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
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ABSTRACT |
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Present address: Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle, UK.
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INTRODUCTION |
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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.
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METHODS |
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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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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 -[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 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.
-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 ml1) 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.
-Galactosidase activity.
-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 ml1 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 ml1 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 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 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 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
).
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RESULTS AND DISCUSSION |
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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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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 fur mutant
In order to further investigate transcriptional regulation by fur in B. cereus and its role in virulence, a B. cereus null mutant (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 ml1 iron, while the wild-type was approximately three times lower (1 mg ml1).
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
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
fur strains under iron-rich and iron-depleted growth conditions. B. cereus 569 and
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
-galactosidase activity as described. Fig. 5(b)
shows the expression level of dhbA in the presence and absence of iron in wild-type and
fur strains. Expression of dhbA is induced by iron starvation in the wild-type and strongly repressed under iron-rich conditions. In contrast, the
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
fur mutant.
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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 fur mutant. Whereas wild-type B. cereus has LD50=1859 c.f.u. (11422774), which is comparable to the entomopathogen B. thuringiensis with LD50=1821 c.f.u. (14992245), the mutant strain has a significantly increased LD50 value of 4932 c.f.u. (36086912). 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
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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Agaisse, H., Gominet, M., Okstad, O. A., Kolsto, A. B. & Lereclus, D. (1999). PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol Microbiol 32, 10431053.[CrossRef][Medline]
Badger, J. L. & Kim, K. S. (1998). Environmental growth conditions influence the ability of Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum resistance. Infect Immun 66, 56925697.
Baichoo, N., Wang, T., Ye, R. & Helmann, J. D. (2002). Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol Microbiol 45, 16131629.[CrossRef][Medline]
Bone, E. J. & Ellar, D. J. (1989). Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol Lett 49, 171177.[Medline]
Brennan, M., Thomas, D. Y., Whiteway, M. & Kavanagh, K. (2002). Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunol Med Microbiol 34, 153157.[CrossRef][Medline]
Bsat, N. & Helmann, J. D. (1999). Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo. J Bacteriol 181, 42994307.
Bsat, N., Herbig, A., Casillas-Martinez, L., Setlow, P. & Helmann, J. D. (1998). Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29, 189198.[CrossRef][Medline]
Chen, L., James, L. P. & Helmann, J. D. (1993). Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions. J Bacteriol 175, 54285437.[Abstract]
Finney, D. J. (1952). Probit Analysis. Cambridge: Cambridge University Press.
Forbes, J. R. & Gros, P. (2001). Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions. Trends Microbiol 9, 397403.[CrossRef][Medline]
Franza, T., Sauvage, C. & Expert, D. (1999). Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: role of the Fur repressor protein. Mol PlantMicrobe Interact 12, 119128.[Medline]
Geyid, A., Fletcher, J., Gashe, B. A. & Ljungh, A. (1996). Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene. FEMS Immunol Med Microbiol 14, 1524.[CrossRef][Medline]
Gohar, M., Okstad, O. A., Gilois, N., Sanchis, V., Kolsto, A. B. & Lereclus, D. (2002). Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics 2, 784791.[CrossRef][Medline]
Grifantini, R., Sebastian, S., Frigimelica, E., Draghi, M., Bartolini, E., Muzzi, A., Rappuoli, R., Grandi, G. & Genco, C. A. (2003). Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. Proc Natl Acad Sci U S A 100, 95429547.
Hall, H. K. & Foster, J. W. (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.
Hantke, K. (1987). Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet 210, 135139.[Medline]
Hantke, K. & Braun, V. (2000). The art of keeping low and high iron concentrations in balance. In Bacterial Stress Responses, pp. 275288. Edited by G. Hengge-Aronis & R. Storz. Washington, DC: American Society for Microbiology.
Helgason, E., Okstad, O. A., Caugant, D. A., Johansen, H. A., Fouet, A., Mock, M., Hegna, I. & Kolsto, A. B. (2000). Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis one species on the basis of genetic evidence. Appl Environ Microbiol 66, 26272630.
Herbig, A. F. & Helmann, J. D. (2002). Metal ion uptake and oxidative stress. In Bacillus subtilis and its Closest Relatives, pp. 405414. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Horsburgh, M. J., Ingham, E. & Foster, S. J. (2001a). 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.
Horsburgh, M. J., Clements, M. O., Crossley, H., Ingham, E. & Foster, S. J. (2001b). PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69, 37443754.
