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1 Department of Biological Sciences, Illinois State University, Normal, IL 61790, USA
2 Department of Chemistry, Illinois State University, Normal, IL 61790, USA
Correspondence
Radheshyam K. Jayaswal
drjay{at}ilstu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). S. aureus is capable of growing in a wide range of adverse environmental conditions, including media containing heavy metals. Understanding of metal resistance in staphylococci has progressed rapidly in the past 20 years. It has been well established that some of the genes for resistance to metal ions, such as cadmium, mercury, antimony and arsenic, are encoded by plasmids (Silver & Phung, 1996). However, staphylococcal strains without plasmids also show resistance to heavy metal ions, such as zinc, cobalt and copper. We have previously identified genes involved in iron, zinc and cobalt transport in S. aureus (Xiong & Jayaswal, 1998; Singh et al., 1999; Xiong et al., 2000; Cabrera et al., 2001). Some of these heavy metals, such as copper, iron and zinc, are necessary for life. However, little is known about genes involved in copper transport of S. aureus.
Copper is an essential trace element required by most organisms as a cofactor for numerous catabolic pathways and electron transport (Mason, 1976; Nicholas et al., 2002; Massaro, 2002). However, copper is toxic to cells at concentrations higher than physiological levels (Gaetke & Chow, 2003). Therefore, the intracellular concentration of copper is controlled by copper transport systems encoded by cop operons, which maintain copper homeostasis (Cooksey, 1993). Copper transport systems have been studied in several micro-organisms. Among bacteria the best-characterized system is that of Enterococcus hirae, where the cop operon consists of four genes: copY, copZ, copA and copB. copA and copB encode P-type ATPases involved in copper uptake and efflux, respectively. The expression of copA and copB is induced by copper and further regulated by CopY, a repressor and CopZ, an activator (Solioz & Stoyanov, 2003).
Recently we have identified genes involved in copper transport in S. aureus ATCC 12600 (Sitthisak et al., 2005). This strain contained a mco gene which encoded a multicopper oxidase and a copper-ATPase (cop) gene with 57 % sequence similarity to copB from Ent. hirae. However, these mco and cop genes were found only in S. aureus MRSA252 and ATCC 12600 but not in the other strains examined. Most of the sequenced S. aureus genomes contained another gene designated copA which encodes a copper-translocating P-type ATPase followed by a copper metallochaperone (copZ).
Copper-ATPase belongs to a large superfamily of CPx-P-type ATPases (Lutsenko & Kaplan, 1995; Solioz & Vulpe, 1996; Rensing et al., 2000; Gatti et al., 2000). Translocation of cations across the cell membrane by copper-ATPases is achieved by utilizing the energy of hydrolysis of the terminal phosphate bound in ATP (Lutsenko & Kaplan, 1995). The structure of CPx-type ATPase contains transmembrane helices and conserved structural elements. These elements are the phosphorylation domain (DKTGS/T), the phosphatase domain (TGES/A), the ATP-binding domain (MXGDGXNDXP), and the conserved putative metal-binding cysteine-proline-x sequence (CPx) in the intramembrane domain. The hydrophilic N-terminal part of CPx-type ATPases encompasses one to six heavy-metal-binding domains (MBDs) containing a GMTCXXC motif (Lutsenko et al., 1997; Deigweiher et al., 2004). The paired cysteine residues in this domain play an important role in heavy metal binding (Walker et al., 2002, 2004).
The copZ-encoded copper chaperone is a small protein involved in the intracellular delivery of copper to copper-utilizing enzymes. It functions by protecting the intracellular milieu from copper toxicity and releasing copper to its partner proteins. In Ent. hirae, CopZ delivers copper to the CopY repressor. In a number of bacteria, the copZ gene is located adjacent to the genes encoding copper-ATPases (Pufahl et al., 1997). CopZ has also been shown to interact with CopA, which may be the site of copper loading of CopZ (Multhaup et al., 2001). Sequence similarities with the N-terminal metal-binding (CXXC) motif from copper-ATPases and metal chaperones have been identified in both eukaryotes and prokaryotes (Harrison et al., 2000).
