Bacillus cereus Fur regulates iron metabolism and is required for full virulence

Duncan R. Harvie{dagger}, Susana Vílchez, James R. Steggles and David J. Ellar

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK

Correspondence
Duncan R. Harvie
d.r.harvie{at}ncl.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a {beta}-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The {Delta}fur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.


Abbreviations: EMSA, electrophoresis mobility shift assay

{dagger}Present address: Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle, UK.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Iron is an essential nutrient for bacterial growth, but its availability in the natural environment is limited by the rapid aerobic oxidation of Fe2+ to Fe3+. It is estimated that the concentration of free iron in the natural environment may be as low as 10–18 M, but bacteria require intracellular concentrations of 10–7 M for growth. To overcome this concentration difference, bacteria express receptor proteins on their surface that can bind a wide range of iron-containing compounds, and in addition they secrete high-affinity iron chelators (siderophores) into the environment which are then imported back into the cell as ferri-siderophore complexes (Neilands, 1993; Ratledge & Dover, 2000). Despite the need for bacteria to accumulate intracellular iron, tight regulation of this process is essential to prevent the accumulation of iron to a level which is toxic to the cell. Excess intracellular iron creates an oxidizing environment via Fenton chemistry which can cause irreparable cell damage (Touati, 2000).

In many bacteria, the control of iron uptake and storage is regulated by the transcriptional repressor Fur (ferric uptake repressor). In several bacteria, Fur has been shown to regulate 40 or more genes in many metabolic pathways (Grifantini et al., 2003; McHugh et al., 2003) and has also been shown to play a role in the response to acid and oxidative stress (Hall & Foster, 1996; Horsburgh et al., 2001a). The crystal structure of Fur from Pseudomonas aeruginosa has recently been solved (Pohl et al., 2003). The Fur protein appears to function as pairs of dimers, with two winged helix motifs responsible for DNA interaction.

In Gram-positive organisms, the archetypal iron-responsive regulatory protein was believed to be DtxR, which regulates toxin expression in Corynebacterium diphtheriae (Herbig & Helmann, 2002). Analysis of the Bacillus subtilis genome identified three fur homologues, which encode a ferric uptake repressor (Fur), a zinc uptake repressor (Zur) and a peroxide regulon repressor (PerR) (Bsat et al., 1998). A similar situation has been identified in the genome of Staphylococcus aureus, where Fur and PerR are interacting regulators of genes involved in the oxidative stress response, with the katA catalase gene being co-regulated by both Fur and PerR (Horsburgh et al., 2001a, b).

As well as controlling iron metabolism, Fur has been implicated in the control of virulence factors in a number of organisms. When pathogenic microbes are growing within a higher organism, iron is likely to be of even lower availability, with all available iron tightly bound by host proteins, including transferrin, ferritins, haemoglobins and myoglobulins. In a number of bacterial pathogens, the expression of virulence factors is linked to the availability of iron via the Fur repressor. For example, haemolysin expression in enteropathogenic Escherichia coli and toxin expression in P. aeruginosa (Prince et al., 1993) have been shown to be controlled by Fur homologues. A number of Fur null mutants have been shown to have reduced virulence, highlighting the importance of the Fur regulator for pathogenic microbes and the potential of this protein as a target for chemotherapeutic intervention (Prince et al., 1993; Thomas & Sparling, 1996).

The Bacillus cereus group of organisms includes the entomopathogen Bacillus thuringiensis, the mammalian pathogen Bacillus anthracis and the opportunistic human pathogen B. cereus (Kotiranta et al., 2000). These three have been suggested to be the same species with their host-specific toxins being carried upon plasmids (Helgason et al., 2000). B. cereus causes emetic and enteric food poisoning and accounts for up to 25 % of all reported food-poisoning cases in some countries (Kramer & Gilbert, 1989). B. cereus is also the second most common cause of endophthalmitis and has the potential to cause blindness within 24 h (Kotiranta et al., 2000). The increase in the number of immunocompromised patients has led to an increase in the number of systemic B. cereus infections, which are often nosocomially acquired. Most worryingly, antibiotic-resistant strains of B. cereus have recently been isolated from hospital material, highlighting the requirement for novel classes of antimicrobial agent (Meena et al., 2000).

