Department of Microbiology and Immunology, Faculty of Medicine1, and Department of Applied Oral Sciences, Faculty of Dentistry2, Dalhousie University, Halifax, Nova Scotia, CanadaB3H 3J5
Author for correspondence: Song F. Lee (Applied Oral Sciences). Tel: +1 902 494 8799. Fax: +1 902 494 6621. e-mail: Song.Lee{at}Dal.Ca
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ABSTRACT |
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Keywords: copper transport, copper resistance, P-type ATPase, heavy metals
Abbreviations: CAT, chloramphenicol acetyltransferase; THA, ToddHewitt agar; THB, ToddHewitt broth; TYG, tryptone/yeast extract glucose broth
The GenBank accession number for the cop operon sequence reported in this paper is AF296446.
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INTRODUCTION |
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Streptococcus mutans is the bacterial aetiological agent in dental caries (Loesche, 1986 ). The oral cavity, in which Strep. mutans dwells, is an environment of highly fluctuating levels of nutrients and essential elements. Metal ions such as those of copper can come from consumed foods and from material, such as amalgam, used in dental restorations (Morrier et al., 1998
; Wataha & Lockwood, 1998
). To date, there have been no reports on the mechanism by which Strep. mutans deals with the fluctuating concentrations of these toxic metal ions.
We have previously described the isolation of a Tn917 mutant (mutant A) that was defective in its ability to effect detachment of adherent Strep. mutans cells (Vats & Lee, 2000 ). Upon mapping the Tn917 insertion site, we found that the transposon had inserted adjacent to the promoter region of a small protein that shared extensive homology with the CopY protein of Ent. hirae. This observation led us to the discovery of the cop operon in Strep. mutans. In this paper, we describe the cloning and characterization of the copYAZ operon from Strep. mutans JH1005 and provide evidence that it plays a role in copper resistance in this bacterium.
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METHODS |
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DNA isolation.
Chromosomal DNA was isolated from Strep. mutans as described previously (Vats & Lee, 2000 ). Briefly, cells were harvested by centrifugation from 10 ml of an 18-h-old culture in THB, washed in GTE (50 mM glucose; 25 mM Tris, pH 8·0; 10 mM EDTA) and incubated with 10 kU lysozyme and 1 kU mutanolysin per ml GTE at 37 °C for 12 h. Cells were lysed with 2% SDS at room temperature for 10 min. The cell lysates were extracted with phenol once, then with phenol:chloroform (1:1, v/v) and chloroform. RNA was removed by RNase treatment and DNA was precipitated by ethanol and redissolved in 20 mM Tris buffer, pH 8, containing 1 mM EDTA. Plasmids were isolated from E. coli by the alkaline lysis method (Sambrook et al., 1989
).
RNA isolation.
RNA was isolated from Strep. mutans by a modification of the method of Lunsford (1995) . Briefly, 200 ml prewarmed THB was inoculated (1:10) with an overnight culture and the culture was grown to early exponential phase (OD600 0·25). Glycine (5%, w/v) was added and the culture was further incubated for 1·5 h at 37 °C. The cultures were then divided in half. CuSO4 was added to one aliquot at a sub-inhibitory concentration (1 mM) and incubated for a further 20 min at 37 °C to induce cop transcription. The second aliquot was the non-induced control. Cells were harvested by centrifugation, washed with PBS and resuspended in 1 ml PBS containing 30% raffinose and 1 kU mutanolysin (Sigma). After incubation at 37 °C for 15 min, cells were centrifuged, vortexed in 700 µl Trizol reagent (Gibco-BRL Life Technologies), cooled on ice for 5 min and extracted with chloroform. RNA was precipitated from the aqueous layer with 2-propanol and washed with 70% ethanol. The pellet was resuspended in 200 µl diethyl-pyrocarbonate-treated water and treated with 20 U RNase-free DNase (Promega); this was followed by extraction with phenol and then chloroform. The RNA was dissolved in 200 µl diethyl-pyrocarbonate-treated water and stored at -70 °C.
Mapping of the Tn917 insertion site in Strep. mutans mutant A.