In this study, we have characterized CopA, a copper-ATPase, and CopZ, a copper chaperone, with respect to their roles in copper transport, their metal-binding domains, and their expression in response to various heavy metals. We have shown that CopA is involved in copper efflux. Disruption of copA resulted in the accumulation of intracellular copper and a copper-sensitive phenotype compared to the parent S. aureus strain. Northern blot analysis showed induction of copA and copZ transcription by copper and, to some extent, by iron and lead. We have cloned and overexpressed the MBDs of CopA and CopZ and have shown by mutagenesis that the cysteine residues in the CXXC metal-binding motif in CopA are involved in copper binding and thus play an important role in copper transport in S. aureus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. S. aureus was grown in defined medium (DM) (Townsend & Wilkinson, 1992) or tryptic soy broth (TSB) and E. coli was grown in Luria–Bertani broth (LB). When needed, ampicillin (50 µg ml–1), carbenicillin (50 µg ml–1), chloramphenicol (10 µg ml–1), erythromycin (20 µg ml–1) and tetracycline (10 µg ml–1) were added to the growth medium.
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. The upstream and downstream regions of copA were amplified by PCR using primers copA-N1/copA-N2 and copA-C1/copA-C2 (Table 2), with S. aureus SH1000 DNA as a template and Taq polymerase enzyme (Promega). The two amplified fragments digested at the BamHI site were ligated. The ligated DNA was PCR amplified using primers N1and C2, which contained att sites, and integrated into pKOR1 using site-specific recombination. The recombinant plasmid was transformed into E. coli DH5
. Plasmids were purified and verified by sequencing. The resulting plasmids were transformed into S. aureus RN4220 and then SH1000. Co-integration was induced by growing the bacteria at 43 °C. The integration mutants were subcultured at 30 °C and screened for the loss of tetracycline resistance. The partially deleted copA mutant (
copA) was further confirmed by PCR and DNA sequencing using copA-N1 and copA-C2 primers. As shown in Fig. 1, there was a deletion of 1009 bp from the copA gene.
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). Two primers, Com-F and Com-B (Table 2
) were designed and the PCR was performed using S. aureus SH1000 DNA as a template. The amplified fragment was cloned into pCR2.1 vector (Invitrogen) and subsequently into plasmid pLI50. The resulting plasmid was transformed into S. aureus RN4220. The plasmid was reisolated from S. aureus RN4220 and then transformed into the SH1000 copA mutant. The transformants were tested for their ability to grow in TSB containing 1.5 mM CuSO4.
Northern blot analysis.
RNA was isolated using a Qiagen kit. Ten micrograms of total RNA was electrophoresed in a 1 % agarose/0.66 M formaldehyde gel. RNA was transferred to nylon membrane (Millipore) and the blot was probed with radiolabelled copA (700 bp) or copZ probe (200 bp) under aqueous phase conditions at 65 °C. The probe was prepared using the Prime-a-Gene labelling system (Promega) in the presence of [-32P]dCTP.
Determination of intracellular copper, iron and lead concentration.
To measure the copper, iron and lead content of the cells, overnight-grown cultures were diluted 1 : 100 in TSB supplemented with 0.5 mM CuSO4, 0.5 mM FeCl3 or 0.5 mM PbSO4 and incubated at 37 °C until the OD600 reached 0.6. Cells were harvested and washed three times with 10 mM Tris/HCl (pH 7.4) containing 1 mM EDTA and once with MilliQ H2O. Cells were dried overnight at 80 °C then dissolved in 30 % nitric acid and the copper, iron and lead contents were measured by inductively coupled argon plasma atom emission spectrometry (Radford et al., 2003).
