ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/00/$04.0010

June 2000, p. 1549–1555 Vol. 44, No. 6

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

A New Class of Genetic Element, Staphylococcus CassetteChromosome mec, Encodes Methicillin Resistance

in Staphylococcus aureusY. KATAYAMA, T. ITO, AND K. HIRAMATSU*

Department of Bacteriology, Juntendo University, Tokyo, Japan

Received 10 December 1999/Returned for modification 30 January 2000/Accepted 10 March 2000

We have previously shown that the methicillin-resistance gene mecA of Staphylococcus aureus strain N315is localized within a large (52-kb) DNA cassette (designated the staphylococcal cassette chromosome mec[SCCmec]) inserted in the chromosome. By sequence determination of the entire DNA, we identified two novelgenes (designated cassette chromosome recombinase genes [ccrA and ccrB]) encoding polypeptides having apartial homology to recombinases of the invertase/resolvase family. The open reading frames were found tocatalyze precise excision of the SCCmec from the methicillin-resistant S. aureus chromosome and site-specificas well as orientation-specific integration of the SCCmec into the S. aureus chromosome when introduced intothe cells as a recombinant multicopy plasmid. We propose that SCCmec driven by a novel set of recombinasesrepresents a new family of staphylococcal genomic elements.

Methicillin-resistant Staphylococcus aureus (MRSA) wasfirst isolated in England in 1961 shortly after the developmentof methicillin, the first penicillinase-resistant semisyntheticpenicillin (15). Since then, MRSA has become the most prev-alent pathogen causing hospital infection throughout theworld, and MRSA incidence is still increasing in many coun-tries (1). MRSA is resistant to practically all b-lactam antibi-otics, a class of antibiotics represented by penicillins and ceph-alosporins (3).

The b-lactam resistance of MRSA is caused by the produc-tion of a novel penicillin-binding protein (PBP) designatedPBP 29 (or PBP 2a), which, unlike the intrinsic set of PBPs(PBP 1 to 4) of S. aureus, has remarkably reduced bindingaffinities to b-lactam antibiotics (9, 24, 30). Despite the pres-ence of otherwise inhibitory concentrations of b-lactam anti-biotics, MRSA can continue cell wall synthesis solely de-pending upon the uninhibited activity of PBP 29 (21). PBP 29is encoded by a mecA gene located on the chromosome ofMRSA. In 1987, the mecA gene was cloned from a JapaneseMRSA strain, and its sequence was determined (20, 26). ThemecA gene is widely distributed among S. aureus as well ascoagulase-negative staphylococci (13, 28). Therefore, it hasbeen speculated that the methicillin resistance determinant(mec determinant) is freely transmissible among staphylococ-cal species. However, with a detailed molecular epidemiolog-ical study, Kreiswirth et al. have proposed that MRSA origi-nated from a single or two ancestral clones (16). This led to theview that the frequency of inter- or intraspecies transmission ofmecA is a rather limited process and that mec transmission maynot be due to specialized transmission machinery, such as atransposon.

We have recently cloned and sequenced the entire chromo-somal region surrounding the mecA gene, which is additionallypresent in the MRSA chromosome and is absent from thechromosome of methicillin-susceptible S. aureus (MSSA)(referred to herein as mecDNA), from a Japanese pre-MRSA

strain, N315 (10). We identified the mecDNA-S. aureus chromo-some junction points and the overall structure of mecDNA,and revealed that mecDNA constitutes a genomic island orantibiotic resistance island of S. aureus (14). In this study,based on the structure of mecDNA, we report that mecDNA isa new class of genomic element designated SCCmec (for staph-ylococcal cassette chromosome mec [12]) driven by two site-specific recombinase genes designated ccrA and ccrB (for cas-sette chromosome recombinases A and B).

MATERIALS AND METHODS

Bacteria and growth condition. Pre-MRSA strain N315 and its SCCmec ex-cising strain N315ex used in this study have been described previously (14). Allthe strains and their transformants were cultivated in brain heart infusion (BHI)broth (Becton Dickinson Microbiology Systems, Sparks, Md.). The antibioticstetracycline (Sigma Chemical Co., St. Louis, Mo.) and tobramycin (Shionogi Co.,Osaka, Japan) were used at the concentration of 10 mg/ml.