Chromosomal DNA from the Tn917 mutant, mutant A, was restricted by HindIII and separated on a 0·7% agarose gel. DNA fragments of 23 kb were recovered from the gel by electroelution and cloned into E. coli XL-1 Blue, using the vector pBluescript (Stratagene). Transformants were screened by colony hybridization using a [32P]dCTP-labelled probe prepared by random priming of a 1·5 kb PCR product of the 3' end of Tn917 (Shaw & Clewell, 1985 ). Colony hybridization was performed as described by Sambrook et al. (1989)
. The Tn917 PCR product was generated by PCR using primers 5'-TACGGATCCATCGAAATATTCAT-3' and 5'-TACGAATTCTAAACCAATGTTTCAAGG-3' under conditions described previously (Homonylo-McGavin & Lee, 1996
). The positive clone (NV4) was confirmed, by restriction mapping and Southern hybridization, as carrying the 2·5 kb HindIII insert. DNA sequencing analysis identified the Tn917 insertion site (shown in Fig. 1b
) and an ORF with a marked similarity to CopY of Ent. hirae downstream from the Tn917 insertion site.
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The entire cop operon was reconstructed from the cloned DNA fragments. A 2·5 kb PstIXbaI fragment from pWH4 containing upstream sequences, the promoter, copY and part of copA was cloned into pNV5 which had been cut with the same enzymes. The resultant plasmid, pNV6, carried on it all but the last half of copZ. The second half of copZ contained within a 0·5 kb BamHIEcoRI fragment was isolated from pNV46 and ligated into pNV6 cut with the same enzymes. The new plasmid, pNV7, consisted of 450 bp of sequence upstream of copY, the 2·9 kb operon and 280 bp of sequence downstream of the stop codon of copZ.
Construction of a cop operon knock-out mutant.
pNV7 was restricted with HincII to drop out the three ORFs, and a 4 kb HincII fragment containing the tetracycline-resistance fragment from pVA981 (Tobian et al., 1984 ) was ligated into pNV7. The resulting construct, pNV/S4, carried 454 bp of sequences upstream of copY and 377 bp of sequence downstream of copZ. These sequences served as homologous DNA for allelic exchange of the cop operon with the tetracycline cassette. pNV/S4 was linearized with ScaI and used to transform Strep. mutans JH1005 by using a previously described method (Homonylo-McGavin & Lee, 1996
). Transformants were selected by plating on tetracycline-containing THA. Chromosomal insertion of the tetracycline cassette and loss of the operon sequences were verified by Southern blotting with two different probes: the first probe was specific for the 2 kb SstIXbaI fragment, pWH4, encompassing copYA, and the second probe was produced by labelling pVA981.
Construction of plasmids for complementation of cop knock-out mutant S4 and Tn917 mutant A.
A plasmid with the entire cop operon was constructed by subcloning the SphI (on the multiple cloning site of pUC18)EcoRI fragment from pNV7 into the E. coli/Streptococcus shuttle vector pDL276 (Dunny et al., 1991 ), yielding pNV7B. A plasmid carrying copY was constructed by taking the SphIXbaI fragment from pWH4 and ligating it into similarly digested pDL276 to produce pWH4/PDL.
The construction of a recombinant plasmid carrying copZ was a multi-step process. In the first step, the cop-operon promoter was amplified using the forward primer SL147 (5'-GCATGCCAGAAGATGAGCGAATGA-3') and the reverse primer SL148 (5'-GTCGACGCTCCTTTCATCTACATT-3') and ligated into SmaI-digested dephosphorylated pUC18 to make pNV8. The promoter was then excised from pNV8 with BamHI and EcoRI and subcloned into pDL276, producing pPNV8. Next, copZ was amplified using the forward primer SL150 (5'-GTCGACATGGAAAAAACATATCATAT-3') and the reverse primer SL146 (5'-GTAGAATTCAAAAGCGTGCCATC-3'). The PCR product was then ligated into EcoRV-digested and dephosphorylated pBluescript, producing pZ1. Finally, pZ1 was cut with SalI, blunt-ended with Klenow (Gibco-BRL) and digested with EcoRI. copZ was then cloned into pPNV8 previously digested with KpnI, blunt-ended with the Klenow fragment and digested with EcoRI to make pNV10.
The plasmids were transformed into the Strep. mutans cop knock-out mutant S4 and Tn917 mutant A by natural transformation. Transformants were selected on kanamycin-containing THA.
Southern, Northern and RNA dot blottings.
