA, which appeared as two spots on 2-DE gels, both spots were significantly lower in intensity in the fur mutant than in the wild type but the spot ratio remained constant as a function of the growth phase. In contrast, only one spot for ATP-dependent protease (Lon) was regulated in a growth-phase-dependent manner in the fur mutant, suggesting the differential regulation of the PTM of this protein in response to the growth phase in the two strains. Similarly, HP0697 exhibited an alteration in the relative spot ratio as a function of the growth phase in the fur strain. The opposite case was observed with FrdB, for which the relative ratio of its spots was modulated as a function of the growth phase in the wild type, but this was abolished in the fur mutant.

image

Figure 4.  Comparison of protein spot levels in Helicobacter pylori 26695 wild type and fur mutant. The mean intensities of the protein spots obtained from 2-DE gels (bar) and their mRNA levels determined by RT-PCR (line) are shown. Immunoblots of two proteins are shown below graphs.

Download figure to PowerPoint

Of particular interest is the quinone-reactive Ni/Fe hydrogenase large subunit (HydB), which was detected as four spots on 2-DE gels (Fig. 2a). In the wild-type strain, two spots, 8709R and 8717R, gradually increased in intensity during the culture period, whereas the intensity of the other two remained relatively constant (Choi et al., 2008). However, in the fur mutant, the pattern of the spot intensities of HydB was significantly different from that of the wild type. Three of the four spots (8709R, 8716R, and 8717R) were under growth-phase-dependent regulation, and 8716R and 8717R were regulated in opposite ways in response to the growth phase (Table 1). Moreover, all four spots were under Fur-mediated regulation. On 2-DE gels, the four HydB spots were aligned as a parallelogram. The differences in the observed molecular weights (MW) and pIs between each pair of two spots appeared to be the same, suggesting that 8709R and 8717R undergo identical type of modification(s) and 8709R and 8707R another type of modification(s).

Table 1.   Relative intensity of Helicobacter pylori HydB protein spots
Spot no.Relative spot intensity to total intensity (%)*Significance in intensity (P value)
Wild type (h)fur mutant (h)Growth phaseFur
6122436486061224364860
  • *

    The intensity of four HydB spots was measured on 2-DE gels obtained from three independent experiments, and the relative ratio of each spot intensity to the total HydB spot intensity was calculated.

  • One-way anova was performed using the avadis program to determine the growth phase dependency of protein spots in the fur mutant.

  • Two-way anova was used to determine the significance of the difference in spot intensity between the two strains.

8707R8945642823211710110.2720.000
8709R1417172722265471318190.0200.001
8716R20171371074156492816160.0080.000
8717R5857666162632617234256540.0030.002
8707R+8709R2226213228303327283028300.4520.657
8716R+8717R7874796872706773727072700.0830.703
8707R+8716R2826171216116979704526270.0210.000
8709R+8717R7274838884893121305574730.0020.001
Sum of four spots1001001001001001001001001001001001000.1570.666

In the fur mutant, 8717R initially represented only 26% of the total intensity of the HydB protein spots detected on 2-DE gels but gradually increased to 56% of the protein at 48 h, whereas 8716R showed the opposite response to the growth phase (Table 1). The spots 8707R and 8709R also showed growth-phase-dependent patterns in their relative ratios, which were similar to those of 8716R and 8717R, respectively. Interestingly, the relative ratio of 8707R+8709R to 8716R+8717R remained constant throughout the culture in both strains, although the intensity of 8716R+8717R showed growth-phase-dependent regulation. Furthermore, neither combination of spots exhibited any Fur dependence. These results suggest that the PTM of 8707R and 8709R, which yielded 8716R and 8717R, respectively, may not be influenced by Fur or the growth phase. In contrast, two other pairs of HydB spots, 8707R/8716R and 8709R/8717R, were under Fur-mediated regulation in a growth-phase-dependent manner, and their relative ratios were inversely correlated throughout the in vitro culture of both strains, suggesting the growth-phase-regulated PTM of 8709R/8717R to generate 8707R/8716R. These data clearly indicate that the HydB protein undergoes two types of PTM, which are under different kinds of regulation. The exact type(s) of modification is being investigated.

Accumulation of intracellular iron in the H. pylori fur mutant

Helicobacter pylori pfr encoding an iron-storage protein is negatively regulated by Fur in an iron-dependent manner, and the Pfr protein is synthesized in excess in the absence of fur expression (Bereswill et al., 1998). The accumulation of Pfr should increase the capacity of the mutant to store iron. Because Fur represses the expression of genes involved in iron uptake in response to environmental iron, a fur mutation would also cause the deregulation of iron uptake. Thus, we presume that the intracellular iron content of a fur mutant would be changed and assessed the iron levels of the two strains harvested at 12 h. Our results show that the intracellular iron level of the fur mutant (18.70±2.93 mg g−1 protein) was twice as high as that of the wild type (9.29±0.02 mg g−1, P<0.05), which is consistent with the previous report by van Vliet et al. (2002). The wild type tended to remain constant in the iron level throughout the culture period, while the fur mutant showed the highest level at 6 h and decreased thereafter (data not shown). The breakdown of iron homeostasis would cause substantial changes in the intracellular redox status and increase oxidative stress. We have not determined whether the high iron content in the fur mutant results from an increase in free iron or in the bound form. However, it is likely that uncontrolled iron uptake and low antioxidant activity result in increased oxidative stress in the fur mutant, which is unfavorable for bacterial cells in maintaining their normal cellular processes.