Construction of recombinant plasmids. Recombinant plasmid pSR harboringintact ccrA and ccrB genes was constructed by cloning the BamHI-digested DNAfragment containing ccr genes into the unique BamHI site of the plasmid vectorpYT3 (8). The DNA fragment containing ccr genes was prepared by PCR usingthe DNA extracted from N315 as a template. The two primers used were59-AAAAGGATCCATTAGCCGATTTGGTAATTGAA-39 and 59-AAAAGGATCCTCTGCTTCTTCGAATCTGCAAAT-39 (introduced BamHI sites areunderlined), which correspond to the nucleotides from base positions 24,004 to24,025, and to the complementary nucleotides from positions 27,365 to 27,343 ofthe SCCmec sequence (accession no. D86934), respectively. To constructpSRA*, the BamHI fragment of pSR was subcloned into the BamHI site ofpUC119DH, a derivative of pUC119 whose HindIII site had been eliminated bytreatment with the Klenow fragment of DNA polymerase I and self-ligation. Theresultant plasmid, pUCSR, was digested with BalI and HindIII and flush endedby Klenow treatment, which was followed by ligation to delete the BalI-HindIIIfragment (344 bp) of the ccrA gene. Then, the BamHI fragment was cut out ofthe plasmid and was ligated into the BamHI site of pYT3 to obtain pSRA*. Toconstruct pSRB*, pUCSR was cut with BstXI and MluI to remove the 252-bpBstXI-MluI fragment of the ccrB gene, and this was followed by Klenow treat-ment and self-ligation. The BamHI fragment of the resultant plasmid was cut outand ligated into the BamHI site of pYT3 to obtain pSRB*.

To construct attSCC-containing recombinant plasmids, the DNA fragmentcontaining the attSCC sequence on the putative closed circular SCCmec cassettewas amplified by PCR using the two primers mR7 and mL2 (see below), and theDNA was extracted from N315 (pSR) and used as a template. The amplifiedDNA was digested with SalI, and the resultant 933-bp fragment was cloned intoa unique SalI site of plasmid pYT3 to obtain pYT3att. Then, the attSCC frag-ment was cut out of pYT3att with SalI and introduced into the SalI sites ofpSR, pSRA*, and pSRB* to obtain pSRatt, pSRA*att, and pSRB*att, respec-tively.

* Corresponding author. Mailing address: Department of Bacteriol-ogy, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8421. Phone: 81-3-5802-1040. Fax: 81-3-5684-7830. E-mail: hiram@med.juntendo.ac.jp.

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Nucleotide sequencing. The DNA fragments for sequencing were obtained byPCR amplification with genomic DNA as templates. After purification using aQIAquick PCR purification kit (QIAGEN, Hilden, Germany), the fragments ofPCR products were directly sequenced with a set of synthetic oligonucleotideprimers (see below) using a dye-labeled terminator–Taq DNA polymerase cyclesequencing kit (Applied Biosystems Inc., Foster City, Calif.). The sequence wasread on a 373A automated fluorescent DNA sequencing system (Perkin-Elmer,Foster City, Calif.). All the computer analyses of nucleotide sequences werecarried out using programs in The Wisconsin Package (version 9.0; GeneticsComputer Group [GCG], Madison, Wis.). A homology search was performedusing BLAST and TFastA programs accessed via the EMBL (release no. 55.0)and GenBank (release no. 107.0) databases and the FastA program accessed viathe SWISS-PROT database. (release no. 35.0).

PFGE. Pulsed-field gel electrophoresis (PFGE) was performed with a modi-fication as described previously (32). For preparation of sample plugs, ca. 2 3 106

cells were embedded in 37.5 ml (1.5 by 5 by 5 mm) of 1% (wt/vol) low-temper-ature-melting agarose (Agarose Low Melt Preparative Grade; Bio-Rad Labora-tories, Hercules, Calif.) containing 40 mg of lysostaphin (Sigma Chemical Co.)per ml. The sample plugs were incubated with 1% (wt/vol) N-lauroylsarcosine(Sigma Chemical Co.)–0.5 M EDTA, pH 7.5, at 37°C for 1 h and furtherincubated with a solution containing 2 mg of proteinase K (Sigma Chemical Co.)per ml, 1% (wt/vol) N-lauroylsarcosine (Sigma Chemical Co.), and 0.5 M EDTA,pH 7.5, at 50°C for 24 h. The plugs were then incubated with 1 mM phenylmeth-ylsulfonyl fluoride (Sigma Chemical Co.) at 50°C for 30 min, followed by washingthree times with Tris-EDTA (20 min each at 4°C). The samples were then treatedwith 60 U of SmaI (Takara Shuzo Co. Ltd., Kyoto, Japan) at 30°C for 24 h andnext were treated with 200 mg of proteinase K at 37°C for 1 h. The finalconcentrations of SmaI and proteinase K were 0.4 U/ml and 100 mg/ml, respec-tively. They were further washed three times with Tris-EDTA at 4°C for 20 minand subjected to PFGE with 1.2% agarose gel (high-strength analysis-gradeagarose; Bio-Rad Laboratories). The PFGE was performed at 4°C for 20 h in0.53 Tris-borate-EDTA running buffer using a CHEF DRII apparatus (Bio-Rad Laboratories) with an electric field strength of ;6 V/cm and a pulse timeof 20 s.

