Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Simon J. Foster. Tel: +44 114 222 4411. Fax: +44 114 272 8697. e-mail: s.foster{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: metals, transcription, zinc, fur, regulation
The GenBank accession number for the sequence reported in this paper is AF101263.
a Present address: Division of Infectious Disease, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
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INTRODUCTION |
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Fur is found in virtually all pathogenic bacteria and also in many non-pathogens. More recently, it has been shown that some organisms contain multiple fur homologues (Bsat et al., 1998 ; Escolar et al., 1999
). The Gram-positive soil microbe Bacillus subtilis is related to Staphylococcus aureus, and encodes three fur homologues (fur, perR and zur) and a homologue of the Corynebacterium diphtheriae iron-regulator dtxR (mntR). B. subtilis Fur binds Fe2+ and regulates the expression of iron-uptake genes (Bsat et al., 1998
). PerR regulates the peroxide regulon and binds to Mn2+ or Fe2+ (Bsat et al., 1998
), while Zur down-regulates two Zn2+-uptake pathways in the presence of Zn2+ (Gaballa & Helmann, 1998
). mntR binds Mn2+ and regulates Mn2+ transport (Que & Helmann, 2000
).
S. aureus is an important bacterial pathogen causing significant morbidity and mortality, particularly in hospitals (where antibiotic-resistant strains are common). Under iron stress, S. aureus produces siderophores for the uptake of iron (Meiwes et al., 1990 ; Haag et al., 1994
; Lindsay & Riley, 1994
; Trivier et al., 1995
; Courcol et al., 1997
), unique cell-associated and exo-proteins which may play a specific role in uptake (Domingue et al., 1989
; Lindsay & Riley, 1994
; Trivier et al., 1995
; Courcol et al., 1997
; Cockayne et al., 1998
), and can access iron bound to transferrin (Modun et al., 1994
, 1998
; Lindsay et al., 1995
; Modun & Williams, 1999
). S. aureus has ORFs corresponding to fur, perR, zur and dtxR which are predicted to be involved in the regulation of these iron responses, and in the regulation of responses to other metal ions. It has been demonstrated recently that fur contributes to virulence and controls siderophore expression and the iron repression of several promoters (Horsburgh et al., 2001
). perR is involved in oxidative stress and iron storage, and is also necessary for virulence (M. J. Horsburgh, M. O. Clements, H. M. Crossley, E. Ingham & S. J. Foster, unpublished). Previously, the S. aureus Fur protein had been overexpressed in Escherichia coli, and bound Fe and putative iron-responsive promoters (Xiong et al., 2000
).
The characterization of the S. aureus zur locus is described here. The role and regulation of the genes mreA, mreB and zur have been investigated. Although we can demonstrate that the genes are involved in Zn2+ regulation, their expression is poor and they are not responsible for all of the S. aureus responses to zinc.
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METHODS |
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Construction of JL100 (zur::tc).
A PCR product spanning 1·46 kb of the zur gene and downstream regions was amplified using pSPW100 (Clements et al., 1999 ), as a template, and the primers 5'-CGTAGTGGATCCGTTGTAAGGCGTTATCACG-3' and 5'-CGTCATGAATTCGCCAATGTAGTCAGGGC-3'. Using the attached restriction endonuclease sites (underlined), the PCR product was digested with BamHI and EcoRI and cloned into pUBS1 (Foster, 1991
) to generate plasmid pJL10 (Table 1
). Two divergent primers internal to the zur gene and 125 bp apart were designed: 5'-GCTAGTAGATCTGGTTTGTACAAGCGATTC-3' with a BglII restriction endonuclease site, and 5'-GCTACTACGCGTGGTGTATGTGAGTCTTGC-3' with a MluI site. By using these primers, a 4·5 kb PCR product was generated, employing pJL10 as the template. This product was cleaved with BglII and MluI and ligated to the tc fragment. A 1·5 kb fragment spanning the tc (tetracycline resistance) cassette was purified from pDG1513 (Guerout-Fleury et al., 1995
) after restriction digestion with BglII and MluI. Ligation of the tc cassette with the 4·5 kb pJL10-derived PCR product and transformation into E. coli DH5
resulted in pJL11 (Table 1
); this contains a copy of the zur region with a 125 bp deletion in the zur gene which had been filled with a tc marker. The mutated zur region was religated from pJL11 by restriction digestion with BamHI and EcoRI and was cloned into similarly digested pMUTIN4 (Vagner et al., 1998
) to generate pJL12.