DNA was separated on 0·8% agarose gels. RNA (20 µg) was separated on formaldehyde agarose gels prepared as described by Kroczek & Siebert (1990) . Nucleic acids were transferred from the gels to nylon membranes (Biodyne A; Gibco-BRL) by using a Vacu-Blot apparatus according to the manufacturers instructions (Amersham Pharmacia Biotech). For RNA dot blotting, a 96-well dot blotter (Bio-Rad) was used. After the transfer, nucleic acids were cross-linked to the nylon membranes by incubation at 80 °C for 2 h. DNADNA hybridization was performed as described by Southern (1975)
, and DNARNA hybridization was performed as described by Ausubel et al. (1990)
.
Primer extension.
The reverse primer SL159 (5'-GCAATAATTTCACTGCTG-3'), corresponding to nucleotide positions 813830 (140 nt downstream of the -35 region) of the cop operon, was end-labelled with [-32P]ATP by using T4 polynucleotide kinase (Sambrook et al., 1989
). The full-length labelled oligonucleotide was separated by electrophoresis on a 20% polyacrylamide gel and purified using a SEP-PAC Light C18 column (Waters) according to the manufacturers instructions. Total RNA (80 µg) was added to 12x106 c.p.m. radiolabelled oligonucleotide in a total volume of 30 µl in annealing buffer (0·25 M KCl, 10 mM Tris/HCl, pH 8·3). Annealing was carried out in a thermocycler: 80 °C for 5 min, 65 °C for 5 min, 42 °C for 10 min and 37 °C for 20 min. Annealed material was precipitated with 2 vols 95% ethanol, washed with 70% ethanol, vacuum-dried and resuspended in 20 µl reverse transcriptase buffer (30 mM Tris buffer, pH 8·3; 20 mM MgCl2; 10 µM DTT; 1 mM dGTP; 0·5 mM each of dATP, dCTP and dTTP; and 15 µg actinomycin D ml-1). Two hundred units of Moloney murine leukaemia virus reverse transcriptase (Gibco-BRL) was added and the reaction was incubated at 37 °C for 1 h. At the end of the incubation, 1 µl 0·5 M EDTA and 1 µl RNase (10 mg ml-1) were added prior to incubation for 30 min at 37 °C. The volume was brought to 100 µl with water and then extracted with phenol/chloroform/isoamyl alcohol and precipitated, washed and analysed by electrophoresis on a denaturing polyacrylamide sequencing gel.
DNA sequencing.
DNA sequences of cloned cop fragments were determined using an automated DNA sequencer (Applied Biosystems/Dalhousie UniversityNRC Joint Laboratory facilities). For primer extension experiments, the DNA sequence was generated from pWH4 with a DNA sequencing kit (T7 Sequenase version 2; Amersham Pharmacia Biotech), using the same primer for the primer extension.
Fusion of the cop-operon promoter with a reporter CAT gene.
Plasmid pNV8 carrying the cop-operon promoter was digested with KpnI, blunt-ended with Klenow and digested with XbaI to yield a 1·7 kb fragment carrying the promoter. This 1·7 kb fragment was ligated into the SmaI and XbaI sites of pMH109 (Hudson & Stewart, 1986 ). This ligation placed the promoter immediately upstream of the promoterless chloramphenicol acetyltransferase (CAT) gene. The ligated plasmid (pHTL1) was transformed into E. coli and expression of the CAT gene was confirmed by selection with chloramphenicol. The 4 kb HindIII (blunted with Klenow)XbaI fragment from pHTL1 was isolated and ligated to a 6·5 kb fragment of pSL2 generated by digestion with BamHI (followed by Klenow treatment) and XbaI. Plasmid pSL2 was constructed in our laboratory and contained a 0·4 kb DNA fragment encoding a non-essential oligopeptide-transport gene (hppG) originating from Streptococcus gordonii DL1 (Jenkinson et al., 1996
). Plasmid pSL2 also carried a 1·5 kb fragment of the spaP gene which encodes the major surface-protein antigen (P1) of Strep. mutans NG5 (Lee et al., 1988
). The presence of the hppG and spaP fragment would facilitate the integration of pSL2 into the streptococcal chromosome via homologous recombination. The resulting plasmid, pHSL2, carried a kanamycin-resistance gene which would hamper attempts to complement the strain created with shuttle vectors (pDL276, Kanr) carrying the cop genes. Hence, plasmid pHSL2/pUC was constructed by ligating the fusion gene and a fragment of the spaP gene fragment into the pUC18 backbone. pHSL2/pUC was produced by digestion of pHSL2 with HindIII, blunt-ending of the overhang with the Klenow enzyme, followed by digestion with XbaI, yielding a 4 kb fragment encompassing the promoterCAT fusion and a fragment of spaP. The 4 kb fragment was ligated to the 1·7 kb ScaIXbaI fragment of pUC18, which provided an origin of replication. pHSL2/pUC was naturally transformed into Strep. mutans S4. Chloramphenicol-resistant transformants were selected on THA. One of the transformants obtained, S4/CAT, was then used as the recipient for plasmids pNV7B, pWH4/PDL and pNV10. These plasmids carried the entire cop operon, copY, and copZ, respectively.
