A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus - PubMed (original) (raw)
A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus
Y Katayama et al. Antimicrob Agents Chemother. 2000 Jun.
Abstract
We have previously shown that the methicillin-resistance gene mecA of Staphylococcus aureus strain N315 is 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 novel genes (designated cassette chromosome recombinase genes [ccrA and ccrB]) encoding polypeptides having a partial homology to recombinases of the invertase/resolvase family. The open reading frames were found to catalyze precise excision of the SCCmec from the methicillin-resistant S. aureus chromosome and site-specific as well as orientation-specific integration of the SCCmec into the S. aureus chromosome when introduced into the cells as a recombinant multicopy plasmid. We propose that SCCmec driven by a novel set of recombinases represents a new family of staphylococcal genomic elements.
Figures
FIG. 1
Identification of ccr genes. (A) Genomic structure of SCC_mec_. Locations of the essential genes are illustrated. The left and right chromosome-SCC_mec_ junctions, 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. The MSSA chromosomal parts of the attL and attR were defined as attB-L and attB-R, delimited by primers cL1 and cR2, respectively. The SCC_mec_ parts of these elements were defined as att_SCC-L and att_SCC-R, delimited by the primers mL1 and mR8, respectively: i.e., attL is attB-L plus att_SCC-L, and attR is attB-R plus att_SCC-R. Transposon Tn_554 (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 IS_431 (or IS_257) (2, 7, 14, 22, 27). Nucleotide sequences around the left and right boundaries of SCC_mec, attL and attR, are shown at the bottom of the panel. Inverted repeats, IRscc-L and IRscc-R, at both extremities of SCC_mec_ are indicated by thin arrows. Thick arrows indicate the direct repeats, DR-B and DR-SCC. The nucleotide sequence of the N315 chromosome containing the entire SCC_mec_ (56,939 bp) is deposited in DDBJ/EMBL/GenBank under accession number D86934. (B) Deduced amino acid sequences of ccrA and ccrB. CcrA and CcrB 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 250 in 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 the invertase/resolvase family. Small bullets indicate amino acid substitution within the same class of amino acids. Of the 64 amino acid positions well conserved among the recombinases of the invertase/resolvase family (shown in pink) (25), 44 and 47 amino acids, respectively, were conserved in CcrA and CcrB. An arrowhead indicates the presumptive serine involved in the 5′ phosphoseryl linkage of the recombinase to DNA characteristically conserved in the NH2-terminal catalytic domain of site-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 of transcription of the structural gene ccr and tetL. The respective set of ccrA and ccrB genes was inserted at the _Bam_HI site of pYT3.
FIG. 1
Identification of ccr genes. (A) Genomic structure of SCC_mec_. Locations of the essential genes are illustrated. The left and right chromosome-SCC_mec_ junctions, 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. The MSSA chromosomal parts of the attL and attR were defined as attB-L and attB-R, delimited by primers cL1 and cR2, respectively. The SCC_mec_ parts of these elements were defined as att_SCC-L and att_SCC-R, delimited by the primers mL1 and mR8, respectively: i.e., attL is attB-L plus att_SCC-L, and attR is attB-R plus att_SCC-R. Transposon Tn_554 (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 IS_431 (or IS_257) (2, 7, 14, 22, 27). Nucleotide sequences around the left and right boundaries of SCC_mec, attL and attR, are shown at the bottom of the panel. Inverted repeats, IRscc-L and IRscc-R, at both extremities of SCC_mec_ are indicated by thin arrows. Thick arrows indicate the direct repeats, DR-B and DR-SCC. The nucleotide sequence of the N315 chromosome containing the entire SCC_mec_ (56,939 bp) is deposited in DDBJ/EMBL/GenBank under accession number D86934. (B) Deduced amino acid sequences of ccrA and ccrB. CcrA and CcrB 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 250 in 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 the invertase/resolvase family. Small bullets indicate amino acid substitution within the same class of amino acids. Of the 64 amino acid positions well conserved among the recombinases of the invertase/resolvase family (shown in pink) (25), 44 and 47 amino acids, respectively, were conserved in CcrA and CcrB. An arrowhead indicates the presumptive serine involved in the 5′ phosphoseryl linkage of the recombinase to DNA characteristically conserved in the NH2-terminal catalytic domain of site-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 of transcription of the structural gene ccr and tetL. The respective set of ccrA and ccrB genes was inserted at the _Bam_HI site of pYT3.