Ismail, N., Olano, J. P., Feng, H. M. & Walker, D. H. (2002). Current status of immune mechanisms of killing of intracellular microorganisms. FEMS Microbiol Lett 207, 111120.[CrossRef][Medline]
Kaito, C., Akimitsu, N., Watanabe, H. & Sekimizu, K. (2002). Silkworm larvae as an animal model of bacterial infection pathogenic to humans. Microb Pathog 32, 183190.[CrossRef][Medline]
Kotiranta, A., Lounatmaa, K. & Haapasalo, M. (2000). Epidemiology and pathogenesis of Bacillus cereus infections. Microbes Infect 2, 189198.[CrossRef][Medline]
Kramer, J. M. & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In Foodborne Bacterial Pathogens, pp. 2170. Edited by M. P. Doyle. New York: Marcel Dekker.
Kurz, C. L., Chauvet, S., Andres, E. & 13 other authors (2003). Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening. EMBO J 22, 14511460.
Lereclus, D., Agaisse, H., Gominet, M. & Chaufaux, J. (1995). Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant. Biotechnology 13, 6771.[Medline]
Lereclus, D., Agaisse, H., Gominet, M., Salamitou, S. & Sanchis, V. (1996). Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase. J Bacteriol 178, 27492756.
Lereclus, D., Agaisse, H., Grandvalet, C., Salamitou, S. & Gominet, M. (2000). Regulation of toxin and virulence gene transcription in Bacillus thuringiensis. Int J Med Microbiol 290, 295299.[Medline]
McHugh, J. P., Rodriguez-Quinones, F., Abdul-Tehrani, H., Svistunenko, D. A., Poole, R. K., Cooper, C. E. & Andrews, S. C. (2003). Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J Biol Chem 278, 2947829486.
Meena, B. S., Kapoor, K. N. & Agarwal, R. K. (2000). Occurrence of multi-drug resistant Bacillus cereus in foods. J Food Sci Technol 37, 289291.
Midura, T., Gerber, M., Wood, R. & Leonard, A. R. (1970). Outbreak of food poisoning caused by Bacillus cereus. Public Health Rep 85, 4548.[Medline]
Neilands, J. B. (1993). Siderophores. Arch Biochem Biophys 302, 13.[CrossRef][Medline]
Nicholson, W. L. & Setlow, P. (1990). Sporulation, germination and outgrowth. In Molecular Biological Methods for Bacillus, pp. 391450. Edited by C. R. Harwood & S. M. Cutting. New York: Wiley.
Panina, E. M., Mironov, A. A. & Gelfand, M. S. (2001). Comparative analysis of FUR regulons in gamma-proteobacteria. Nucleic Acids Res 29, 51955206.
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 24442448.[Abstract]
Pohl, E., Haller, J. C., Mijovilovich, A., Meyer-Klaucke, W., Garman, E. & Vasil, M. L. (2003). Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol Microbiol 47, 903915.[CrossRef][Medline]
Pospiech, A. & Neumann, B. (1995). A versatile quick-prep of genomic DNA from gram-positive bacteria. Trends Genet 11, 217218.[CrossRef][Medline]
Prince, R. W., Cox, C. D. & Vasil, M. L. (1993). Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol 175, 25892598.[Abstract]
Ratledge, C. & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881941.[CrossRef][Medline]
Rowan, N. J., Deans, K., Anderson, J. G., Gemmell, C. G., Hunter, I. S. & Chaithong, T. (2001). Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl Environ Microbiol 67, 38733881.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Stojiljkovic, I., Baumler, A. J. & Hantke, K. (1994). Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a Fur titration assay. J Mol Biol 236, 531545.[CrossRef][Medline]
Thomas, C. E. & Sparling, P. F. (1996). Isolation and analysis of a fur mutant of Neisseria gonorrhoeae. J Bacteriol 178, 42244232.
Touati, D. (2000). Iron and oxidative stress in bacteria. Arch Biochem Biophys 373, 16.[CrossRef][Medline]
Trieu-Cuot, P. & Courvalin, P. (1983). Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3',5''-aminoglycoside phosphotransferase type III. Gene 23, 331341.[CrossRef][Medline]
Xiong, A., Singh, V. K., Cabrera, G. & Jayaswal, R. K. (2000). Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus. Microbiology 146, 659668.[CrossRef][Medline]
Yoshiga, T., Hernandez, V. P., Fallon, A. M. & Law, J. H. (1997). Mosquito transferrin, an acute-phase protein that is up-regulated upon infection. Proc Natl Acad Sci U S A 94, 1233712342.
Received 28 October 2004;
revised 24 August 2004;
accepted 3 November 2004.
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