Electroporation. Exponentially growing cultures of strain N315 or N315exwere harvested at an optical density at 600 nm of 0.5 and washed with prechilled1.1 M sucrose three times. The pellet was resuspended in 100 ml of EP buffer (1.1M sucrose, 2 mM MgCl2, 14 mM KH2PO4-Na2HPO4 [pH 7.4]). To the cellsuspension, 5 mg of the plasmid DNA was added, and the mixture was kept onice for 25 min. An electric pulse of 25 mF, 2.5 kV, and 100 V was delivered by aGene Pulser (Nippon Bio-Rad Laboratories, Tokyo, Japan). The cells were thentransferred into 0.5 ml of 1.1 M sucrose in BHI broth and incubated for 2 h at30°C (permissive temperature for plasmid replication) before aliquots wereplated on BHI agar plates containing tetracycline (10 mg/ml). After a 48-hincubation at 30°C, mature colonies were picked and subjected to rapid plasmidanalysis by digestion with various restriction enzymes to confirm the integrity of

the introduced plasmid. The plasmid preparation procedure has been describedpreviously (32). The transformants were further colony purified by streakingthem onto the BHI agar plate with tetracycline (10 mg/ml) and incubating theplate overnight before establishing the transformant strains as those used in thisstudy.

Southern blot hybridization. Southern transfer of DNA from the PFGE gel toa nylon membrane was performed as described previously (32). Hybridizationtook place in a hybridization solution (50% formamide, 53 SSC [13 SSC is 0.15M NaCl plus 0.015 M sodium citrate], 0.3% sodium dodecyl sulfate [SDS], 2%blocking reagent [Boehringer, Mannheim, Germany]) containing 20 ng of digoxi-genin-labeled DNA probe per ml. Incubation was carried out for 16 h at 42°C.The orfX probe was prepared by using primers 59-CCACGCATAATCTTAAATGCTCT-39 and 59-AAACGACATGAAAATCACCAT-39 (primer cR2 [14]),which corresponded to the nucleotides from base positions 56,357 to 56,379 andcomplementary nucleotides from base positions 56,824 to 56,804 of the reportednucleotide sequence of orfX (accession no. D86934), respectively. The probe forthe ermA gene was prepared using synthetic oligonucleotides 59-TGAAACAATTTGTAACTATTGA-39 and 59-TGAACCAGAAAAACCCTAAAGA-39 asprimers, which corresponded to the nucleotides from positions 33,804 to 33,825and the complementary nucleotides from position 34,530 to 34,509, respectively,of the reported nucleotide sequence of the N315 SCCmec (accession no. D86934[14]). The cloned DNA fragment SJ2, which contains Tn554 of SCCmec, wasused as the template for the PCR amplification of the ermA gene (14). Theseprobes were labeled with digoxigenin using a DNA labeling kit (Boehringer).The hybridized filter (Biodyne A; Pall Biosupport Co., Glen Cove, N.Y.) waswashed twice in 23 SSC with 0.1% SDS for 5 min at room temperature and thenin 13 SSC with 0.1% SDS for 15 min at 68°C. Visualization of the signal wasachieved using an alkaline phosphatase-conjugated antidigoxigenin antibody(Boehringer) and the chemiluminescent substrate CSPD (Boehringer) accordingto the procedure recommended by the manufacturers.

