Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC

Abstract

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-DeltamecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome.

FIG. 1.

Genetic structure of the type V SCCmec element of strain WIS. The structure of the type V SCCmec element illustrated is based on the nucleotide sequence deposited in the DDBJ/EMBL/GenBank databases under accession no. AB121219. (a) Essential structure of type V SCCmec. The locations of the essential genes are illustrated. The colors of the essential genes are based on those for the ORFs described for panel b. The HindIII (H) and XbaI (X) restriction sites are indicated. Black arrowheads indicate primer-specific locations. The nucleotide sequences of primers cR1, mA2, mA3, is1, and cLs1 have been described previously (17, 26). The nucleotide sequences of primers V1, V2, and V3 are listed in Table 2. The locations of direct repeats are indicated by red arrowheads. (b) ORFs in and around type V SCCmec. The ORFs corresponding to sequences more than 100 bp are indicated by arrows, which also indicate the directions of the ORFs. Light gray arrows, ORFs conserved in the five types of SCCmec elements with identities of more than 99%; gray arrows, ORFs conserved in the five types of SCCmec elements with identities of 48.1 to 93.4%; blue arrows, ORFs commonly found in both the type V SCCmec element and the J region of type III SCCmec; yellow arrows, ORFs unique to type V SCCmec; orange arrow, orfX.

FIG. 2.

Characteristics of deduced amino acid sequences of CcrC. (a) The deduced amino acid sequences of the three ccr genes were aligned with that of ccrB2 of strain N315. The alignment was performed by using ClustalX software with the protein weight matrix PAM series. The three Ccr proteins were CcrC_WIS (CcrC of WIS), CcrC₀₃₄₂ (CcrC of strain 81/0342; the sequence of the strain 91/2674 CcrC was exactly identical to that of CcrC₀₃₄₂), and CcrC₂₀₈₂ (CcrC of strain 85/2082). The three Ccr proteins were highly homologous. Of the 517 amino acids corresponding to CcrC₂₀₈₂, 484 amino acids were conserved in the three Ccr proteins (pale blue) and 30 amino acids were conserved in two of three Ccr proteins. One hundred thirty-eight amino acids were conserved in CcrC₀₃₄₂, Ccr_WIS, and CcrB2 (blue). The consensus sequence shown above the amino acid sequences indicates conserved amino acids, as follows: asterisks, identical or conserved residues in all sequences in the alignment; colons, conserved substitutions; dots, semiconserved substitutions; hyphens, gaps. A black arrowhead indicates the presumptive serine involved in the phosphoseryl linkage conserved in the NH₂-terminal catalytic domain of site-specific recombinases of the invertase-resolvase family. (b) Phylogenetic relationships among ccrA genes, ccrB genes, ccrC genes, and two site-specific recombinases. The two site-specific recombinases used were the integrase (int) of bacteriophage φ FC1 found in E. faecalis (1,216 bp; DDBJ/EMBL/GenBank accession no. AF124258) and the site-specific recombinase found in C. acetobutylicum ATCC 824 (1,635 bp; DDBJ/EMBL/GenBank accession no.AE007636). The ccrA and ccrB genes used were as follows: ccrA1 and ccrB1*, respectively, found in strain NCTC10442 (DDBJ/EMBL/GenBank databases accession no. AB033763); ccrA2 and ccrB2, respectively, found in strains N315 (DDBJ/EMBL/GenBank databases accession no. D86934) and CA05 (DDBJ/EMBL/GenBank databases accession no. AB063172); ccrA3 and ccrB3, respectively, found in strain 85/2082 (DDBJ/EMBL/GenBank databases accession no. AB037671); and ccrA4 and ccrB4*, respectively, found in strain HDE288 (DDBJ/EMBL/GenBank databases accession no. AF411935). Since ccrB1 of NCTC10442 and ccrB4 of HDE288 were truncated, we reconstituted large ORFs of 1,626 bp (ccrB1*) and 1,629 bp (ccrB4*) by adding an adenine and used them for the comparison. The nucleotide sequences of five ccrA genes, five ccrB genes, four ccrC genes, and two site-specific recombinases were aligned by using the ClustalX program. The phylogenetic tree was generated by the neighbor-joining method by creating 2,000 bootstrap replicates. The tree was visualized with Tree View software, which was obtained from the website http://taxonomy.zoology.gla.ac.uk/rod/treeview.htm. The branch length indicates the distance, which is expressed as the number of substitutions per 100 bases.

FIG. 2.