Cloning, expression and site-directed mutagenesis of the MBD encoded by copA.
Two oligonucleotide primers, MBD-F and MBD-B (Table 2), were designed with a BamHI site at the 5' end and a HindIII site at the 3' end of the DNA fragment. The PCR was performed using S. aureus genomic DNA as template and the PCR product was first cloned into pCR2.1 vector (Invitrogen) and subsequently into the BamHI and HindIII sites of pRSETa (Invitrogen). The resulting plasmid, pSA-MBD, was transformed into E. coli BL21(DE3)(pLysS) (Novagen) by electroporation. To overexpress the cloned gene product, the transformants were grown in LB containing ampicillin and chloramphenicol at 37 °C until the OD600 reached 0.5 and then the cells were induced for the expression of protein by the addition of 2.0 mM IPTG for 3 h. The induced cells were harvested, washed, and resuspended in lysis buffer (20 mM Tris/HCl, pH 7.4, containing 145 mM NaCl), Pellets were chilled on ice and homogenized by sonicator (Branson Sonifier 450) and cell debris was removed by centrifugation 10 000 g at 4 °C. The supernatants were applied to nickel-charged agarose-affinity columns (Novagen) and eluted with 200–400 mM imidazole. Eluted fractions were subjected to 12.5 % SDS-PAGE analysis. Fractions containing the overexpressed His-tag protein were pooled and dialysed against 25 mM Tris/HCl, pH 8.0, containing 100 mM sucrose, 50 mM NaCl and 1 mM DTT.
PCR-based site-directed mutagenesis was performed in order to replace Cys residues by Ala in the MBD of CopA as described by Allemandou et al. (2003). The four primers MBD-N1, MBD-N2, MBD-C1 and MBD-C2 were used to exchange Cys at positions 16, 19, 83 and 86 for Ala residues. Mutations were confirmed by DNA sequencing. The fragment corresponding to the mutated MBD was gel purified and subcloned into the BamHI and HindIII sites of pRSETa, and the resulting plasmid, pSA-MBD mutant, was overexpressed in E. coli BL21(DE3)(pLysS) as described above. His-tag fragments from both the recombinant proteins were removed by using an enterokinase kit (Invitrogen) before performing the binding assay.
Bicinchoninic acid (BCA)-based copper-binding assay.
The stoichiometry of copper binding by the putative wild-type MBD or the mutated MBD was determined by a BCA-based assay (Brenner & Harris, 1995; Lutsenko et al., 1997). To determine the amount of copper bound to the protein in vitro, purified copper-free MBD or mutated MBD proteins at a concentration of 100 µg ml–1 were mixed with CuCl2 (20 µmol copper per µmol protein). After 10 min incubation at room temperature, unbound copper was removed by dialysis at 4 °C overnight and bound copper was measured by the BCA assay as described by Lutsenko et al. (1997).
Cation-binding specificity of the MBD by iminodiacetic acid-agarose (IAA) chromatography.
IAA columns equilibrated with different heavy metals were used to determine the cation-binding specificity as described by Lutsenko et al. (1997). Columns containing 100 µl IAA (Sigma) were extensively washed with 50 mM sodium phosphate buffer (pH 7.5) and then separately equilibrated with 10 volumes of the same buffer containing one of several heavy metal chloride compounds (CdCl2, CuCl2, CoCl2, MnCl2 and FeCl3) at a final concentration of 1 mM. Excess metal ions were removed by extensive washing with sodium phosphate buffer and then 100 µg purified MBD protein or mutated MBD protein (MBD) was added to the resin and incubated for 10 min at room temperature. Columns were centrifuged to remove unbound proteins. Columns were washed with 500 µl sodium phosphate buffer and bound proteins were eluted from the column with 50 mM EDTA in sodium phosphate buffer. Both eluted and unbound proteins were concentrated and analysed by 12.5 % SDS-PAGE.