Synthetic oligonucleotides used as primers for PCR and nucleotide sequenc-ing. The primers used in this study and their nucleotide sequences with refer-ences, if not specified above, are listed below in the order of appearance in thetext: cL1, 59-ATTTAATGTCCACCATTTAACA-39 (14); mL1, 59-GAATCTTCAGCATGTGATTTA-39 (corresponds to the complementary nucleotides frompositions 4,977 to 4,957 of the nucleotide sequence [accession no. D86934]);mR8, 59-ATGAAAGACTGCGGAGGCTAACT-39 (corresponds to the nucle-otides from base position 56,636 to 56,658 [accession no. D86934]); cR2, 59-AAACGACATGAAAATCACCAT-39 (14); mA1, 59-TGCTATCCACCCTCAAACAGG-39 (10); mA2, 59-AACGTTGTAACCACCCCAAGA-39 (10); mR7, 59-AAAAAGTCGACACTGCTTGGGTAACTTATCATGGA-39; mL2, 59-AAAAAGTCGACATCACAGTAGTGCAAAGCACGTCGA-39; a, 59-TTTCACACAGGAAACAGCTATGAC-39; and b, 59-ATCACGATATTGCTTATAAGCA-39(corresponds to the nucleotides from base positions 26,673 to 26,694 of thereported sequence [accession no. D86934]).

FIG. 1. Identification of ccr genes. (A) Genomic structure of SCCmec. Locations of the essential genes are illustrated. The left and right chromosome-SCCmecjunctions, attL and attR, were tentatively and operationally defined in this study by PCR primer combinations of cL1 and mL1 for attL, and mR8 and cR2 for attR. TheMSSA chromosomal parts of the attL and attR were defined as attB-L and attB-R, delimited by primers cL1 and cR2, respectively. The SCCmec parts of these elementswere defined as attSCC-L and attSCC-R, delimited by the primers mL1 and mR8, respectively: i.e., attL is attB-L plus attSCC-L, and attR is attB-R plus attSCC-R.Transposon Tn554 (23), encoding resistance to erythromycin and spectinomycin, was located in upstream of the mecI-mecR1-mecA gene complex (14). Plasmid pUB110,encoding resistance for kanamycin-tobramycin and bleomycin, was inserted between two insertion sequences IS431 (or IS257) (2, 7, 14, 22, 27). Nucleotide sequencesaround the left and right boundaries of SCCmec, attL and attR, are shown at the bottom of the panel. Inverted repeats, IRscc-L and IRscc-R, at both extremities ofSCCmec are indicated by thin arrows. Thick arrows indicate the direct repeats, DR-B and DR-SCC. The nucleotide sequence of the N315 chromosome containing theentire SCCmec (56,939 bp) is deposited in DDBJ/EMBL/GenBank under accession number D86934. (B) Deduced amino acid sequences of ccrA and ccrB. CcrA andCcrB amino acid sequence were aligned with that of TP901-1 integrase (4). The alignment was performed using the Pile-Up program with a scoring matrix of pam 250in the Wisconsin Package (version 9.0; Genetics Computer Group). Amino acids shared by the three peptides are boxed in red. CcrA and CcrB had 26.5 and 37.4%amino acid identity, respectively, with the TNP901-1 integrase. Large bullets indicate amino acid residues of CcrA or CcrB shared by site-specific recombinases of theinvertase/resolvase family. Small bullets indicate amino acid substitution within the same class of amino acids. Of the 64 amino acid positions well conserved amongthe recombinases of the invertase/resolvase family (shown in pink) (25), 44 and 47 amino acids, respectively, were conserved in CcrA and CcrB. An arrowhead indicatesthe presumptive serine involved in the 59 phosphoseryl linkage of the recombinase to DNA characteristically conserved in the NH2-terminal catalytic domain ofsite-specific recombinases of the invertase/resolvase family (25). (C) Construction of plasmids carrying intact and disrupted ccr genes. Restriction maps of pSR, pSRA*(a derivative of pSR with a partially deleted ccrA), and pSRB* (a derivative of pSR with a partially deleted ccrB) are shown. Arrows indicate the direction oftranscription of the structural gene ccr and tetL. The respective set of ccrA and ccrB genes was inserted at the BamHI site of pYT3.

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RESULTS

ccrA and ccrB gene-mediated excision of SCCmec. Two novelORFs, ccrA and ccrB, were located in the midst of SCCmec(Fig. 1A). CcrA and CcrB polypeptides of 449 and 542 aminoacids were potentially encoded by the ORFs. A Shine-Dal-garno sequence and 210 and 235 presumptive promoter se-quences were identified immediately upstream of ccrA. On theother hand, although Shine-Dalgarno sequence was found forccrB, no candidates for promoter sequences were identified inthe adjacent upstream region of the gene. This suggests thatthe two ORFs may be transcribed as a single mRNA.