To date, the only transcriptional regulator of virulence genes identified in B. cereus is PlcR (phospholipase C regulator), which regulates the expression of a number of secreted virulence factors during late-exponential-phase growth (Lereclus et al., 1996). Little research has been carried out on the importance of environmental signals such as iron availability to virulence in this organism. The recent availability of the B. cereus genome sequence has facilitated a systematic approach to this question. In this paper we identify and characterize the iron-responsive regulator Fur from B. cereus and demonstrate that it plays a key role in the virulence of this human pathogen.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The bacterial strains used in this study are shown in Table 1. Bacteria were routinely grown in LB medium with antibiotics at the following concentrations: carbenicillin, 250 µg ml–1; erythromycin, 10 µg ml–1; kanamycin, 20 µg ml–1 for E. coli and 200 µg ml–1 for B. cereus. Minimal medium for siderophore assays was prepared as previously described (Chen et al., 1993). Minimal medium for {beta}-galactosidase assays contained 5 g Casamino acids l–1, 6 g Na2HPO4 l–1, 0·5 g NaCl l–1, 1 g NH4Cl l–1, 1 mg thiamine l–1, 0·1 mM CaCl2, 1 mM MgSO4, 20 mM glucose, 12 µM H3BO3, 0·9 µM ZnCl2, 0·38 µM MnCl2, 2·1 µM CoCl2, 0·15 µM CuCl2, 0·21 µM NiSO4 and 0·31 µM Na2MoO4. Minimal medium was prepared with MilliQ water to avoid iron contamination.


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Table 1. Bacterial strains and plasmids used in this study

 
Subcloning was routinely carried out in E. coli DH5{alpha}. Plasmids were subcloned into E. coli Et12567 prior to transformation into B. cereus 569 as previously described (Bone & Ellar, 1989).

DNA manipulation.
DNA cloning and transformation was carried out according to standard methods (Sambrook & Russell, 2001) using primer sequences described in Table 2. B. cereus genomic DNA was isolated as previously described (Pospiech & Neumann, 1995). Plasmids were purified using the QIAprep spin miniprep kit (Qiagen) and DNA fragments were purified using the QIAquick PCR purification kit. DNA sequencing was carried out on an ABI Prism system (Perkin Elmer).


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Table 2. Oligonucleotides used in this study

Restriction endonuclease sites are underlined.

 
Sequence analysis.
DNA sequence analysis was carried out using the GCG and Vector NTI bioinformatics packages. Whole genome analysis was carried out using the Artemis package (www.sanger.ac.uk/artemis). B. cereus genome data were accessed and analysed using the ERGO-Light package (www.integratedgenomics.com).

Fur overexpression and purification.
The B. cereus fur ORF was amplified using primers DRH45 and DRH46 (sequences given in Table 2) and then cloned into expression vector pET-16b (Novagen) to form plasmid pETfur. Protein expression was carried out in strain BL21(DE3) pLysS according to standard protocols, cells were lysed in Y-Per (Pierce) and recombinant BcFur was purified using Ni-NTA spin columns (Qiagen) according to the manufacturer's instructions. The identity of purified proteins was confirmed by N-terminal sequencing. Recombinant BcFur could be purified in levels of approximately 0·85 mg (g wet cell weight)–1.

Electrophoresis mobility shift assay (EMSA).
Synthetic Fur box sequences were produced by annealing oligonuclotides DRH37 and DRH38 together by heating to 70 °C and then cooling in 1x TE buffer. Double-stranded DNA fragments were radio-labelled using T4 polynucleotide kinase (Promega) and {gamma}-[32P]ATP (Amersham) according to the manufacturer's instructions. EMSAs with purified BcFur were carried out as previously described (Xiong et al., 2000).