pJL12 was transformed into S. aureus RN4220, and a single crossover Campbell insertion of the plasmid into the chromosome was confirmed using Southern blotting. A phage lysate was generated from this strain and transduced into 8325-4. Clones were selected for tetracycline resistance and erythromycin sensitivity, indicating an outcross event. The correct construct containing a single copy of the region with a tc insertion in the zur gene was confirmed by Southern blotting (results not shown).
Construction of JL110, JL111, JL120 and JL121.
JL110 (mreB::pMUTIN4) and JL120 (mreA::pMUTIN4) were constructed in a similar manner and are illustrated in Fig. 1. The primers 5'-TCGAGCAAGCTTCGTTGTTAGACGACTATCAC-3' and 5'-TCAGCAGGATCCCAACACGCATTGAGGCAG-3' were used to PCR-amplify a 502 bp fragment internal to mreB. The primers had restriction sites incorporated (underlined), allowing the product to be digested with HindIII and BamH1. Similarly, the primers 5'-TCGACGAAGCTTGTTACTAGGAGTTGTATAG-3' and 5'-TCAGCAGGATCCCTCATCAGTTGTACCATGG-3' were used to amplify a 717 bp fragment of the 5' end of the mreA gene, including the putative ribosome-binding site. The digested fragments were cloned into HindIII- and BamHI-cut pMUTIN4, generating plasmids pJL110 and pJL120, respectively (Table 1
).
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The zur::tc mutation was transduced into JL110 and JL120, generating JL111 and JL121, respectively (Table 1).
Specialist media and growth conditions.
CL medium was developed for this study. It is a completely defined medium based on CDM (Watson et al., 1998 ). The components of the media are (mg l-1): glucose (1000), Na2HPO4 (7000), KH2PO4 (300), adenine sulphate (20), guanine.HCl (20), L-glutamic acid (2220), L-aspartic acid (2220), L-proline (2220), glycine (2220), L-threonine (2220), L-serine (2220), L-alanine (2220), L-lysine.HCl (560), L-isoleucine (560), L-leucine (560), L-histidine (440), L-valine (440), L-arginine (330), L-cystine (220), L-phenylalanine (190), L-tyrosine (170), L-methionine (170), L-tryptophan (60), pyridoxal (0·8), pyridoxamine.2HCl (0·8), D-pantothenic acid (0·4), riboflavin (0·4), nicotinic acid (0·4), thiamin.HCl (0·4) and biotin (0·02). Chelex 100 resin (10 g l-1; Sigma) was mixed for 6 h with the medium to remove ions, and the resin was then removed by filtration. S. aureus has an absolute requirement for magnesium, so CL medium was supplemented with 400 µM MgSO4. Staphylococcal siderophore detection medium (SSD) was prepared and used as previously described (Lindsay & Riley, 1994
). When appropriate, SSD and CL media were supplemented with various ions, typically at the following concentrations: ZnCl2, 20 µM (or 0·02 or 0·2 µM); CaCl2, 400 µM; FeCl3, 20 µM; MnCl2, 20 µM; CoCl2, 200 µM; CuSO4, 200 µM; MbCl2, 200 µM; NiCl2, 200 µM.