Characteristics of deduced amino acid sequences of CcrC. (a) The deduced amino acid sequences of the three ccr genes were aligned with that of ccrB2 of strain N315. The alignment was performed by using ClustalX software with the protein weight matrix PAM series. The three Ccr proteins were CcrC_WIS (CcrC of WIS), CcrC₀₃₄₂ (CcrC of strain 81/0342; the sequence of the strain 91/2674 CcrC was exactly identical to that of CcrC₀₃₄₂), and CcrC₂₀₈₂ (CcrC of strain 85/2082). The three Ccr proteins were highly homologous. Of the 517 amino acids corresponding to CcrC₂₀₈₂, 484 amino acids were conserved in the three Ccr proteins (pale blue) and 30 amino acids were conserved in two of three Ccr proteins. One hundred thirty-eight amino acids were conserved in CcrC₀₃₄₂, Ccr_WIS, and CcrB2 (blue). The consensus sequence shown above the amino acid sequences indicates conserved amino acids, as follows: asterisks, identical or conserved residues in all sequences in the alignment; colons, conserved substitutions; dots, semiconserved substitutions; hyphens, gaps. A black arrowhead indicates the presumptive serine involved in the phosphoseryl linkage conserved in the NH₂-terminal catalytic domain of site-specific recombinases of the invertase-resolvase family. (b) Phylogenetic relationships among ccrA genes, ccrB genes, ccrC genes, and two site-specific recombinases. The two site-specific recombinases used were the integrase (int) of bacteriophage φ FC1 found in E. faecalis (1,216 bp; DDBJ/EMBL/GenBank accession no. AF124258) and the site-specific recombinase found in C. acetobutylicum ATCC 824 (1,635 bp; DDBJ/EMBL/GenBank accession no.AE007636). The ccrA and ccrB genes used were as follows: ccrA1 and ccrB1*, respectively, found in strain NCTC10442 (DDBJ/EMBL/GenBank databases accession no. AB033763); ccrA2 and ccrB2, respectively, found in strains N315 (DDBJ/EMBL/GenBank databases accession no. D86934) and CA05 (DDBJ/EMBL/GenBank databases accession no. AB063172); ccrA3 and ccrB3, respectively, found in strain 85/2082 (DDBJ/EMBL/GenBank databases accession no. AB037671); and ccrA4 and ccrB4*, respectively, found in strain HDE288 (DDBJ/EMBL/GenBank databases accession no. AF411935). Since ccrB1 of NCTC10442 and ccrB4 of HDE288 were truncated, we reconstituted large ORFs of 1,626 bp (ccrB1*) and 1,629 bp (ccrB4*) by adding an adenine and used them for the comparison. The nucleotide sequences of five ccrA genes, five ccrB genes, four ccrC genes, and two site-specific recombinases were aligned by using the ClustalX program. The phylogenetic tree was generated by the neighbor-joining method by creating 2,000 bootstrap replicates. The tree was visualized with Tree View software, which was obtained from the website http://taxonomy.zoology.gla.ac.uk/rod/treeview.htm. The branch length indicates the distance, which is expressed as the number of substitutions per 100 bases.

FIG. 3.

Chromosome-SCC junction sequences. The nucleotide sequences at the left and right boundaries of the SCCmec element of strain WIS are aligned with those of six previously reported SCC elements: type I SCCmec of S. aureus NCTC10442 (DDBJ/EMBL/GenBank databases accession no. AB033763), type II SCCmec of S. aureus N315 (DDBJ/EMBL/GenBank databases accession no. D86934), type III SCCmec of S. aureus 85/2082 (DDBJ/EMBL/GenBank databases accession no. AB037671), type IV SCCmec of S. aureus CA05 (DDBJ/EMBL/GenBank databases accession no. AB063172), SCCcap1 of S. aureus M (DDBJ/EMBL/GenBank databases accession no. U10927), and SCC₁₂₂₆₃ of S. hominis GIFU12263 (DDBJ/EMBL/GenBank databases accession no. AB063171). Two sets of nucleotide sequences are listed for the type III SCCmec of S. aureus 85/2082. One set is composed of the nucleotide sequence of the left extremity (direct repeat 1) and the nucleotide sequence in the midst of the element flanked by direct repeat 2. The other set is composed of the nucleotide sequence containing direct repeat 2 and the right extremity of the element flanked by direct repeat 3 (15). Thin arrows indicate inverted repeats at both extremities of SCCmec elements carrying the ccrA and ccrB genes. Dotted lines indicate the characteristic nucleotide sequence conserved at both extremities of type V SCCmec or SCC elements carrying the ccrC gene. The 3′ ends of orfX are indicated with light green shading and lowercase bases. The consensus sequences of attBscc and ISS are boxed. Direct repeat sequences in ISS and the left end of the SCC element are indicated by thick lines. Nucleotide sequences of both extremities of SCC or SCCmec elements are colored as follows: red, type I, II, and IV SCCmec elements and the region between direct repeat 2 and direct repeat 3 in type III SCCmec element; orange, SCC12263; blue, type V SCCmec and the region between direct repeat 1 and direct repeat 2 in type III SCCmec element; green, SCCcap1.

FIG. 4.