Null strain construction.
B. cereus 569 {Delta}fur was constructed by allelic exchange. The 5' and 3' ends of the B. cereus fur gene were amplified by PCR using oligonucleotide primers DRH70, DRH71, DRH72 and DRH73. The kanamycin resistance cassette was amplified from aphA3 of Enterococcus faecalis plasmid pDG783 using primers DRH74 and DRH75 (Trieu-Cuot & Courvalin, 1983). Amplified DNA fragments were cut with appropriate restriction enzymes and then ligated into pUC19 to form pfurkan. The disrupted Fur gene was then amplified using primers DRH70 and DRH73 and ligated into the EcoRV site of the temperature-sensitive suicide vector pRN5101 (Lereclus et al., 1995) to form pRNfur. pRNfur was transformed into B. cereus 569 and transformants were selected on LB media containing kanamycin and erythromycin. Transformants were repeatedly grown at 39 °C in liquid media containing no antibiotics and then replica plated on media containing either erythromycin or kanamycin. Successful knockouts grew only in the presence of kanamycin. The disruption of the fur gene was confirmed by PCR analysis.

Construction of the dhbAlacZ fusion.
{beta}-Galactosidase assays were carried out using the promoterless lacZ plasmid pHT304-18Z (Agaisse & Lereclus, 1994). An approximately 1 kb DNA fragment containing the upstream region of the dhbA gene was amplified using primers SV127 and SV126 from B. cereus 569 chromosomal DNA. The purified fragment was digested with HindIII and BglII and ligated into pHT304-18Z previously digested with HindIII and BamHI. XL-1 Blue chemically competent cells were transformed with the ligation mixture and selected on erythromycin, carbenicillin, X-Gal (40 µg ml–1) LB plates. Transformants were screened by colony PCR using universal and SV120 primers designed to anneal upstream of the polylinker and within the lacZ gene, respectively. A positive clone was designated pSVC15 and confirmed by DNA sequencing.

{beta}-Galactosidase activity.
{beta}-Galactosidase activity was determined as previously described (Nicholson & Setlow, 1990), with minor modifications. Briefly, 100 µl bacterial culture was diluted in 540 µl buffer Z in an Eppendorf tube. Then 160 µl of 2·5 mg lysozyme ml–1 in buffer Z was added and incubated for 5 min at 37 °C; 8 µl of 10 % Triton X-100 was added and the suspensions were briefly vortexed. Then, 200 µl of 4 mg ONPG ml–1 in buffer Z was added and the reaction was incubated at room temperature. Reactions were stopped by the addition of 400 µl of 1 M Na2CO3. Activity was expressed in Miller units.

Siderophore expression measurement.
Wild-type B. cereus 569 and B. cereus 569 {Delta}fur were grown to mid-exponential phase in minimal medium. The yield of siderophores into the culture supernatant was measured as previously described (Chen et al., 1993).

Peroxide resistance.
The peroxide resistance of vegetative B. cereus 569 and B. cereus 569 {Delta}fur was determined as previously described (Horsburgh et al., 2001b). Mid-exponential-phase cells were exposed to 0·25 mM H2O2, aliquots were taken at regular intervals and excess catalase was added to remove any remaining H2O2. At each time point, the number of viable cells was determined and expressed as a percentage of the total cell number at time point zero.

Insect infection.
The virulence of the wild-type and {Delta}fur mutant B. cereus strains was compared by injecting known quantities of vegetative cells into 5th instar Manduca sexta lepidopteran larvae and observing mortality over 48 h. Each dosage was repeated on at least 20 larvae. LD50 values for each strain were calculated by Probit analysis of mortality data (Finney, 1952).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification and cloning of B. cereus fur
The sequence of the B. subtilis fur gene was searched against the B. cereus genome data using the FASTA algorithm (Pearson & Lipman, 1988). A corresponding ORF was identified and predicted to encode a protein 151 amino acids in length. Alignment of the B. cereus and B. subtilis Fur protein sequences (Fig. 1) showed them to be highly conserved, with an identity of 80 % and a similarity of 91 %. The B. cereus Fur (BcFur) sequence also displays a predicted helix–turn–helix motif as is found in many DNA-binding proteins. BcFur contains an HXH motif which has been shown to be essential for metal cofactor binding and the subsequent binding of the protein to the DNA Fur box sequence (Hantke & Braun, 2000). Genes homologous to B. subtilis perR and zur were also identified, and their predicted protein sequences are included in Fig. 1.



