Characterization of a copper-transport operon, copYAZ, from Streptococcus mutans

Neeraj Vats1 and Song F. Lee2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A copper-transport (copYAZ) operon was cloned from the oral bacterium Streptococcus mutans JH1005. DNA sequencing showed that the operon contained three genes (copY, copA and copZ), which were flanked by a single promoter and a factor-independent terminator. copY encoded a small protein of 147 aa with a heavy-metal-binding motif (CXCX4CXC) at the C-terminus. CopY shared extensive homology with other bacterial negative transcriptional regulators. copA encoded a 742 aa protein that shared extensive homology with P-type ATPases. copZ encoded a 67 aa protein that also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Northern blotting showed that a 3·2 kb transcript was produced by Cu2+-induced Strep. mutans cells, suggesting that the genes were synthesized as a polycistronic message. The transcriptional start site of the cop operon was mapped and shown to lie within the inverted repeats of the promoter–operator region. Strep. mutans wild-type cells were resistant to 800 µM Cu2+, whereas cells of a cop knock-out mutant were killed by 200 µM Cu2+. Complementation of the cop knock-out mutant with the cop operon restored Cu2+ resistance to wild-type level. The wild-type and the mutant did not show any differences in susceptibility to other heavy metals, suggesting that the operon was specific for copper. By using a chloramphenicol acetyltransferase reporter gene fusion, the cop operon was shown to be negatively regulated by CopY and could be derepressed by Cu2+.

Keywords: copper transport, copper resistance, P-type ATPase, heavy metals

Abbreviations: CAT, chloramphenicol acetyltransferase; THA, Todd–Hewitt agar; THB, Todd–Hewitt broth; TYG, tryptone/yeast extract glucose broth

The GenBank accession number for the cop operon sequence reported in this paper is AF296446.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Copper is an essential element for growth because of its role as a cofactor for enzymes. However, copper is also very toxic to both eukaryotic and prokaryotic cells. Hence, regulation of the intracellular copper concentration is crucial for cell viability. In bacteria, the cytoplasmic copper concentration can be controlled by a decrease in copper transport, by copper complexation by cell components, or by an energy-dependent efflux system (Cervantes & Gutierrez-Corona, 1994 ). The latter is a common mechanism, not only for the homeostasis of copper but also for that of other transition metals (Silver & Phung, 1996 ; Rensing et al., 1999 ). The best-understood copper transport and resistance system is that of Enterococcus hirae (Wunderli-Ye & Solioz, 1999a ). The chromosomally located copYABZ operon of Ent. hirae encodes two P-type ATPases, CopA and CopB, which respectively transport copper into and out of the cells, and CopY and CopZ, which regulate the expression of the cop operon in response to both copper starvation and copper excess (Odermatt & Solioz, 1995 ). Among Gram-negative bacteria, copper transport and resistance systems conferred by plasmids have been identified in Pseudomonas, Escherichia coli and Xanthomonas (Silver & Phung, 1996 ). These systems are highly homologous. In Pseudomonas sp., the system contains four structural genes (copABCD), the expression of which is regulated by the two-component regulatory system genes (copR and copS). The four structural genes encode an inner-membrane protein (CopD), an outer-membrane protein (CopB) and two copper-binding periplasmic proteins (CopA and CopC).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and culture conditions.
Strep. mutans strains NG8 and JH1005 (serotype c) were grown in Todd–Hewitt broth (THB), in TYG broth (1% K2HPO4, 1% trypticase peptone, 0·5% yeast extract and 1% glucose, w/v) without agitation, or on Todd–Hewitt agar (THA) in candle jars. E. coli was grown in Luria–Bertani broth. All strains were cultivated at 37 °C. Where required, antibiotics (Sigma) were added to the media as follows: ampicillin, 100 µg ml-1 (for E. coli); kanamycin, 50 µg ml-1 (for E. coli) and 400 µg ml-1 (for Strep. mutans); erythromycin, 300 µg ml-1 (for E. coli) and 10 µg ml-1 (for Strep. mutans); tetracycline, 10 µg ml-1 (for E. coli) and 15 µg ml-1 (for Strep. mutans); and chloramphenicol, 10 µg ml-1 (for E. coli) and 5 µg ml-1 (for Strep. mutans).