In conclusion, to investigate the role of Fur in global cellular protein regulation in response to the growth phase, we compared the proteome profiles of the H. pylori wild type and the fur mutant grown in in vitro culture. Our results reveal that the fur mutation induced major changes in the temporal regulation of proteins in H. pylori. Several H. pylori proteins also exhibited changes in their PTM patterns according to the growth phase. The fur mutation also caused a premature switch in the transcription of several genes and abolished the temporal regulation of the mRNA transcripts of H. pylori genes. Although Fur is known to be a transcriptional repressor, and transcription is thought to be the major step at which gene expression is regulated, the data collected in this study suggest that H. pylori Fur plays a critical role in controlling, directly or indirectly, the overall cellular processes by regulating both proteins and mRNAs in response to changing growth environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are very grateful to Dr A. van Vliet of Erasmus MC University, the Netherlands, for providing the H. pylori fur mutant strain. This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare (no. A080323).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Alamuri P, Mehta N, Burk A & Maier RJ (2006) Regulation of the Helicobacter pylori Fe–S cluster synthesis protein NifS by iron, oxidative stress conditions, and fur. J Bacteriol 188: 53255330.
  • Bachman MA & Swanson MS (2001) RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase. Mol Microbiol 40: 12011214.
  • Baichoo N & Helmann JD (2002) Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence. J Bacteriol 184: 58265832.
  • Bereswill S, Waidner U, Odenbreit S, Lichte F, Fassbinder F, Bode G & Kist M (1998) Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori. Microbiology 144: 25052516.
  • Bijlsma JJ, Waidner B, Van Vliet AH, Hughes NJ, Hag S, Bereswill S, Kelly DJ, Vandenbroucke-Grauls CM, Kist M & Kusters JG (2002) The Helicobacter pylori homologue of the ferric uptake regulator is involved in acid resistance. Infect Immun 70: 606611.
  • Blaser MJ & Atherton JC (2004) Helicobacter pylori persistence: biology and disease. J Clin Invest 113: 321333.
  • Cho MJ, Jeon BS, Park JW et al. (2002) Identifying the major proteome components of Helicobacter pylori strain 26695. Electrophoresis 23: 11611173.
  • Choi YW, Park SA, Lee HW, Kim DS & Lee NG (2008) Analysis of growth phase-dependent proteome profiles reveals differential regulation of mRNA and protein in Helicobacter pylori. Proteomics 8: 26652675.
  • Crosa JH (1997) Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol R 61: 319336.
  • Danielli A, Roncarati D, Delany I, Chiarini V, Rappuoli R & Scarlato V (2006) In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis. J Bacteriol 188: 46544662.
  • Delany I, Spohn G, Rappuoli R & Scarlato V (2001) The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol Microbiol 42: 12971309.
  • Dubrac S & Touati D (2000) Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter. J Bacteriol 182: 38023808.
  • Ernst FD, Bereswill S, Waidner B, Stoof J, Mader U, Kusters JG, Kuipers EJ, Kist M, Van Vliet AH & Homuth G (2005) Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression. Microbiology 151: 533546.
  • Gaynor EC, Wells DH, MacKichan JK & Falkow S (2005) The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes. Mol Microbiol 56: 827.
  • Hall HK & Foster JW (1996) The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition. J Bacteriol 178: 56835691.
  • Hassett DJ, Sokol PA, Howell ML, Ma JF, Schweizer HT, Ochsner U & Vasil ML (1996) Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J Bacteriol 178: 39964003.
  • Horsburgh MJ, Ingham E & Foster SJ (2001) In Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J Bacteriol 183: 468475.
  • Jungblut PR, Bumann D, Haas G, Zimmy-Arndt U, Holland P, Lamer S, Siejak F, Aebischer A & Meyer TF (2000) Comparative proteome analysis of Helicobacter pylori. Mol Microbiol 36: 710725.
  • Lee HW, Choe YH, Kim DK, Jung SY & Lee NG (2004) Proteomic analysis of a ferric uptake regulator mutant of Helicobacter pylori: regulation of Helicobacter pylori gene expression by ferric uptake regulator and iron. Proteomics 4: 20142027.
  • Merrell DS, Thompson LJ, Kim CC, Mitchell H, Tompkins LS, Lee A & Falkow S (2003) Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect Immun 71: 65106525.
  • Ochsner UA & Vasil ML (1996) Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. P Natl Acad Sci USA 93: 44094414.
  • Thompson LJ, Merrell DS, Neilan BA, Mitchell H, Lee S & Falkow S (2003) Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression. Infect Immun 71: 26432655.
  • Van Vliet AH, Stoof J, Vlasblom R et al. (2002) The role of the Ferric Uptake Regulator (Fur) in regulation of Helicobacter pylori iron uptake. Helicobacter 7: 237244.
  • Van Vliet AH, Stoof J, Poppelaars SW, Bereswill S, Homuth G, Kist M, Kuipers EJ & Kusters JG (2003) Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor. J Biol Chem 278: 90529057.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fig S1. Growth curves of H. pylori 26695 wild type and its isogenic fur mutant strains. Shown are the mean ± SD of values obtained from three independent experiments. The arrows indicate the time points at which there are marked decreases in mRNA transcript levels for a number of genes in each strain.

Fig S2. Two-DE profiles of whole-cell lysates of an H. pylori 26695 fur mutant harvested during in vitro culture. A, 6 h; B, 12 h; C, 24 h; D, 36 h; E, 48 h; F, 60 h. Gels shown are representative of three independent sets of six cultures, and the observed protein MW and pI are shown

Table S1. Summary of protein spots on 2-DE gels of a Helicobacter pylori 26695 fur mutant

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
FML_1557_sm_suppFigs.doc14413KSupporting info item
FML_1557_sm_TableS1.xls48KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.