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Fig. 1. CLUSTALW alignment of the predicted B. cereus Fur, PerR and Zur amino acid sequences with B. subtilis Fur. Highly conserved residues believed to be involved in the metal binding of each protein are marked with an asterisk. The predicted helix–turn–helix region essential for DNA binding is labelled.

 
The ORFs surrounding fur are identical to those on the B. subtilis genome, but the promoter regions show some sequence divergence. In most studied bacteria, a link between oxidative stress and fur expression is provided by the presence of either an OxyR or PerR binding domain in the promoter region of the fur gene (Touati, 2000). B. cereus fur contains no clear PerR or Fur binding domain. It may be the case, however, that the B. cereus PerR binding box shows significant divergence from the B. subtilis consensus sequence. Until the exact B. cereus PerR binding sequence is determined, the mechanism of regulation of Fur remains to be elucidated.

Complementation of an E. coli fur mutant
The B. cereus fur gene was amplified using primers DRH41 and DRH42. The resulting DNA fragment was ligated to plasmid pGEM-T (Promega) to form plasmid pBcfur. Plasmid pBcfur was transformed into E. coli H1681. This strain is fur and contains a genetic fusion of a fur-controlled gene, fhu, to lacZ (Hantke, 1987). This strain contains no fur gene that can repress fhu expression under iron-rich conditions. When a functional fur gene is transformed into the bacterium, iron-regulated expression of the fhulacZ fusion is restored. When grown under iron-rich conditions, H1681 expresses lacZ in an unregulated fashion. If a functional fur gene is present, lacZ expression will be reduced. Plasmid pBcfur was transformed into E. coli H1681 and lacZ expression levels were determined under iron-rich and iron-depleted conditions. Results are given in Table 3. It can be seen that pBcFur1 partially restored iron-regulated behaviour to the fur mutant, with lacZ expression levels being reduced under iron-rich conditions but returning to similar levels to those of H1681 under iron-depleted conditions. This confirms that the cloned gene does function as a ferric uptake repressor, as predicted from the nucleotide sequence.


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Table 3. Confirmation of function of cloned B. cereus fur gene

A plasmid-borne copy of the gene partially restored iron-regulated gene expression to an E. coli fur mutant. Expression levels are the means of three experiments and are given in Miller units±SD.

 
Purification and characterization of B. cereus Fur
His-tagged BcFur was overexpressed and purified as described in Methods. Eluted proteins were separated on 17 % SDS-PAGE gels, as shown in Fig. 3, with purity of the eluted protein judged to be greater than 90 %. It can be seen that a protein doublet was consistently eluted from the protein purification column, despite the inclusion of protease inhibitor cocktails in all purification buffers. N-terminal sequencing confirmed that the proteins were identical at the N-terminus and therefore likely to be truncated at the C-terminus. As the protein was being used purely to demonstrate DNA binding, no further purification was carried out.



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Fig. 3. Purification of B. cereus Fur on a Ni-NTA spin column. M, molecular mass markers; F, column flow through; W1, wash one (250 mM imidazole); W2, wash two (200 mM imidazole); E1, E2, E3, protein elutions.

 
Recombinant BcFur was used in DNA-binding experiments with radio-labelled synthetic Fur box sequences, as previously described (Xiong et al., 2000). BcFur was shown to bind specifically the Fur box sequence in a metal-dependent manner which was disrupted when the protein sample was incubated with Chelex-100 resin prior to addition to the DNA sample (Fig. 4). This activity is consistent with that observed for S. aureus Fur, in which the addition of Mn2+ to the binding reaction was also found to be essential for DNA binding (Xiong et al., 2000). B. subtilis Fur binding has been observed not to be inhibited by the addition of the metal-chelating agent EDTA to the binding reaction (Bsat & Helmann, 1999). It may be the case that purification of the protein on an immobilized metal affinity chromatography (IMAC) column results in stripping of all cations from the protein which then have to be restored by the addition of Mn2+ to the binding buffer. Purification of the protein without the use of a histidine tag may result in a protein sample which more closely mimics the in vivo situation.