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 1–2 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 2–3 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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Fig. 1. The Strep. mutans cop operon. (a) Genetic organization of the cop operon with the three ORFs. The relevant restriction sites are shown below the operon: B, BamHI; E, EcoRI; H, HindIII; Hc, HincII; P, PstI; S, SstI; X, XbaI. The DNA fragments carried by the plasmids described in the text are depicted below the operon. (b) The promoter–operator region of the copYAZ operon. The position of the Tn917 insertion is indicated by a vertical arrowhead, the inverted repeats within the -35 and -10 regions are represented by horizontal arrows above the sequence, the Shine–Dalgarno (SD) sequences and the ATG start codon are shown in bold type, and the transcriptional start site is indicated by an asterisk.

 
Cloning of the cop operon from Strep. mutans.
Southern blot analysis of HindIII-digested Strep. mutans JH1005 chromosomal DNA, using a probe prepared from the 2·5 kb HindIII insert on pNV4, yielded a signal of approximately 3 kb. To clone this 3 kb fragment, DNAs of 2–5 kb were recovered from an agarose gel and ligated into HindIII-digested and dephosphorylated pBluescript and transformed into E. coli XL-1 Blue. The transformants obtained were screened by colony hybridization, using the same 2·5 kb HindIII insert from pNV4 as probe. A positive clone (NVD20) was obtained. DNA sequencing showed that the DNA fragment in clone NVD20 did not contain the entire gene. Therefore, a partial library consisting of 3–6 kb HindIII fragments of partially digested Strep. mutans JH1005 chromosomal DNA, which were isolated by a sucrose density gradient (10–40%; Sambrook et al., 1989 ) was prepared using pUC18 as the vector. By screening the library, using colony blotting with the 2·5 kb insert of pNVD20 as a probe, a positive clone (WH4) was found to carry the same 2·5 kb HindIII insert of pNVD20 and an additional 1·5 kb HindIII fragment. By using the 1·5 kb HindIII fragment as the probe in Southern blotting, a 1·4 kb HincII fragment was identified and subsequently cloned into SmaI-restricted and dephosphorylated pUC18 (Amersham Pharmacia Biotech) to obtain clone NV5. From a homology search of the database of the partial genome sequence of Strep. mutans UAB159 (www.genome.ou.edu), using our sequence as a query, a positive match within a 7·5 kb DNA fragment was identified. The sequence data from strain UAB159 were used to design the forward primer, SL145 (5'-GGACCTTTGCTGGATCC-3'), and the reverse primer, SL146 (5'-GTAGAATTCAAAAGCGTGCCATC-3'), for PCR amplification of a 500 bp PCR product carrying the last portion of the cop operon. The PCR product was blunt-end cloned into SmaI-restricted and dephosphorylated pUC18 to obtain the plasmid pNV46.

The entire cop operon was reconstructed from the cloned DNA fragments. A 2·5 kb PstI–XbaI 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 BamHI–EcoRI 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 SstI–XbaI 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 SphI–XbaI 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 manufacturer’s 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. DNA–DNA hybridization was performed as described by Southern (1975) , and DNA–RNA hybridization was performed as described by Ausubel et al. (1990) .

Primer extension.
The reverse primer SL159 (5'-GCAATAATTTCACTGCTG-3'), corresponding to nucleotide positions 813–830 (140 nt downstream of the -35 region) of the cop operon, was end-labelled with [{gamma}-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 manufacturer’s 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 University–NRC 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 promoter–CAT fusion and a fragment of spaP. The 4 kb fragment was ligated to the 1·7 kb ScaI–XbaI 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 20–24 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequence analysis of the Strep. mutans cop operon
The genes within the Strep. mutans JH1005 cop operon were cloned in the following four overlapping fragments: a 2·5 kb HindIII fragment (pNVD20); a 4 kb HindIII fragment (pWH4); a 1·4 kb HincII fragment (pNV5); and a 0·5 kb fragment (pNV46) (Fig. 1a). From the DNA sequence of these fragments, three ORFs of 147, 743 and 67 aa were identified. A single promoter was found upstream of the first ORF, each ORF was preceded by a Shine–Dalgarno sequence, and a factor-independent terminator was identified approximately 100 bp downstream of the stop codon of the third ORF. These features suggest that the three ORFs form an operon. At the promoter–operator region of the operon was a pair of inverted repeats; the first repeat overlapped the -35 region and the second immediately followed the -10 region (Fig. 1b). Similar repeats were also present at the promoter–operator region of the Ent. hirae copYZAB (Odermatt & Solioz, 1995 ) and Staphylococcus aureus cadAC operons (Endo & Silver, 1995 ; Nucifora et al., 1989 ), where they function as binding sites for the regulatory proteins CopY and CadC, respectively.