Colonies from overnight cultures grown on CL agar plates (CL broth without Chelex treatment, supplemented with 400 µM MgSO4 and 1%, w/v, agar) were inoculated into a CL preculture. Test cultures of 10 ml in 250 ml acid-washed glass flasks were inoculated with preculture to an OD600 of 0·002 and incubated at 250 r.p.m. and 37 °C. All experiments were repeated at least three times.
Assays.
ß-Galactosidase assays were performed as described by Chan & Foster (1998) . One unit of ß-galactosidase activity was defined as the amount of enzyme that catalysed the production of 1 pmol MU min-1 per OD600 unit. Siderophores were assayed by using the chrome azurol S liquid assay on supernatants from SSD cultures, as described by Lindsay & Riley (1994)
.
Pathogenicity study.
A mouse-abscess model of infection was used, as described elsewhere (Chan et al., 1998 ). Briefly, bald mice were inoculated subcutaneously with 1x108 bacteria, either 8325-4 (10 mice) or JL100 (10 mice). After 7 d, the mice were killed, their lesions were removed, and viable bacteria within the lesions were counted. Differences were compared using the MannWhitney U test.
Mass spectrometry.
Cells (12 ml) from 24 h cultures were centrifuged, washed three times with deionized water, autoclaved and then dried. Cells were resuspended in 1 ml 6 M HCl, heated to 60 °C for 3 h, then left at room temperature overnight. Samples were spun in a microfuge for 10 min and supernatants were tested for ion concentrations by MS. The ions tested were Li, Na, Mg, Al, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Sr, Mo, Ba and Pb.
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RESULTS |
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Upstream of zur are two genes predicted to encode the ATP-binding and membrane-bound components of an ABC transporter system, the strongest homology being to Zn2+ transporters and then to other metal-ion transporters (Fig. 1; Table 2
). These have been designated mreA and mreB (metal-responsive elements). The mreAB zur operon has a hairpin-loop structure downstream of zur (
G -6·2 kJ mol-1), suggesting that it is not transcriptionally linked to the next downstream ORF, sodA, encoding a manganese-dependent superoxide dismutase (Clements et al., 1999
). Upstream of mreA is the 3' end of an ORF showing strong homology to the endonuclease IV family of Saccharomyces cerevisiae (40% amino acid homology over 105 aa) and E. coli (38% identity over 101 aa). Endonuclease IV proteins are involved in DNA repair of free-radical damage, and contain Zn2+ and Mn2+ ions (Levin et al., 1991
). The hairpin loop (
G -5·4 kJ mol-1) between the putative exonuclease IV gene and mreA suggests they are in separate operons.
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A consensus sequence upstream of mreA corresponding to the Fur box (Escolar et al., 1999 ) or to the putative B. subtilis Zur box (Gaballa & Helmann, 1998
) was not identified.
Characterization of the roles of zur and mreAB
The zur gene was inactivated by partial deletion and insertion of a tetracycline-resistance cassette. First, the role of zur in Zn2+ susceptibility was tested. If the zur gene product bound Zn2+ and repressed Zn2+ uptake, zur mutant strains would be expected to be more susceptible to Zn2+ toxicity at high concentrations. However, there was no difference in the Zn2+ susceptibility of the two strains (data not shown). Secondly, the addition of 20 µM Zn2+ enhanced the growth of S. aureus 8325-4 in CL medium, suggesting that the growth of cells in unsupplemented media was limited by Zn2+ (Fig. 2). However, this response was not affected in the zur::tc mutant, even when tested with a range of Zn2+ concentrations. Furthermore, the addition of 20 µM Zn2+ to 8325-4 cultures resulted in the repression of two cell wall proteins and the induction of at least one exoprotein (results not shown). Again, the zur::tc mutant strain showed expression patterns identical to that of the wild-type. These results are in contrast to phenotypic differences seen in B. subtilis, in which the Zn2+ repression of cell wall proteins and Zn2+-transport genes is alleviated in the zur mutant strain (Gaballa & Helmann, 1998
).