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Fig. 4. EMSA analysis of purified Fur protein binding to the synthetic DNA Fur box. Protein was bound to radiolabelled DNA in 1x EMSA buffer at 25 °C for 30 min and then visualized by separation on a 5 % native polyacrylamide gel. Lanes: 1, labelled Fur box, no protein; 2, labelled Fur box, 10 nM BcFur protein; 5, Chelex-100 treated BcFur; 3, 4, empty lanes.

 
Identification of B. cereus Fur-regulated genes
As Fur is a regulatory protein which has been shown to repress the expression of a large number of genes, the identification of its regulon is of prime importance. This has previously been carried out by a range of methods, including the Fur titration assay (Stojiljkovic et al., 1994), genomic analysis (Panina et al., 2001) and microarray expression analysis (Baichoo et al., 2002). As no DNA microarray is currently available for B. cereus, the identification of Fur box sequences was carried out by genomic analysis. The completed B. cereus genome sequence was searched for occurrences of the consensus Fur box sequence (GATAATGATAATCATTCT) as identified in the promoter region of the dhbA gene (Fig. 2b), which is known to be regulated by Fur in B. subtilis (Bsat & Helmann, 1999). Hits were only accepted if they were within 250 bp of an identified ORF and contained a maximum of two mismatches from the consensus sequence. Identified Fur box regions and their associated ORFs are shown in Table 3.



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Fig. 2. Proposed B. cereus Fur box region, as identified in the promoter region of the dhbA gene, aligned with the highly conserved Fur box of the B. subtilis dhbA gene. The identified Fur box sequences are situated 83 bp and 48 bp upstream of the translational start site of the B. subtilis and B. cereus genes, respectively.

 
The genes predicted to be regulated by Fur are responsible for a number of metabolic functions, including iron uptake and storage and secondary cellular metabolism, and also encompass a number of genes putatively involved in virulence. DNA microarray analysis of the Fur regulon of B. subtilis has revealed that up to 40 genes are altered in expression level in a B. subtilis fur mutant, several of which have no identifiable Fur box region upstream. It has also been found that a number of genes with distinct Fur box sequences in the promoter region are not upregulated in a fur null strain (Baichoo et al., 2002). This suggests that more than one transcriptional regulator is involved in controlling the expression of some predicted members of the Fur regulon. Fur has also been proposed to act as a transcriptional activator with respect to a small number of genes, including the katA catalase gene in S. aureus (Horsburgh et al., 2001b), although this has not been observed in B. subtilis. Interestingly, the hemH ferrochelatase gene, identified as having an upstream Fur box (Table 4), is situated directly upstream of the katX catalase gene on the B. cereus genome. This configuration would suggest negative regulation by Fur, although this remains to be experimentally verified.


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Table 4. Genes identified in the B. cereus genome sequence as having upstream Fur box motifs

The identified Fur box sequence for each gene is given, along with the contig (ORF) number containing each gene and the relative position (in bp) of each Fur box upstream of the proposed start codon. Proposed functions are taken from FASTA searches of each ORF against all available databases.