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 {gamma}-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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Fig. 2. Comparison of the amino acid sequences of Strep. mutans CopZ (SmCopZ) with similar proteins. CopZ was aligned, using CLUSTAL W (Thompson et al., 1994 ), with CopZ of Ent. hirae (EhCopZ, accession no. Z46807), H. pylori CopP (HpCopP, accession no. Q48271), H. felis CopP (HfCopP, accession no. O032620), P. alcaligenes MerP (PaMerP, accession no. AAC33271) and a Synechocystis sp., SynCh (SynCh, accession no. BAA17240). Identical residues are represented by white letters on a black backgound and are indicated by exclamation marks, conserved residues found in at least 60% of the sequences are indicated by asterisks, and similar residues are shown as lower-case letters. Helicobacter CopP is a metal-binding protein postulated to regulate the expression of ATPase. Enterococcal CopZ is an anti-repressor of the cop operon. MerP is a mercury-binding protein that facilitates mercury resistance. SynCh is a hypothetical chaperone protein.

 
Generation of a cop-operon knock-out strain
An isogenic mutant, S4, of Strep. mutans JH1005 devoid of the cop operon was produced by homologous recombination using the construct pNV/S4. The results of Southern blotting showed that HindIII-restricted chromosomal DNA from mutant S4 did not hybridize to the probe made from copYA DNA, whereas the wild-type DNA showed an expected 1·8 kb band with this probe (data not shown). When the DNAs were hybridized with a probe made from the tetracycline cassette specific to pNV7/S4, two bands (7 kb and 2·5 kb) were noted in the S4 DNA, but were absent in the wild-type DNA (data not shown). These results indicate that the tetracycline-resistant transformant, S4, lacked the cop operon and had the tetracycline cassette in its place.

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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Fig. 3. Northern hybridization of the Strep. mutans cop transcript. (a) RNA dot blotting. Total RNA from JH1005 (non-induced, column 1), JH1005 (Cu2+-induced, column 2), S4 (non-induced, column 3) and S4 (Cu2+-induced, column 4) were blotted at 20, 12 or 6 µg. (b) Northern blotting of 20 µg total RNA from JH1005 (Cu2+-induced, lane 1) and S4 (lane 2) as described in (a). The RNA size markers were from Gibco-BRL. DNA probes were prepared against copYA from pWH4 by the Bio-Nick labelling kit and were detected by using the Photogene detection system (Gibco-BRL). The arrow indicates the 3·2 kb transcript.

 
The transcriptional start site of the cop operon was determined by primer extension using RNA prepared from the Cu2+-induced wild-type cells. As shown in Fig. 4, the first base of the transcript was identified as the adenine located 6 nt downstream of the -10 region but within the second pair of inverted repeats.



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Fig. 4. Mapping of the transcriptional start site of the Strep. mutans cop operon by primer extension. The sequence of the promoter region of the cop operon was generated from pWH4. The DNA sequence is shown on the left and the transcriptional start site is indicated by an asterisk. P, primer extension product from the RNA of Cu2+-induced JH1005 cells.