Involvement of cysteine residues in the MBD.
Involvement of the cysteine residues was demonstrated by the ability of copper to protect the cysteine residues in the MBDs against labelling with the cysteine-directed fluorescent reagent 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), as described by Lutsenko et al. (1997) and Walker et al. (2002). Briefly, 50 µg of purified MBD or MBD protein was incubated in the presence of different concentrations of copper for 10 min, and then pulse-labelled with a 20 molar excess of CPM for 1 min in the dark. Proteins were separated by 15 % SDS-PAGE. The CPM-labelled proteins were then monitored under UV light.
Molecular genetic procedures.
Plasmid and chromosomal DNA isolation, DNA manipulation, digestion of DNA with restriction enzymes, DNA ligation, Northern blot analysis and PCRs were performed as described by Sambrook & Russell (2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Upstream of copA, potential –35 (TTGCAC) and –10 (TAAAAT) sequences, and a putative Shine–Dalgarno (GGGAGG) sequence, were identified. The molecular masses of the putative proteins encoded by copA and copZ were predicted to be 86.7 and 7.2 kDa, respectively. The copA gene encodes a copper-translocating P-type ATPase protein exhibiting features of a CPx-type ATPase that pumps copper out of the cytoplasm (Huffman & O'Halloran, 2001). CopA is a transmembrane protein that contains two heavy-metal-binding sites with CXXC motifs (MBD) in the N-terminal region, six conserved intramembrane helices, a phosphorylation domain, phosphatase domains and two additional transmembrane helices at the N-terminus. copZ encodes a copper-binding chaperone protein which contains only an MBD at the N-terminus. The putative characterization of CopA protein was achieved by using a computer program provided by San Diego Super Computer Center (http://workbench.sdsc.edu/).
The genome database analyses suggested that the genes involved in copper transport are conserved in S. aureus. copA and copZ showed almost 100 % sequence identity among S. aureus strains and about 75 % with other staphylococcal species. We also performed PCR using primers from copA and copZ, confirming that these genes are also present in other S. aureus strains such as ATCC 12600, H and Wood 46 whose genomes have not been sequenced (data not shown). The CopA from S. aureus showed 49 % sequence identity with CopA of Ent. hirae. In addition, the CopA of S. aureus showed a similar CPC motif and two metal-binding motifs (CXXC). CopA, which is responsible for the copper influx in Ent. hirae, has a similar CXXC domain (Solioz & Vulpe, 1996).
Copper tolerance in S. aureus
To determine the tolerance level of S. aureus SH1000 to copper, cells were grown in either chemically defined medium (DM) or TSB containing various concentrations of CuSO4. As shown in Fig. 2, the MIC of CuSO4 in TSB was around 2.5 mM whereas in DM it was about 0.2 mM. The higher level of copper tolerance in TSB may be due to the faster growth rate and some factor(s) not available in DM.
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2.4 kb and 2.0 kb, when copA was used as probe (Fig. 3a). The 2.4 kb band represents the copA transcript whereas the 2.0 kb transcript might represent multiple transcripts from the same gene, a degradation product of copA or the transcript of some homologous gene. The expression of copZ was also dependent on copper ion concentration and it was induced by as low as 5 µM copper (Fig. 3b). A 0.2 kb transcript was detected when copZ was used as probe, which seemed to be in good agreement with that predicted from the sequence data.