Two ORFs, ccrA and ccrB, were subcloned into a shuttlevector, pYT3, which has a temperature-sensitive origin of rep-lication (copy numbers: 14 at 30°C, 1 at 37°C, and 0 at 42°C).

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The recombinant plasmids obtained were pSR, pSRA* (with adeletion introduced in ccrA), and pSRB* (with a deletion in-troduced in ccrB) (Fig. 1C). These recombinant plasmids wereintroduced into N315 by electroporation (17).

After cultivation of these strains in drug-free broth for 20 hat 30°C, we evaluated the proportion of the cells which had lostSCCmec in the culture by plating onto agar with and withouttobramycin. The cultures of strains N315, N315(pYT3), N315(pSRA*), and N315(pSRB*) yielded essentially equal numbersof colonies on the BHI agar plates with or without tobramycin.On the other hand, the culture of strain N315(pSR), harbor-ing both intact ccrA and ccrB genes, generated tobramycin-susceptible cells, which constituted 51 to 62% of the entire cellpopulation in repeated experiments. A representative tobra-mycin-susceptible subclone, N315ex was established from theexperiment.

Antibiotic susceptibility patterns of N315 and N315ex werecompared on the basis of MICs determined using a standardprocedure (17). The MICs of various antibiotics (in micro-grams per milliliter) for N315 and N315ex were, respectively,64 and 2 (oxacillin), 64 and 4 (ceftizoxime), 256 and 0.5 (to-bramycin), 256 and 2 (kanamycin), and .512 and 16 (bleomy-cin). The data showed that all the resistance phenotypes wereassociated with the presence of SCCmec except for erythromy-cin resistance. Southern blot hybridization analysis of SmaI-digested DNAs of N315 and N315ex using the ermA gene-specific probe detected four restriction fragments (.436.5,420, 320, and 110 kb in size) in both strains in addition to the215-kb fragment of N315 on which SCCmec was localized(data not shown; see below for the PFGE-separated SmaI-restriction fragment pattern).

To confirm that the loss of resistance was caused by theexcision of SCCmec, DNAs were extracted from transformantsN315(pYT3), N315(pSR), N315(pSRA*), and N315(pSRB*),as well as from control strains N315 and N315ex. PCR usingthe combination of primers cL1 and cR2, which theoreticallyamplifies the 0.5-kb DNA fragment containing attB whenSCCmec is excised from the chromosome [Fig. 2A), did notamplify any DNA fragment with N315, N315(pYT3), N315(pSRA*), and N315(pSRB*) DNAs as templates but the0.5-kb DNA fragment was amplified with DNAs extractedfrom N315(pSR) and N315ex (Fig. 2A). The nucleotide se-quencing of the amplified 0.5-kb DNA fragments from bothN315(pSR) and N315ex DNAs showed that they contained theattB sequence theoretically expected to be generated by theprecise excision of SCCmec from the chromosome (Fig. 2B).The SCCmec excision was considered to be precise since thenucleotide sequencing using the total DNA extracted fromN315(pSR) as a template yielded a readable nucleotide se-quence of attB which was identical to that yielded when theN315ex DNA was used as a template.

Concomitant appearance of a rearranged attachment se-quence, attSCC, with SCCmec excision. Upon the precise ex-cision, the excised SCCmec was expected to form an extrachro-mosomal closed circular DNA. To test this possibility, PCRwas performed using a pair of primers set divergently at bothtermini of SCCmec (primers mR8 and mL1 [Fig. 1A]). Figure2A shows that a DNA fragment of 0.5 kb was specificallyamplified from N315(pSR) but not from the other test strains.Nucleotide sequencing of the fragment showed a novel nucle-otide sequence presumably generated by a head-to-tail ligationof attSCC-R and attSCC-L of both termini of SCCmec, forminga novel attachment sequence designated attSCC (Fig. 2B). Thedivergently arranged 27-bp inverted repeats, IR-L and IR-R,with a core of four nucleotides, TTCT (underlined in Fig. 2B),between them and the presence of direct repeats, DR-SCC,

were characteristic of the structure of the attSCC. The datasuggested that a closed circular form of SCCmec was producedas a result of the precise excision of SCCmec from the N315chromosome.