 
Role of the cop operon in the copper resistance of Strep. mutans
The sequence similarities between the ORFs and the Ent. hirae cop operon suggested that the Strep. mutans cop operon was also involved in copper transport and resistance. Hence, the effect of copper on growth of the Strep. mutans wild-type, S4, and mutant A was determined. As shown in Fig. 5(a), the wild-type cells were resistant to 400 µM Cu2+ and exhibited impaired growth at 600 µM Cu2+. Growth was inhibited at 800 µM Cu2+. Interestingly, the Tn917 mutant, mutant A, was able to tolerate higher Cu2+ concentrations than the wild-type. Although growth was impaired, mutant A was able to grow better in the presence of 800 µM Cu2+ than was JH1005 (Fig. 5b). Complementation of mutant A with copY carried on plasmid pWH4/PDL resulted in growth inhibition by 400 µM Cu2+ (Fig. 5b, open triangles). In comparison, growth of the cop knock-out mutant, S4, was severely impaired by 400 µM Cu2+ and completely inhibited by 600 µM Cu2+ (Fig. 5c). As expected, the ability of S4 to grow in higher Cu2+ concentrations was restored by complementation with the cop operon (Fig. 5d).



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Fig. 5. Effects of copper on growth of Strep. mutans JH1005 (a), mutant A (b), cop knock-out mutant S4 (c) and cop-operon-complemented S4 (d). The Cu2+ concentrations were as follows: 0 ({square}), 400 ({bullet}), 600 ({blacksquare}) and 800 µM ({circ}). In (b), {triangleup} represents mutant A complemented with copY in the presence of 400 µM Cu2+.

 
The observations on the effects of Cu2+ on growth were further confirmed by results from susceptibility studies. The MIC values of Cu2+ for JH1005, mutant A, and cop knock-out mutant S4 were 800, 800, and 200 µM, respectively. Complementing S4 cells with the cop operon restored the MIC value to the wild-type level. The sensitivity of JH1005 and the mutants to other heavy metal ions was also examined. There was no difference between JH1005 and the mutants in the MICs of Ag+ (50 µM), Cd2+ (3 µM), Zn2+ (14 mM) and Hg2+ (6 µM).

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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Fig. 6. Growth of Strep. mutans S4/CAT and cop-gene-complemented strains in the presence of copper and chloramphenicol. Each strain was grown in TYG, with or without chloramphenicol, for each of tested copper concentrations. Percentage growth was calculated as (OD600 of cultures with chloramphenicol/OD600 of cultures without chloramphenicol)x100. {blacksquare}, S4/CAT, the cop-operon knock-out carrying the cop promoter/CAT reporter gene; {square}, copZ-complemented S4/CAT; , cop-operon-complemented S4/CAT; , copY-complemented S4/CAT.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present work, we have identified and isolated a copper-transport and -resistance operon in Strep. mutans. To the best of our knowledge, this is the first report of the isolation and characterization of such an operon from the genus Streptococcus. The operon consists of three genes: copY, copA and copZ. Overall, the Strep. mutans cop operon is most similar to the Ent. hirae cop operon. In other characterized bacterial operons, such as the Staph. aureus cadmium-resistance (cadAC) operon or the Helicobacter copper-transport (copAP) operon, similar P-type ATPases were encoded but, respectively, lacked a copZ or a copY homologue (Bayle et al., 1998 ; Endo & Silver, 1995 ; Ge et al., 1995 ; Nucifora et al., 1989 ). The Strep. mutans cop operon differs from that of Ent. hirae in the following ways. First, the Strep. mutans operon dose not have a copper-import ATPase. In the enterococcal system, CopA is one such ATPase (Odermatt et al., 1993 ). Second, the genetic organization of the Strep. mutans copYAZ operon is slightly different from that of Ent. hirae (copYZAB). Third, the Strep. mutans cop operon appears to be specific for copper, unlike the Ent. hirae cop operon (which is also involved in silver resistance). Fourth, the metal-binding motif of the Strep. mutans CopA is more similar to the Ent. hirae copper-import pump, CopA, than the copper-efflux pump, CopB.

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 protein–DNA 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.


   ACKNOWLEDGEMENTS
 
We thank H. Wang and H. Loung for their technical assistance, and R. J. Lamont for providing pMH109. This study was supported by the Medical Research Council of Canada.


   REFERENCES
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DISCUSSION
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Received 29 August 2000; revised 13 November 2000; accepted 27 November 2000.