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). There was no effect of cobalt, cadmium, manganese, silver and zinc (Fig. 3c and data not shown). The induction of copA gene by other metal ions such as silver has been reported (Rensing et al., 2000). We also measured the expression of copA and copZ during the growth cycle. The expression of copA and copZ was growth phase dependent: both genes were expressed to a higher level in the exponential phase than in the stationary phase (data not shown). As described above we have not found either any potential regulatory gene or possible regulatory sequences upstream of copA. Therefore, it is difficult to predict the mechanism by which copper regulates the expression of copA and copZ in S. aureus
copA mutation induces copper, iron and lead sensitivity in S. aureus
A partial deletion in the copA gene was constructed as described in Methods. To examine the effect of mutation in copA, S. aureus strains SH1000 (parent) and the copA partial deletion mutant (copA) strains were grown in DM with or without copper. There was no difference in the growth rate between parent and copA strains in DM without copper (data not shown). However, in the presence of 75 µM copper sulfate the copA strain showed increased copper sensitivity (Fig. 4a). There was a significant difference in growth rate between the wild-type and the mutant strain (P=0.004). The copA mutant was also tested for ferric iron and lead sensitivity, as the Northern analysis suggested that the expression of copA is also induced by ferric and lead ions. As shown in Fig. 4(b, c), the copA mutant was also sensitive to ferric and lead ions. There was a significant difference in growth rate between the wild-type and the mutant strain (P=0.004 and 0.023 respectively). In B. subtilis, mutation in copA sensitized the cells to copper but not to other metal ions (Gaballa & Helmann, 2003).
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copA mutant. The complemented strain was tested for growth in the presence of copper, ferric ions and lead. As shown in Fig. 4(a, b), the complemented strain was able to grow at the same rate as the parent strain in the presence of copper and iron but it grew slightly slower in the presence of lead (Fig. 4c). These results suggested that the copper-, ferric- and lead-sensitive phenotype of the mutant was due to the mutation in the copA gene. The capacity to transport diverse metals by CPX-ATPase has been shown in Archaeoglobus fulgidus, B. subtilis and other micro-organisms (Arguello et al., 2003; Gaballa & Helmann, 2003). In addition, association between genes for copper transport and genes for iron transport has been found in Saccharomyces cerevisiae (van Bakel et al., 2004, 2005). Since both lead and iron can induce the transcription of the copA, this further suggests that copA may function as a co-transporter for these ions.
Mutation in copA causes hydrogen peroxide sensitivity
Another phenotype of the copA mutant was found to be its increased sensitivity to hydrogen peroxide. As shown in Fig. 4(d), the growth of copA mutant in DM containing 1.5 mM H2O2 was slower compared to the parent strain. The copA mutant's hydrogen peroxide tolerance was restored to the normal level, as observed in the parent strain, by complementation. We also checked the tolerance levels of the copA mutant to another oxidative agent, paraquat, and found no difference (data not shown). The precise mechanism by which the copA mutant becomes sensitive to hydrogen peroxide is not clear. However, as mentioned above CopA is a Cu(I)-translocating P-type ATPase, and mutation in copA causes the accumulation of Cu+ in the mutant cells. In addition, the copA mutant showed increased iron sensitivity (Fig. 4b) and a higher concentration of intracellular iron (Table 3). Iron and copper are both capable of catalysing the formation of hydroxyl radicals (OH
) from H2O2 via the Haber–Weiss reaction (Bremner, 1998; Pierre & Fontecave, 1999), which may be the cause of the H2O2-sensitive phenotype.
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, intracellular copper concentrations increased over sevenfold in the copA mutant when cultures were grown in TSB containing 0.5 mM CuSO4 compared to the parent strain (P=0.0001). In the copA complemented strain the concentrations of intracellular copper were equivalent to the concentrations in the parent strain. We also determined the colony count of cells grown in the presence of various ions. There were slightly fewer c.f.u. ml–1 in the mutant compared to wild-type and the complemented strains (data not shown). However, this difference cannot account for a several-fold increase in the concentration of intracellular ions. The accumulation of high concentrations of copper ions in the copA mutant cells is indicative of their inability to efflux copper. The lower concentration in the complemented strain also supports the idea that CopA functions as a copper transporter. Under normal physiological conditions, copA and copZ are not transcribed in either the mutant or the parent strain. This suggests that the copA is not essential for the growth of S. aureus under normal conditions but is required under conditions of high copper.