Loss of SCCmec during passage of N315(pSR). To see therate of loss of SCCmec from the culture of N315(pSR) andconfirm the precision of excision mediated by ccr genes, PFGEanalysis on the total DNAs that were consecutively extractedfrom the culture was performed (Fig. 3A). Compared to theculture of N315 and N315(pYT3) (lanes 1 and 9, respectively),N315(pSR) had an extra band of about 160-kb (Fig. 3A, lane2). The band was indistinguishable from that of N315ex, fromwhich a 215-kb band of N315 was missing (lane 8). As long asthe N315(pSR) was cultured in tobramycin-containing BHI

FIG. 2. Precise excision of SCCmec mediated by ccrA and ccrB. (A) Detec-tion of ccr-mediated SCCmec excision and appearance of attSCC. TemplateDNAs for PCR were extracted from overnight culture of the colony purifiedtransformant strains in BHI broth with tetracycline (10 mg/ml). Four sets ofprimers were used to detect precise excision and the closed circle form ofSCCmec. Primers cR2 and cL1 were used to detect attB (549 bp). Primers mL1and mR8 were used to identify attSCC (456 bp). Primers cL1 and mL1 were usedto detect attL (371 bp). Primers mA1 and mA2 were used to detect a part ofmecA gene (286 bp). Note that precise excision of SCCmec occurred only in N315(pSR) and N315ex and that attSCC was detected only in N315(pSR). The mo-lecular weight marker was a 1-kb ladder (Gibco-BRL, Gaithersburg, Md.) andonly the relevant sizes are indicated. (B) Generation of attSCC and attB. Thepresumptive closed circular form of SCCmec is illustrated with attSCC, whichappeared to be generated by head-to-tail ligation of attSCC-L (stippled box) andattSCC-R (striped box) (see Fig. 1A). Nucleotide sequences of attB in amplifiedDNA fragments from N315ex and N315(pSR) are also illustrated. The primersmR7 and mL2 were used for the cloning of attSCC into pYT3.

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broth, the proportions of the two bands were the same: den-sitometric measurement of the 215-, and 160-kb bands yieldeda ratio of about 7 to 8:1 (data not shown). When, the cells werewashed and resuspended in BHI broth without tobramycin, theintensity of the 215-kb band of N315(pSR) started decreasingand continued to do so during the course of the passage, whilethe 160-kb band continued increasing in intensity (Fig. 3A,lanes 2 to 7). Southern blot hybridization of the PFGE bandsusing orfX DNA as a probe showed that both the 215- and160-kb bands carried orfX (Fig. 3B). Rehybridization of themembrane using the mecA gene probe showed that the 215-kbband hybridized with the probe, but the 160-kb band did not(data not shown). This clearly showed that the loss of mecAgene was caused by the precise excision of SCCmec. Greaterthan 99% of the cells in culture became susceptible to tobra-mycin and methicillin after 10 days’ passage of N315(pSR)without tobramycin.

Site-specific, orientation-specific integration of experimen-tal plasmid mediated by ccr genes. To investigate whether theputative closed circular form of SCCmec serves as a substratefor integration when introduced into a MSSA cell, we con-structed experimental plasmids pSRatt and pSRtta on whichboth ccr genes and the presumptive attachment sequence,attSCC, are subcloned (14). The plasmid pSRtta was identicalwith pSRatt except that the attSCC fragment was cloned in theopposite orientation (Fig. 4). The resultant recombinant plas-mids were introduced into strain N315ex by electroporation,and transformants were selected by overnight incubation onBHI agar plates containing tetracycline (10 mg/ml) at 30°C.The colony of each transformant was respread onto BHI agarplates containing tetracycline (10 mg/ml) and incubated over-night at 30 or 43°C (nonpermissive temperature for the plas-mid replication) before enumeration of the generated colonies.

Two strains, N315ex(pSRatt) and N315ex(pSRtta), generatedsignificant number of colonies at 43°C, i.e., 17.7 and 21.2%,respectively, of those grown at 30°C, whereas the other strainsgenerated only small number of colonies; i.e., ,0.001% ofthose grown at 30°C. The result showed that the chromosomalintegration of the plasmids occurred only when attSCC and anintact set of ccrA and ccrB genes were present on the plasmids.