Determination of MICs for heavy-metal ions.
Strep. mutans was grown overnight in TYG and diluted in fresh TYG to a concentration of approximately 106 c.f.u. ml-1. Aliquots (0·5 ml) of the cultures were then added to aliquots (0·5 ml) of TYG containing twofold dilutions of the metal ions being tested. The tubes were then incubated at 37 °C for 2024 h and scored visually for growth. The tube containing the lowest concentration of metal ions still able to inhibit growth was recorded as the MIC.
Effect of Cu2+ on growth of Strep. mutans.
Fresh TYG was dispensed into sterile cuvettes and inoculated (1:10) with overnight cultures grown in the same medium. The cultures were placed in a 37 °C water bath and growth was monitored by measuring the OD600 at hourly intervals. Strep. mutans S4/CAT and S4/CAT carrying pNV7B, pWH4/PDL or pNV10 in the presence of chloramphenicol or copper were grown in TYG over a 20 h period.
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RESULTS |
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The first ORF encoded a small protein of 147 aa named CopY. A putative heavy-metal-binding motif (CXCX4CXC) was identified at the C-terminus of CopY. A similar motif was also present in Ent. hirae CopY and Lactobacillus sake OrfY (a putative regulator of unknown function). At the N-terminus of CopY were two stretches of conserved sequence (IX3EXEVMX2W and WX3TX2TX3RLX2K) which shared high homology with Ent. hirae CopY, L. sake OrfY, Staph. aureus BlaI, Staphylococcus epidermidis MecI and Bacillus licheniformis PenI. The enterococcal CopY protein is the negative transcriptional regulator of the copYABZ operon, and the staphylococcal and Bacillus proteins are negative regulators for the penicillinase and penicillin-binding protein expression, respectively. The amino acid identities between Strep. mutans CopY and Ent. hirae CopY, L. sake OrfY, Staph. aureus BlaI, B. licheniformis PenI and Staph. epidermidis MecI were 35, 33, 24, 23 and 19%, respectively.
The second ORF encoded a 742 aa protein (CopA). Analysis of the deduced sequence of CopA revealed several features that are hallmarks of P-type ATPases. These include: (1) an N-terminal heavy-metal-binding motif (GMXCXXC); (2) eight transmembrane regions; (3) a TGES motif that forms part of the phosphatase domain which mediates phosphorylation of an aspartic acid within the DKTGT motif, allowing the protein to transduce ions via a CPC motif located in a hydrophobic transmembrane region; and (4) a C-terminal VGDGINDAP motif that is predicted to form a Mg2+ salt bridge with the -phosphate of ATP, placing the phosphate in a position from which it can be transferred to the aspartic acid. Sequence alignment showed extensive homology to metal-ion-transporting ATPases. The amino acid identities between CopA and known copper/silver ion-translocating ATPases Ent. hirae CopA, Ent. hirae CopB, E. coli CopA, Helicobacter pylori CopA, Synechocystis PacS, Salmonella typhimurium SilP, Synechococcus CtaA were 42, 28, 36, 34, 39, 35 and 31, respectively. Sequence identities between CopA and known zinc/lead/cadmium-ion-transporting ATPases E. coli ZntA, Proteus mirabilis ZntA, H. pylori CadA, Bacillus firmus CadA and Synechocystis ZiaA were 28, 27, 24, 31 and 28, respectively. As expected, the highest sequence identity was observed in regions associated with biological functions, such as phosphorylation and ion transduction. The N-terminal GMXCXXC motif was also conserved in all of the above-mentioned ATPases except Ent. hirae CopB and Sal. typhimurium SilP, which contain a histidine-rich motif.