 
With respect to the pathogenesis of B. cereus, it was interesting to note that a number of predicted cell surface proteins with putative roles in cellular adhesion and invasion were found to have Fur box sequences in their promoter regions. These were two predicted internalins and two cell surface proteins with predicted roles in cellular adhesion and collagen adhesion. The iron-regulated expression of cellular invasion proteins has been observed in a number of bacterial pathogens, including Yersinia pestis and E. coli O157 : H7 (Badger & Kim, 1998; Geyid et al., 1996). Exposure to an iron-deficient environment may be the signal that the bacterium has entered a host, and this is used in turn to trigger expression of proteins involved in adhering to and invading eukaryotic cells. Intracellular growth increases the available pool of nutrients and provides protection from the host immune system. The ability to invade cells and cross physiological barriers is of particular importance in bacterial meningitis, with which B. cereus is being increasingly associated (Kotiranta et al., 2000). In vitro studies have shown that B. cereus can invade a range of mammalian cell types including CaCo-2 and Hep-2 cells (Rowan et al., 2001). Until now, nothing was known about the molecular basis of cellular adhesion and invasion by B. cereus. The analysis of the B. cereus genome sequence presented here suggests that a number of different cell surface proteins may be involved in this process, with iron levels playing a vital role in the regulation of these proteins.

Fur is predicted to regulate the haemolysin hlyII. This is one of the few secreted virulence factors of B. cereus which does not appear to be regulated directly by PlcR (Gohar et al., 2002). Despite the potential regulation of a haemolysin by Fur, no difference in haemolysis between the wild-type and fur mutant strains was detected on human blood agar (results not shown). B. cereus possesses a number of haemolysins, which differ in specificity and regulation (Kotiranta et al., 2000; Lereclus et al., 2000), and therefore a difference in haemolytic potential between the two strains might not be detected by this assay.

Analysis of a B. cereus {Delta}fur mutant
In order to further investigate transcriptional regulation by fur in B. cereus and its role in virulence, a B. cereus null mutant ({Delta}fur) was constructed by allelic replacement, as described in Methods. The viability of Gram-positive fur mutants may result from the possession of a number of other Fur homologues, such as PerR, which still afford the bacterium some capability to respond to peroxide stress (Bsat et al., 1998).

Growth of the fur mutant was found to be slightly reduced in rich media compared to that of the wild-type (data not shown). The iron concentration within the mutant and wild-type bacteria was measured using the Ferrozine dye binding kit (Sigma), according to the manufacturer's instructions. Diluted crude cell lysates of the fur mutant were estimated to be approximately 3 mg ml–1 iron, while the wild-type was approximately three times lower (1 mg ml–1).

Increased intracellular iron is likely to result from deregulated iron uptake by the mutant. Siderophore biosynthesis by the mutant and wild-type was measured, and the results are shown in Fig. 5(a). B. cereus 569 {Delta}fur constitutively expressed siderophores even in the presence of abundant iron in the growth medium, while the wild-type strain did not produce siderophores under iron-replete conditions. The regulation of siderophore production by Fur was confirmed by measuring expression from the dhbA promoter in wild-type and {Delta}fur strains under iron-rich and iron-depleted growth conditions. B. cereus 569 and {Delta}fur-containing plasmid pSVC15 were grown overnight in minimal media containing 0·1 mM FeCl3. Cultures were diluted into identical media to an OD600 of 0·025. Growth was continued for 4 h before 2',2'-dipyridyl was added to one culture at a final concentration of 0·5 mM. After 1 h, each culture was tested for {beta}-galactosidase activity as described. Fig. 5(b) shows the expression level of dhbA in the presence and absence of iron in wild-type and {Delta}fur strains. Expression of dhbA is induced by iron starvation in the wild-type and strongly repressed under iron-rich conditions. In contrast, the {Delta}fur mutant shows derepressed dhbA expression under all growth conditions. These results confirm Fur regulation of dhbA, as predicted by bioinformatics, and also unregulated iron uptake in the {Delta}fur mutant.



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Fig. 5. (a) Siderophore production by B. cereus 569 and B. cereus 569 {Delta}fur under iron-rich and iron-deficient (–Fe) conditions. Cells were grown to mid-exponential phase in defined media, and the level of siderophore secretion was determined from cell-density measurements, as described by Chen et al. (1993). Error bars, 1 SEM. (b) dhbA promoter-dependent {beta}-galactosidase expression in B. cereus 569 and B. cereus {Delta}fur in iron-replete (white bars) and iron-limited (black bars) conditions. Error bars, 1 SEM. (c) Hydrogen peroxide resistance of B. cereus 569 (hatched bars) and B. cereus 569 {Delta}fur (black bars). Viable cells were enumerated along a time-course following the addition of 25 mM H2O2 to mid-exponential-phase vegetative cells. Viable cell numbers are expressed as a percentage of the cell number at time point 0.