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To determine if zur or mreAB was involved in the response to metals other than zinc, similar experiments using Fe3+, Mg2+, Ca2+, Mn2+, Cu2+, Co2+, Ni2+ and Mo2+ and various combinations of these ions were conducted, and siderophore production was measured (Lindsay & Riley, 1994 ). In no case could an altered phenotypic response between the wild-type and the zur::tc mutant strain or mreAB mutant strains be demonstrated.
S. aureus also possesses a Zn2+-efflux mechanism (ZntRA) which is induced by very high concentrations of zinc (1·5 mM Zn2+; Xiong & Jayaswal, 1998 ). Mutants in zntA are unable to export toxic levels of Zn2+, and have an MIC of approximately 0·5 mM Zn2+; wild-type cells have an MIC of 5 mM. The mreAB zur operon did not appear to interfere with this process, as all strains had MICs in the region of 5 mM Zn2+. Therefore, zur, mreA and mreB genes do not appear to be involved in the export of Zn2+ out of cells.
The role of zur in pathogenesis was evaluated by comparing JL100(zur) with 8325-4 in a mouse skin-lesion model. No attenuation of virulence (compared to the wild-type strain) was seen (results not shown).
Expression of mreA, mreB, zur
Using Northern blotting, the zur transcript could not be detected. Consequently, it was also impossible to map the transcription start site. Strains with a lacZ reporter gene fused to either mreA or mreB were constructed (Fig. 3) and used to measure gene expression levels in growth media containing a range of metal-ion concentrations. Both constructs (JL110 and JL120) showed low-level (<50 MUG units) constitutive expression (results not shown). This suggested that the likely promoter controlling mreA and mreB upstream of mreA is weakly expressed in all of the growth conditions tested.
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The Pspac promoter is repressed by the product of lacI encoded on the integrated plasmid. In the presence of IPTG, LacI inhibition of Pspac is relieved. Therefore, it was possible to induce expression of the operon by adding IPTG.
The entire mreAB zur operon (JL120) was expressed by using IPTG in low-ion conditions or in the presence of 20 µM Zn2+, but had no effect on growth of the culture or on the cellular protein profile (results not shown). However, when lacZ activity was measured, the expression of mreA was strongly repressed by the presence of Zn2+. JL120 grown in the presence of Zn2+ and IPTG produced significantly less ß-galactosidase activity than the same strain grown without Zn2+ (P<0·05; Fig. 3). This suggests that zur may repress transcription of the genes in a Zn2+-dependent manner. No other metal ion was able to cause this repression of transcription.
To confirm that Zn2+-dependent repression of mreA::lacZ is due to zur alone, two experiments were conducted. Using JL110 (mreB::lacZ; Fig. 1), in which mreB is inactivated, expression was still found to be repressed by Zn2+ when zur was induced by the presence of IPTG. Conversely, when mreA mreB zur expression was induced in strain JL121, in which zur has been inactivated, no Zn2+-dependent repression was seen (P<0·05; Fig. 3
). Similar results were obtained using cultures of JL111. Therefore, the Zn2+-dependent repression of mreAB zur is mediated by zur.
Addition of Zn2+ (20 µM) to CL results in an increased (almost double) growth yield of 8325-4 (Fig. 2). The potential role of mreA and mreB in this effect was analysed. All strains grew significantly better when supplemented with Zn2+ (P<0·05); the exception to this was JL121 supplemented with IPTG, which showed enhanced growth without Zn2+ (asterisk in Fig. 2
). In this case, mreA and mreB were induced in the absence of zur, and growth was significantly greater than that in the same experiment when zur was present (double asterisk in Fig. 2
) (P<0·05). JL110 and JL111 did not show enhanced growth in IPTG-induced cultures (Fig. 2
), suggesting that both mreA and mreB are required. Therefore, mreAB may encode components of a ion-uptake pathway that only functions in the absence of zur. To determine if this enhanced growth was due to increased uptake of Zn2+ or another ion, MS on whole washed cells from cultures of JL121 with or without IPTG was carried out. However, there were no apparent differences in ion content in the two cell preparations.