The intracellular concentrations of iron and lead were also measured (Table 3). The concentrations of these metals were higher in the copA mutant when cultures were grown in TSB containing 0.5 mM FeCl3 or 0.5 mM PbSO4 (P=0.001 and 0.029 respectively). The accumulation of iron and lead ions in the copA mutant cells may be due to the inability of these cells to efflux both metal ions.
Characterization of the MBD in copA
It has been shown that the N-terminal domains of the Wilson's and Menkes disease proteins bind copper selectively in vivo as well as in vitro (Lutsenko et al., 1997; DiDonato et al., 1997). Here we have investigated the role of the putative MBD in CopA protein from S. aureus strain SH1000. The MBD, containing C16XXC19 and C83XXC86 motifs, was cloned and overexpressed (Fig. 5a) as described in Methods, and its copper-binding ability was determined in vitro.
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In addition, we tested the binding by using IAA chromatography, equilibrated with different heavy metals (copper, cobalt, cadmium, iron and manganese and lead). As shown in Fig. 5b, A, binding was detected for copper-, cobalt- and cadmium-equilibrated columns eluted by EDTA but no binding was observed for iron-, manganese- or lead-equilibrated columns (Fig. 5b, B). Based on in vivo studies (data presented in Figs 3 and 4) it was expected that iron and lead would bind to the MBD in IAA chromatography. In in vitro studies, IAA chromatography showed that cobalt binds to the MBD, but it does not induce the transcription of copA in Northern blot analysis (Fig. 3). The discrepancies in these results are difficult to interpret, but it might be that in vivo metal ions regulate the transcription of the copA through a separate metal-binding regulatory protein. In vitro binding of various metals to the MBD of the CopA protein may depend on conditions such as pH, ion concentration, binding constant, redox state of metals, etc. The MBD of CopA and other copper P-type ATPases is thought to initially bind copper and then deliver it to a distinctly separate copper-transporting site (probably within the transmembrane domains). It is conceivable that iron binds directly to the copper transporting site without first interacting with the MBD. In this study we wanted to examine the role of the MBD of copA. We have shown that it binds copper in vitro, and a copper–protein complex may play an important role during copper transport. Northern blot data showed that copper regulates copA expression. In vitro the MBD binds cadmium and cobalt, but these metals do not regulate copA expression. It may be that the conformation of the protein when it binds to these non-copper metals is incompatible with gene expression. Of course, metal binding to the MBD and gene expression are two different phenomena and not necessarily correlated. In vitro studies suggest the role of the CXXC motifs in metal binding. When the Cys-mutated MBD protein was used in this experiment no binding was observed under any condition (Fig. 5b, C and D).
To further confirm the role of cysteine residues in the MBD, a cysteine-directed fluorescent reagent, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin was used as described in Methods. As shown in Fig. 5(c, A and C), when the MBDs from copA and copZ were incubated with fluorescent reagent in the presence of various concentrations of copper, a concentration of copper above 50 µM prevented the fluorescent labelling of cysteine residues. There was no effect on fluorescent labelling when the MBD protein was incubated with other heavy metals. The labelling of the Cys-mutated MBD protein with fluorescent reagent was not observed, whether copper was present or not (Fig. 5c, B). These results confirmed that cysteine binds to copper and the formation of a copper–protein complex may play an important role during copper transport.
Concluding remarks
We have characterized a copA gene responsible for the efflux of mainly copper ions and probably also ferric and lead ions in S. aureus. The copZ gene encodes a copper-binding chaperone most probably involved in the intracellular delivery of copper to copper-utilizing enzymes. The expression of copA and copZ is inducible by copper and to some extent by ferric and lead ions. In vitro metal-binding studies showed that copper, cadmium and cobalt can bind to the MBD of CopA. We are currently investigating the regulation aspects of copA and copZ genes in S. aureus using DNA microarray technology.