The third ORF of the operon encoded a 67 aa protein (CopZ) which also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Homology searches provided matches to four known small proteins involved in copper or mercury resistance: (1) CopZ of Ent. hirae, (2) CopP of H. pylori, (3) CopP of Helicobacter felis, and (4) MerP of Pseudomonas alcaligenes (Fig. 2). The amino acid sequence identities between CopZ and these proteins were 17, 23, 30 and 18%, respectively. The Strep. mutans CopZ also showed homology to a small hypothetical chaperone protein, SynCh of Synechocystis sp., having an amino acid identity of 27%.
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Characterization of the cop transcript
Northern dot blotting using a probe made from copYA gave a positive signal with an RNA sample from JH1005 but not with one from the mutant S4 RNA, further indicating that the cop operon has been inactivated in mutant S4 (Fig. 3a). Interestingly, a much stronger signal was observed with RNA prepared from Cu2+-induced JH1005 cells than with RNA from non-induced cells. In Northern hybridizations (Fig. 3b
), two bands of 3·2 kb and 1·3 kb were observed from the wild-type RNA and no transcripts were observed in the S4 RNA samples (Fig. 3b
).
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Regulation of the cop operon
The results of the transcript analysis suggested that the expression of the cop operon is regulated by Cu2+ concentrations. To assist in the study of cop expression, a promoterless CAT gene was fused to the cop promoter. Plasmid pHSL2/pUC carrying the CAT reporter gene was transformed into Strep. mutans cop knock-out mutant S4 and transformants were selected with chloramphenicol. Chloramphenicol-resistant transformants were assumed to have the fusion gene integrated into the chromosome, since the plasmid did not have an origin of replication that is functional in Strep. mutans. The new strain was named S4/CAT.
Plasmids carrying the entire operon, copY or copZ were transformed into S4/CAT and the ability of the strains to grow in the presence of chloramphenicol after the addition of copper was examined. Since copper could also inhibit growth, duplicate cultures were grown in the presence of copper but without chloramphenicol. The results were expressed as percentage growth of cultures in chloramphenicol plus copper relative to that of cultures with copper only. As shown in Fig. 6, the growth of S4/CAT or of copZ-complemented S4/CAT was not inhibited by copper at concentrations of 40 µM or lower. These results indicate that the toxic effect of copper is negligible at 40 µM or lower. Growth of the cop-operon-complemented S4/CAT was not observed in the presence of chloramphenicol when copper was absent, or at 1 µM. Growth of this strain was observed when the copper concentration was higher than 10 µM, indicating the induction of the CAT reporter gene. In contrast, copY-complemented S4/CAT failed to grow with the addition of copper.
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DISCUSSION |
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The Strep. mutans CopA is a P-type ATPase that shares similarities with other bacterial ATPases that have specificity for copper or cadmium (Bayle et al., 1998 ; Ge et al., 1995
; Nucifora et al., 1989
; Odermatt et al., 1993
) and with the eukaryotic copper-transport ATPases (Payne & Gitlin, 1998
; Petrukhin et al., 1994
). These ATPases are members of a large superfamily of P-type ATPases (Axelsen & Palmgren, 1998
). This group of heavy-metal-transporting P-type ATPases contains a number of conserved structural and functional domains, which include eight transmembrane helices, a CPC ion-transduction motif, an ATP-binding site, a DKTG phosphorylation domain, and an N-terminal heavy-metal-binding motif. The CopB of Ent. hirae can transport Cu2+ and Ag2+, the CadA of Staph. aureus transports Cd2+, and the CopA from H. pylori and H. felis transports Cu2+ (Bayle et al., 1998
; Nucifora et al., 1989
; Odermatt et al., 1993
). The results of our susceptibility experiments suggest that the CopA of Strep. mutans mainly transports copper. In this regard, the Strep. mutans CopA is more similar to the Helicobacter CopA than to the enterococcal CopB.
The results from growth studies showed that the ATPase enables Strep. mutans to tolerate extracellular copper concentrations up to 800 µM. Without the ATPase, Strep. mutans mutant S4 failed to grow in copper concentrations greater than 200 µM. The ability of the wild-type Strep. mutans strain to grow in higher copper concentrations than mutant S4 implies that the Strep. mutans CopB functions to transport copper out of the cell. These results are consistent with findings in other systems, in which the disruption of CopB in Ent. hirae (Odermatt et al., 1993 ), CopA in H. pylori (Bayle et al., 1998
) and CopA in E. coli (Rensing et al., 2000
) renders cells more sensitive to copper ions.