To learn whether the integration of the experimental plas-mids was site- and orientation-specific, i.e., whether a strandexchange occurs between the attB site of the chromosome andthe attSCC site of pSRatt in a fixed orientation, a crisscrossPCR experiment was designed (Fig. 4). The cultures of trans-formants N315ex(pSRatt) and N315ex(pSRtta), with N315ex(pYT3att) as a control, were analyzed with PCR amplificationusing crisscross combinations of four primers: L (cL1) and R(cR2) flanking the attB region on the chromosome, and a (RVin pUC119) and b (in the ccrB gene) flanking the attSCCregion on the plasmids (Fig. 4). The results showed that theplasmid pSRatt generated plasmid-chromosome junctionswhich were detectable only by the two combinations of theprimers, L-b and R-a. On the other hand, the plasmid-chro-mosome junctions generated by pSRtta were detected only bythe combinations of the primers, L-a and R-b. Integration inthe opposite orientation could not be detected with the sensi-tivity of PCR employed in this study: the PCR sensitivity wassuch that it was able to detect the integrated template in N315ex(pSRatt) DNA diluted up to 104 times (wt/wt) with N315ex(pYT3att) DNA (data not shown).

The amplified DNAs from N315ex(pSRatt) and from N315ex(pSRtta) contained sequences which were identical with thoseof the chromosome-SCCmec junction regions (attL and attR)of N315 as presented in Fig. 1A.

PCR with any combinations of the primers did not amplify

FIG. 3. Loss of SCCmec during the cultivation of N315(pSR). (A) PFGE banding patterns of SmaI-digested total DNAs extracted from N315(pSR) on differentdays of passage. (B) Southern blot hybridization of the DNA fragments of the gel in panel A after transfer to a sheet of nylon membrane. The probe used was orfX.Lanes: 1, N315 cultivated for 24 h with tobramycin (10 mg/ml); 2, N315(pSR) cultivated for 24 h with tobramycin; 3 to 7, N315(pSR) after cultivation without tobramycinfor 1, 4, 5, 7, and 9 days, respectively; 8, N315ex cultivated for 24 h without tobramycin; 9, N315(pYT3) cultivated for 24 h with tobramycin; 10, lambda DNA markerfor PFGE (lambda ladder; Bio-Rad Laboratories). The sizes of the marker DNA (from top to bottom) were 485.0, 436.5, 388.0, 339.5, 291.0, 242.5, 194.0, 145.5, 97.0,and 48.5 kb. The arrows at the side of each figure indicate the DNA fragments hybridizable with the orfX probe.

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DNA fragments on template DNAs extracted from any of thetransformants [N315ex(pYT3), N315ex(pSRA*att), N315ex(pSRB*att), or N315ex(pSR) (not shown)]. This showed thatall of ccrA, ccrB, and attSCC were required for the integrationof the experimental plasmids.

DISCUSSION

The size of SCCmec (52 kb) is comparable to those of othergenetic elements such as bacteriophages, conjugative trans-posons, and pathogenicity islands, but SCCmec does not haveany structural similarity to those previously identified geneticelements: it is distinguishable from conjugative transposonssince it lacks the tra gene complex (5), and from bacterio-phages because it lacks the structure genes (or their remnants)for head or tail proteins of bacteriophages (29). UnlikeSCCmec pathogenicity islands (18, 19), does not contain anyORFs predictably encoding virulence factors as far as we couldjudge from the homology search of extant gene products (14).Besides these structural characteristics of SCCmec, we dem-onstrated, in this study, that SCCmec carries a set of uniquerecombinase genes, ccrA and ccrB, that are specifically in-volved in the recombination events (integration and excision)of SCCmec with the S. aureus chromosome. The presence ofthe two genes was required for both excision and integrationprocesses of SCCmec. In other genetic elements, such as bac-teriophage lambda and conjugative transposons, two site-spe-cific recombinases, designated integrase and excisionase, arereported to be required for the excision process. However, in

these elements, the integration can be mediated by the inte-grase alone (5, 29).

The Ccr recombinases are also characteristic in that theirNH2-terminal thirds had a substantial homology to the recom-binases of the invertase/resolvase family, whereas the inte-grases of bacteriophage lambda and conjugative transposonsbelong to the integrase family of site-specific recombinases.The characteristic serine residue that is considered to offer thehydroxyl group that attacks the DNA molecule in the strandexchange reaction is conserved among the recombinases of theinvertase/resolvase family and in Ccr proteins as well, but Ccrproteins also differ considerably from the recombinases of theinvertase/resolvase family in that they possess much largerCOOH-terminal domains than those possessed by the recom-binases belonging to this family. A homology search of theCOOH-terminal domains did not find any homologue in theextant proteins. Therefore, the molecular evolutionary rela-tionship of Ccr proteins to other recombinases and their modeof action pose intriguing questions to be explored.