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ACKNOWLEDGEMENTS |
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Edited by: J. A. Lindsay
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Archer, G. L. (1998). Staphylococcus aureus: a well-armed pathogen. Clin Infect Dis 26, 1179–1181.[Medline]
Arguello, J. M., Mandal, A. K. & Mana-Capelli, S. (2003). Heavy metal transport CPx-ATPases from the thermophile Archaeoglobus fulgidus. Ann N Y Acad Sci 986, 212–218.[Medline]
Bae, T. & Schneewind, O. (2006). Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63.[CrossRef][Medline]
Bremner, I. (1998). Manifestations of copper excess. Am J Clin Nutr 67 (5 Suppl.), 1069S–1073S.[Abstract]
Brenner, A. J. & Harris, E. D. (1995). A quantitative test for copper using bicinchoninic acid. Anal Biochem 226, 80–84.[CrossRef][Medline]
Cabrera, G., Xiong, A., Uebel, M., Singh, V. K. & Jayaswal, R. K. (2001). Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus. Appl Environ Microbiol 67, 1001–1003.
Cooksey, D. A. (1993). Copper uptake and resistance in bacteria. Mol Microbiol 7, 1–5.[Medline]
Deigweiher, K., Drell, T. L., Prutsch, A., Scheidig, A. J. & Lubben, M. (2004). Expression, isolation, and crystallization of the catalytic domain of copB, a putative copper transporting ATPase from the thermoacidophilic archaeon Sulfolobus solfataricus. J Bioenerg Biomembranes 36, 151–159.[CrossRef][Medline]
DiDonato, M., Narindrasorasak, S., Forbes, J. R., Cox, D. W. & Sarkar, B. (1997). Expression, purification, and metal-binding properties of the N-terminal domain from the Wilson disease putative copper-transporting ATPase (ATP7B). J Biol Chem 272, 33279–33282.
Gaballa, A. & Helmann, J. D. (2003). Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems. Biometals 16, 497–505.[CrossRef][Medline]
Gaetke, L. M. & Chow, C. K. (2003). Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 189, 147–163.[CrossRef][Medline]
Gatti, D., Mitra, B. & Rosen, B. P. (2000). Escherichia coli soft metal ion-translocating ATPases. J Biol Chem 275, 34009–34012.
Harrison, M. D., Jones, C. E., Solioz, M. & Dameron, C. T. (2000). Intracellular copper routing: the role of copper chaperones. Trends Biochem Sci 25, 29–32.[CrossRef][Medline]
Horsburgh, M. J., Aish, J. L., White, I. J., Shaw, L., Lithgow, J. K. & Foster, S. J. (2002). 
B modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J Bacteriol 184, 5457–5467.
Huffman, D. L. & O'Halloran, T. V. (2001). Function, structure, and mechanism of intracellular copper trafficking proteins. Annu Rev Biochem 70, 677–701.[CrossRef][Medline]
Kreiswirth, B. N., Lofdahl, M. S., Betley, M. J., O' Reilly, M., Schlievert, P. M., Bergdoll, M. S. & Novick, R. P. (1983). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by prophage. Nature 305, 709–712.[CrossRef][Medline]
Lee, C. Y., Schmidt, J. J., Johnson-Winergar, A. D., Spero, L. & Iandolo, J. J. (1987). Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus. J Bacteriol 169, 3904–3909.
Lutsenko, S. & Kaplan, J. H. (1995). Organization of P-type ATPases: significance of structural diversity. Biochemistry 34, 15607–15613.[CrossRef][Medline]
Lutsenko, S., Petrukhin, K., Cooper, M. J., Gilliam, C. T. & Kaplan, J. H. (1997). N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal binding repeat. J Biol Chem 272, 18939–18944.
Mason, H. S. (1976). Binuclear copper clusters as active sites for oxidases. Adv Exp Med Biol 74, 464–469.[Medline]
Massaro, E. J. (2002). Handbook of Copper Toxicology, pp. 624. Totowa, NJ: Humana Press.