Homologous regions in nucleotide sequences of the type V SCCmec and extant SCCmec elements. The nucleotide positions of the type V SCCmec element are indicated on the horizontal axes, and those of extant SCCmec elements (types I, II, III, and IVa) are indicated on the vertical axes. The following regions of the nucleotide sequences of the SCCmec elements were used: type V SCCmec, nucleotides 880 to 28503 of the nucleotide sequence with DDBJ/EMBL/GenBank databases accession no. AB121219; type I SCCmec, the region from nucleotides 4504 to 38867 of the nucleotide sequence with DDBJ/EMBL/GenBank databases accession no. AB033763; type II SCCmec, nucleotides 4687 to 57653 of the nucleotide sequence with DDBJ/EMBL/GenBank databases accession no. D86934; type III SCCmec, nucleotides 899 to 67794 of the nucleotide sequence with DDBJ/EMBL/GenBank databases accession no. AB037671; and type IVa SCCmec, nucleotides 975 to 25222 of the nucleotide sequence with DDBJ/EMBL/GenBank databases accession no. AB063172. The regions showing the high degrees of similarity between two SCCmec elements are indicated with lines. Rising lines signify that homologous regions between two elements are located in the opposite direction. Short homologous regions appear as dots. The regions through A (A1 and A2), B, and C (C1 and C2) were designated by the extent of nucleotide sequence identity described in the text. The locations of the regions in the nucleotide sequence of type V SCCmec are as follows: A1, nucleotide positions 1778 to 7341; A2, nucleotide positions 7342 to 8131; B, nucleotide positions, 16964 to 18296; C1, nucleotide positions 12041 to 17040; and C2, nucleotide positions 18309 to 21540.

FIG. 5.

Phylogenetic relations of the constituents of the hsd system. Type V SCCmec carries three genes, hsdR, hsdM, and hsdS, which encode a type I restriction-modification system. To look for the derivations of those genes, the phylogenetic relationships among the genes found in the type V SCCmec element and previously reported hsd genes were investigated by creating phylogenetic trees for the hsdR (a), hsdM (b), and hsdS (c) genes. Phylogenetic trees were generated as described in the legend to Fig. 2b. The nucleotide sequence of the truncated hsdR gene in type III SCCmec was obtained from the DDBJ/EMBL/GenBank databases (accession no. AB037671). All other nucleotide sequences of the hsd genes analyzed here were obtained from websites that provide the whole genome sequences of the following S. aureus strains: N315 and MW2 (http://www.bio.nite.go.jp/), Mu50 (http://w3.grt.kyushu-u.ac.jp/VRSA/), MSSA 476 and strain 252 (http://www.sanger.ac.uk/Projects/S_aureus/), COL (http://www.tigr.org/tdb/mdb/mdbinprogress.html), and NCTC8325 (http://www.genome.ou.edu/staph.html).

FIG. 6.

Precise excision and site- and orientation-specific integration of SCCmec. (a) Detection of ccr-mediated SCCmec excision and appearance of attSCC. Template DNAs for PCR were extracted from a culture incubated in BHI broth with tetracycline (10 mg/liter) for 20 h. Four sets of primers were used to detect the precise excision and the closed circular form of SCCmec in strain N315 or WIS cells. The locations of the primers used are shown in panel b. For N315, primers cL1 and cR1 were used to detect attBscc (276 bp), and primers mL1 and mR8 were used to detect attSCC (456 bp). For WIS, primers cLs1 and cR1 were used to detect attBscc (314 bp), and primers mVL1 and mVR1 were used to detect attSCC (292bp). Lane MW, molecular size marker (1-kb ladder; Invitrogen, Carlsbad, Calif.), and only the relevant sizes are indicated. (b) Generation of attBscc and attSCC. The nucleotide sequences of attBscc in the DNA fragments amplified from N315ex and N315(pSR2) and from WIS(pSR2), WIS(pSR5_W), and WIS(pSR5_E) are indicated attBscc(N315) and attBscc(WIS), respectively. The nucleotide sequences of attSCC in DNA fragments amplified from N315(pSR2) and from WIS(pSR2), WIS(pSR5_W), and WIS(pSR5_E) are indicated attSCC(N315) and attSCC(WIS), respectively, above the nucleotide sequence of attBscc. (c) Locations of the primers used to detect attBscc generated from N315 (primers cR1 and cL1) and WIS (primers cR1 and cLs1) and attSCC generated from N315 (primers mR8 and mL1) and WIS (primers mVR1 and mVL1). The locations of the primers used to clone attSCC-II (primers mR7 and mL2) and attSCC-V (primers mVR2 and mVL2) are also illustrated. The drawings are not to scale. (d) Identification of site- and orientation-specific integration of recombinant plasmids (mini-SCCs) into the chromosome. The recombinant plasmids carrying ccr gene and attSCC of each type, pSR2attII and pSR5_EattV, were introduced into N315ex by electroporation as models of SCCmec elements. DNAs were extracted from cultures of the transformants grown at 43°C on BHI agar plates containing tetracycline (10 mg/liter). The locations and directions of the four primers (primers α, β, cL1, and cR1) used to detect the integration of plasmid pSR2attII as well as four primers (primers α, γ, cL1, and cR1) used to detect the integration of plasmid pSR5attV are illustrated. We could amplify DNA fragments of the expected sizes by the site- and orientation-specific integration of the plasmids. The expected sizes of the DNA fragments are 1,255 bp (cL1-β) and 735 bp (cR1-α) for pSR2attII and 826 bp (cL1-γ) and 719 bp (cR1-α).