 
The increased intracellular iron concentration is likely to result in the production of an increased number of oxidizing free radicals via Fenton chemistry (Touati, 2000). Accordingly, the mutant strain was found to have increased sensitivity to hydrogen peroxide, as shown in Fig. 5(c). This has implications for the survival of the bacterium in an infection model in which the oxidative burst is one of the major defence mechanisms of the eukaryotic immune system (Ismail et al., 2002).

Virulence of fur mutant against an insect infection model
It has been demonstrated in a number of pathogenic bacteria that Fur is essential for full virulence (Franza et al., 1999; Horsburgh et al., 2001a). To test this in the case of B. cereus, its virulence was measured in an insect model of infection. Lepidopteran models are being increasingly utilized for investigating the virulence of bacterial pathogens and give results comparable to those from mammalian infection models (Brennan et al., 2002; Kaito et al., 2002; Kurz et al., 2003). In previous work with B. cereus using insect infection models, the results were highly comparable to those obtained in a mouse model (Agaisse et al., 1999).

The importance of a tightly regulated iron metabolism in bacterial pathogens was demonstrated by the reduced virulence of the B. cereus 569 {Delta}fur mutant. Whereas wild-type B. cereus has LD50=1859 c.f.u. (1142–2774), which is comparable to the entomopathogen B. thuringiensis with LD50=1821 c.f.u. (1499–2245), the mutant strain has a significantly increased LD50 value of 4932 c.f.u. (3608–6912). Values in parentheses are 95 % confidence limits of each calculated LD50 value. The mutant strain is not entirely avirulent, as observed for B. subtilis (LD50>1x105). This demonstrates the importance of other regulators, such as PlcR, which has previously been demonstrated to be essential for the virulence of B. cereus (Agaisse et al., 1999). It also demonstates that other regulators would still be able to coordinate the expression of many virulence factors. It is known that both insects and mammals actively chelate iron in response to bacterial infection, highlighting the importance of iron to the pathogen and the requirement for high-affinity uptake systems (Yoshiga et al., 1997). The reversal of this ‘iron-withholding response’ by the NRAMP family of proteins within macrophage phagolysosomes means that iron uptake by the bacterium must be able to be rapidly and tightly repressed if necessary (Forbes & Gros, 2001). Fur plays an essential role in ensuring that intracellular iron concentrations are maintained within limits which are commensurate with bacterial growth, survival and pathogenesis. Reduced pathogenicity of the B. cereus 569 {Delta}fur mutant further highlights the suitability of Fur as a target for novel antimicrobial agents. The protein is highly conserved throughout a wide range of pathogenic bacteria, both Gram-positive and Gram-negative, and no known homologue exists in mammalian organisms.

This work demonstrates the first analysis of a global environmental regulator to be investigated in a B. cereus-group organism. The correct regulation of iron metabolism has been shown to be essential for the full virulence of the bacterium. The role of environmental regulators such as Fur may be essential in allowing bacteria to survive long enough for virulence gene regulators such as PlcR to stimulate secreted toxin expression, which further enlarges the available niche for bacterial growth. This work demonstrates the importance of environmental stresses, such as iron limitation, to pathogens such as B. cereus. The further elucidation of transcriptional regulators required for the full virulence of pathogenic microbes such as B. cereus will increase the number of potential targets for novel antimicrobial chemotherapeutic agents.


   ACKNOWLEDGEMENTS
 
This work was funded by the BBSRC. Iron concentration determination was kindly carried out by Dr Gerald Maguire, Department of Clinical Biochemistry, University of Cambridge.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 28 October 2004; revised 24 August 2004; accepted 3 November 2004.



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