Multhaup, G., Strausak, D., Bissig, K. D. & Solioz, M. (2001). Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance. Biochem Biophys Res Commun 288, 172–177.[CrossRef][Medline]
Nicholas, K. M., Wentworth, P., Harwig, C. W., Wentworth, A., Shafton, D. & Janda, K. D. (2002). A cofactor approach to copper-dependent catalytic antibodies. Proc Natl Acad Sci U S A 99, 2648–2653.
Pierre, J. L. & Fontecave, M. (1999). Iron and activated oxygen species in biology: the basic chemistry. Biometals 12, 195–199.[CrossRef][Medline]
Pufahl, R. A., Singer, C. P., Peariso, K. L., Lin, S. J., Schmidt, P. J., Fahrni, C. J., Culotta, C., Penner-Hahn, J. E. & O'Halloran, T. V. (1997). Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science 278, 853–856.
Radford, D. S., Kihlken, M. A., Borrelly, G. P., Harwood, C. R., Le Brun, N. E. & Caver, J. S. (2003). CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA. FEMS Microbiol Lett 220, 105–112.[CrossRef][Medline]
Rensing, C., Bin, F., Sharma, R., Mitra, B. & Rosen, B. P. (2000). CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci U S A 97, 652–656.
Sambrook, J. & Russell, D. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Silver, S. & Phung, L. T. (1996). Bacterial heavy metal resistance: new surprises. Annu Rev Microbiol 50, 753–789.[CrossRef][Medline]
Singh, V. K., Xiong, A., Usgaard, T., Chakrabarty, S., Deora, R., Misra, T. & Jayaswal, R. K. (1999). ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon of S. aureus. Mol Microbiol 33, 200–207.[CrossRef][Medline]
Sitthisak, S., Howieson, K., Amezola, C. & Jayaswal, R. K. (2005). Characterization of a multicopper oxidase gene from Staphylococcus aureus. Appl Environ Microbiol 71, 5650–5653.
Solioz, M. & Stoyanov, J. V. (2003). Copper homeostasis in Enterococcus hirae. FEMS Microbiol Rev 27, 183–195.[CrossRef][Medline]
Solioz, M. & Vulpe, C. (1996). CPx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biochem Sci 21, 237–241.[CrossRef][Medline]
Townsend, D. E. & Wilkinson, B. J. (1992). Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation. J Bacteriol 174, 2702–2710.
van Bakel, H., Huynen, M. & Wijmenga, C. (2004). Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway. Bioinformatics 20, 2644–2655.
van Bakel, H., Strengman, E., Wijmenga, C. & Holstege, F. C. (2005). Gene expression profiling and phenotype analyses of S. cerevisiae in response to changing copper reveals six genes with new roles in copper and iron metabolism. Physiol Genomics 22, 356–367.
Walker, J. M., Tsivkovskii, R. & Lutsenko, S. (2002). Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity. J Biol Chem 277, 27953–27959.
Walker, J. M., Huster, D., Ralle, M., Morgan, C. T., Blackburn, N. J. & Lutsenko, S. (2004). The N-terminal metal-binding site 2 of the Wilson's disease protein plays a key role in the transfer of copper from atox1. J Biol Chem 279, 15376–15384.
Xiong, A. & Jayaswal, R. K. (1998). Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus. J Bacteriol 180, 4024–4029.
Xiong, A., Singh, V. K., Cabrera, G. & Jayaswal, R. K. (2000). Molecular characterization of a ferric uptake regulator, Fur, from Staphylococcus aureus. Microbiology 146, 659–668.
Received 16 May 2007;
revised 11 August 2007;
accepted 15 August 2007.
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) and TSB (
, Wild-type; 
, copA mutant ; 
, copA complemented strain. Each point represents the mean±SD of three experiments.