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DISCUSSION |
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The complex results regarding the ability of mreA and mreB to function in ion uptake can be explained as follows. Inductionof mreAB (in the absence of zur) allows transport of either Zn2+ or another ion that compensates for Zn2+. The fact that zur prevents this uptake could be due to Zur-mediated repression of an essential component of mreAB transport. Since most ABC-type transporters consist of three types of protein (Dintilhac et al., 1997 ; Gaballa & Helmann, 1998
; Patzer & Hantke, 1998
), it seems reasonable to predict that a third protein (a lipoprotein, equivalent to the Gram-negative substrate-binding protein) is encoded elsewhere on the S. aureus chromosome and that its expression is down-regulated by Zur. The fact that znuA encoding the lipoprotein component of an ABC transporter of Zn2+ in H. ducreyi is monocistronic (Lewis et al., 1999
) supports this theory.
If mreA and mreB are components of a high-affinity Zn2+-transport mechanism, the organization of this operon suggests that it forms a regulatory feedback loop. This would have the effect of maintaining expression of the operon at a constant level, with repression occurring in high-Zn2+ conditions when a high-affinity uptake mechanism would be unnecessary or even deleterious.
The role of zur in Zn2+ regulation appears to be markedly different in the Gram-positive bacteria studied so far. Whilst L. monocytogenes has an operon organization similar to that of S. aureus, it is expressed at a much higher level (Dalet et al., 1999 ), suggesting the putative transporter and Zur might play a greater role. Surprisingly, S. epidermidis zur is monocistronic (Heidrich et al., 1996
), implying that the regulation of both itself and other Zn2+ transporters may be more flexible. The B. subtilis zur is also located on the chromosome separately from the two Zn2+-transporter pathways that it regulates (Gaballa & Helmann, 1998
). The implication is that zur is constitutively expressed in B. subtilis, thus allowing it to function solely according to the intracellular Zn2+ concentration. Therefore, these ion-regulatory pathways are more complex than predicted, as well as being species-specific.
S. aureus 8325-4 appears to respond to zinc by altering protein expression (in our assay conditions), although this activity is independent of mreAB zur. Similarly, under Zn-stressed conditions, such as in CL broth or on CL agar plates impregnated with a filter-paper disc containing EDTA, the mreAB locus in 8325-4 offered no growth advantage. We conclude, therefore, that other Zn transporters and regulators function in our wild-type strain independently of mreAB zur.
This work emphasizes the care that must be taken in assigning roles for genes on the basis of sequence similarity or functional activity in related bacteria. If Zur had been overexpressed in E. coli, our results suggest that we would have detected Zn2+-binding and promoter-binding activity, which might have led to the conclusion that Zur plays an important role in S. aureus Zn2+ homeostasis. It is therefore essential to use gene inactivation to determine the importance of such regulators unambiguously.
An understanding of the ability of S. aureus to respond to environmental ions might have implications for its ability to survive or induce infection. Whilst much has been written about the essential role of Fe3+ uptake and signalling in pathogenic bacteria (for a review, see Litwin & Calderwood, 1993 ), Zn2+ is also thought to be an essential nutrient for all cells, having important effects on the immune response and the activity of superantigens such as toxic shock syndrome toxin and enterotoxins produced by S. aureus (Driessen et al., 1995a
, b
). Indeed, Zn2+ appears to be a component of some Fur-like proteins, in which it plays a structural role (Althaus et al., 1999
). Because of the low concentrations of free Zn2+ found in vivo (Magneson et al., 1987
), further study of specific transporters and regulators is warranted and might prove useful in the study of S. aureus survival during infection.
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ACKNOWLEDGEMENTS |
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Received 2 October 2000;
revised 17 January 2001;
accepted 5 February 2001.