Since the 1960s, the spontaneous loss of the mecA gene hasbeen observed during the storage or long-term cultivation ofMRSA strains in antibiotic-free medium (11). Deletion of alarge chromosomal region is identified in such mecA deletionstrains. The deletion starts precisely from the left boundary ofIS431mec and extends leftwards for various distances beyondthe mecA gene. The precise excision of SCCmec demonstratedin this study is unrelated to the transposase-mediated mecAdeletions (14). Curiously, spontaneous precise excision doesnot occur appreciably during cultivation of N315 despite the

FIG. 4. Site-, and orientation-specific integration of experimental plasmids mediated by ccrA and ccrB. PCR detection of the left and right plasmid-chromosomejunctions was performed with four crisscross combinations of primers with DNAs extracted from the culture of N315ex(pSRatt), N315ex(pSRtta), and N315ex(pYT3att)as templates. Four primers L (cL1; see Fig. 1A), R (cR2; see Fig. 1A), a, and b were used. The location and direction of the four primers are illustrated. Only the regionsadjacent to the introduced attSCC on the plasmids and the attB on N315ex chromosome are illustrated. The expected sizes of the amplified DNA fragments generatedby a site-specific integration of the plasmids were 610 bp (L-a), 1,255 bp (L-b), 1,649 bp (R-a), 1004 bp (R-b), and 549 bp (L-R). The results show a strict site andorientation specificity of ccr-mediated integration. The molecular weight marker used was a 1-kb ladder.

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fact that the strain carries an intact pair of ccr genes on itschromosome. The excision does, however, seem to occur at anextremely low frequency and can be detected by nested-PCRamplification of the attB sequence (14). Therefore, it is anintriguing question whether the expression of the intact copyof ccr genes of SCCmec is down regulated to stabilize theSCCmec in the chromosome. Alternatively, even an unrestrict-ed expression of a single copy of the ccr genes on the chromo-some may not be enough to increase the concentration of Ccrproteins to a level high enough to trigger the excision ofSCCmec. A study is under way to clarify these points.

It is well known that the mecA gene is carried by manystaphylococcal species besides S. aureus (13, 28), so it has beensuspected that mecA is transmissible among staphylococcalspecies. In fact, methicillin resistance can be experimentallytransferred between S. aureus cells by phage transduction (6).The DNA element with similar structural features to SCCmecis also identified in the strains of various staphylococcal specieswith the methicillin resistance phenotype (T. Ito, unpublishedobservation). Thus, it seems that the methicillin resistancegene mecA is transferred from cell to cell as a part of theSCCmec across staphylococcal species. However, as far as weare aware, no transducing phage capable of transferring ge-netic information across the staphylococcal species barrier hasbeen described. Thus, it remains to be seen if SCCmec istransmissible by phage transduction or if some other genetictransfer system specific for the movement of SCCmec exists.

In conclusion, we have demonstrated that the methicillinresistance gene of MRSA is carried by a novel genetic element,SCCmec, whose integration into and excision from the S. au-reus chromosome are mediated by a unique set of recombinasegenes, ccrA and ccrB. Further study of the distribution ofSCCmec and its related elements among S. aureus and otherstaphylococcal species will be helpful to elucidate how staph-ylococcal species exchange useful genetic information. Also,future elucidation of the mechanism of regulation of SCCmecexcision may lead to the attractive possibility of the develop-ment of a novel therapeutic measure to aid in antibiotic che-motherapy against MRSA infection by converting MRSAstrains in vivo into MSSA strains against which many extantantibiotics are effective.

ACKNOWLEDGMENTS

We thank K. Tsutsumimoto for her excellent technical assistanceand Yasmin Abu Hanifah for valuable suggestions concerning theworldwide epidemiology of MRSA.

This work was supported by the Core University Program under theauspices of the Japan Society for the Promotion of Science (JSPS),coordinated by the Graduate School of Medicine, University of Tokyo,and School of Medical Sciences, Universiti Sains Malaysia, and by theSpecially Designated Research Promotion of Monbusho, Japan.

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