CopY is similar to a few other bacterial negative transcriptional regulatory proteins, such as the CopY protein of Ent. hirae and proteins that regulate ß-lactam resistance, such as MecI and BlaI of staphylococci and PenI of B. licheniformis. These regulatory proteins are bifunctional, having N-terminal domains that interact with DNA while the C-terminal domains allow the proteins to dimerize and interact with their respective effector molecules or ions. The similarities between Strep. mutans CopY and the above examples include the conserved N-terminal domain, which is common to these proteins, the C-terminal CXCX4CXC motif (which is shared only by CopY of Ent. hirae), and the hypothetical protein encoded by orfY of L. sake. These regulatory proteins have been shown to repress transcription when the effector molecules (ß-lactam antibiotics) or ions (Cu2+) are absent or at physiological levels by binding to inverted repeat sequences overlapping the promoter (Hiramatsu et al., 1992 ; Smith & Murray, 1992
; Strausak & Solioz, 1997
; Wittman et al., 1993
; Wunderli-Ye & Solioz, 1999b
). Our studies on the CAT reporter gene suggest that Strep. mutans CopY is also a repressor (see below). The C-terminal motif may play an important role in derepressing transcription of the cop operon. In the enterococcal system, the binding of copper to CopY, presumably at the CXCX4CXC motif, at high copper concentrations induced disassociation of the proteinDNA complex, thereby allowing transcription (Odermatt & Solioz, 1995
; Strausak & Solioz, 1997
; Wunderli-Ye & Solioz, 1999b
).
CopZ is similar to the putative positive regulator, CopZ, of Ent. hirae. Cobine et al. (1999) recently demonstrated that the Ent. hirae CopZ protein could function as a chaperone delivering copper to CopY bound to the operator. In this capacity, Ent. hirae CopZ appears to function as an anti-repressor of the cop operon. However, previous results from the same group suggest that derepression of the cop operon could be CopZ-independent at high concentrations of Cu2+ (Strausak & Solioz, 1997
). Our experiments with the CAT reporter gene showed that the growth of S4/CAT complemented with copYAZ, but not of S4/CAT complemented with copY, could be restored by Cu2+. This result indicates that either CopB or CopZ can derepress the cop promoter activity. Given the finding of Cobine et al. (1999)
, CopZ is probably the anti-repressor.
The results of transcript analysis suggest that the expression of the cop operon is regulated. This is further supported by the results from the experiments on the CAT reporter gene. In the transcript-analysis experiments, we showed that a higher level of cop transcript was expressed by Cu2+-induced cells than by non-induced cells. In the experiment on the CAT reporter gene, we showed that S4/CAT, but not S4/CAT, complemented with copY or copYAZ could grow in the presence of chloramphenicol. Furthermore, the growth of S4/CAT complemented with copYAZ could be restored by Cu2+. These data collectively suggest that CopY is the negative regulator repressing cop promoter activity. The repression can be derepressed by Cu2+. In general, the mechanism of cop-operon regulation in Strep. mutans is similar to that in Ent. hirae. This model of regulation, however, appears to be different from that of Staph. aureus cadAC and H. pylori copAP, because the staphylococcal and the Helicobacter systems lack a CopZ or CopY analogue, respectively.
It is interesting to note that mutant A is more resistant to Cu2+ than is the wild-type (Fig. 5). The increase in resistance implies an increase in CopA activity. Such an increase may be due to an increase in transcription of the cop operon through the decreased affinity of CopY for the inverted repeats overlapping the promoter. The decreased affinity of CopY to the promoter may be caused by the insertion of Tn917 into the AT-rich region immediately upstream of the promoter. In other systems, it is known that upstream sequences play a role in promoter activation (Lamond & Travers, 1983
). Hence, it is conceivable that the upstream sequence of the cop operon is also important for promoter activity. However, in this case, the upstream sequence is required for efficient binding by CopY.
Northern blotting demonstrated that the operon is transcribed as a single polycistronic message. The 1·3 kb transcript was too small to be the ATPase and too large to be copY, and was probably a degradation product. The 3·2 kb transcript matched the size of the cop operon.
In conclusion, we have demonstrated that Strep. mutans possesses a conserved P-type ATPase which enables the bacterium to tolerate high concentrations of extracellular copper. The expression of the ATPase appears to be regulated by copper via the activity of two other members, copY and copZ, of the operon. The role of this operon in the survival of Strep. mutans in the human oral cavity, where copper ions are known to leach out of dental amalgam, remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 29 August 2000;
revised 13 November 2000;
accepted 27 November 2000.