FIG. 1
Identification of ccr genes. (A) Genomic structure of SCC_mec_. Locations of the essential genes are illustrated. The left and right chromosome-SCC_mec_ junctions, 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. The MSSA chromosomal parts of the attL and attR were defined as attB-L and attB-R, delimited by primers cL1 and cR2, respectively. The SCC_mec_ parts of these elements were defined as att_SCC-L and att_SCC-R, delimited by the primers mL1 and mR8, respectively: i.e., attL is attB-L plus att_SCC-L, and attR is attB-R plus att_SCC-R. Transposon Tn_554 (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 IS_431 (or IS_257) (2, 7, 14, 22, 27). Nucleotide sequences around the left and right boundaries of SCC_mec, attL and attR, are shown at the bottom of the panel. Inverted repeats, IRscc-L and IRscc-R, at both extremities of SCC_mec_ are indicated by thin arrows. Thick arrows indicate the direct repeats, DR-B and DR-SCC. The nucleotide sequence of the N315 chromosome containing the entire SCC_mec_ (56,939 bp) is deposited in DDBJ/EMBL/GenBank under accession number D86934. (B) Deduced amino acid sequences of ccrA and ccrB. CcrA and CcrB 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 250 in 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 the invertase/resolvase family. Small bullets indicate amino acid substitution within the same class of amino acids. Of the 64 amino acid positions well conserved among the recombinases of the invertase/resolvase family (shown in pink) (25), 44 and 47 amino acids, respectively, were conserved in CcrA and CcrB. An arrowhead indicates the presumptive serine involved in the 5′ phosphoseryl linkage of the recombinase to DNA characteristically conserved in the NH2-terminal catalytic domain of site-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 of transcription of the structural gene ccr and tetL. The respective set of ccrA and ccrB genes was inserted at the _Bam_HI site of pYT3.
FIG. 2
Precise excision of SCC_mec_ mediated by ccrA and ccrB. (A) Detection of ccr_-mediated SCC_mec excision and appearance of att_SCC. Template DNAs for PCR were extracted from overnight culture of the colony purified transformant strains in BHI broth with tetracycline (10 μg/ml). Four sets of primers were used to detect precise excision and the closed circle form of SCC_mec. Primers cR2 and cL1 were used to detect attB (549 bp). Primers mL1 and mR8 were used to identify _att_SCC (456 bp). Primers cL1 and mL1 were used to detect att_L (371 bp). Primers mA1 and mA2 were used to detect a part of mecA gene (286 bp). Note that precise excision of SCC_mec occurred only in N315(pSR) and N315ex and that _att_SCC was detected only in N315(pSR). The molecular weight marker was a 1-kb ladder (Gibco-BRL, Gaithersburg, Md.) and only the relevant sizes are indicated. (B) Generation of att_SCC and attB. The presumptive closed circular form of SCC_mec is illustrated with _att_SCC, which appeared to be generated by head-to-tail ligation of attSCC-L (stippled box) and attSCC-R (striped box) (see Fig. 1A). Nucleotide sequences of attB in amplified DNA fragments from N315ex and N315(pSR) are also illustrated. The primers mR7 and mL2 were used for the cloning of _att_SCC into pYT3.
FIG. 3
Loss of SCC_mec_ during the cultivation of N315(pSR). (A) PFGE banding patterns of _Sma_I-digested total DNAs extracted from N315(pSR) on different days 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 μg/ml); 2, N315(pSR) cultivated for 24 h with tobramycin; 3 to 7, N315(pSR) after cultivation without tobramycin for 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 marker for 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.
FIG. 4
Site-, and orientation-specific integration of experimental plasmids mediated by ccrA and ccrB. PCR detection of the left and right plasmid-chromosome junctions 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), α, and β were used. The location and direction of the four primers are illustrated. Only the regions adjacent to the introduced _att_SCC on the plasmids and the attB on N315ex chromosome are illustrated. The expected sizes of the amplified DNA fragments generated by a site-specific integration of the plasmids were 610 bp (L-α), 1,255 bp (L-β), 1,649 bp (R-α), 1004 bp (R-β), and 549 bp (L-R). The results show a strict site and orientation specificity of _ccr_-mediated integration. The molecular weight marker used was a 1-kb ladder.
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