Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus - PubMed (original) (raw)

Comparative Study

Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus

Yuki Katayama et al. J Bacteriol. 2003 May.

Abstract

We previously reported that the methicillin resistance gene mecA is carried by a novel type of mobile genetic element, SCCmec (staphylococcal cassette chromosome mec), in the chromosome of methicillin-resistant Staphylococcus aureus (MRSA). These elements are precisely excised from the chromosome and integrated into a specific site on the recipient chromosome by a pair of recombinase proteins encoded by the cassette chromosome recombinase genes ccrA and ccrB. In the present work, we detected homologues of the ccr genes in Staphylococcus hominis type strain GIFU12263 (equivalent to ATCC 27844), which is susceptible to methicillin. Sequence determination revealed that the ccr homologues in S. hominis were type 1 ccr genes (ccrA1 and ccrB1) that were localized on a genetic element structurally very similar to SCCmec except for the absence of the methicillin-resistance gene, mecA. This genetic element had mosaic-like patterns of homology with extant SCCmec elements, and we designated it SCC(12263) and considered it a type I staphylococcal cassette chromosome (SCC). The ccrB1 gene identified in the S. hominis strain is the first type 1 ccrB gene discovered to retain its function through the excision process as judged by two criteria: (i) SCC(12263) was spontaneously excised during cultivation of the strain and (ii) introduction of the S. hominis ccrB1 into an MRSA strain carrying a type I SCCmec whose ccrB1 gene is inactive generated SCCmec excisants at a high frequency. The existence of an SCC without a mec determinant is indicative of a staphylococcal site-specific mobile genetic element that serves as a vehicle of transfer for various genetic markers between staphylococcal species.

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Figures

FIG. 1.

FIG. 1.

(a) The deduced amino acid sequences of CcrB1 of GIFU 12263 S. hominis were aligned with those of NCTC 10442 MRSA. A black arrowhead indicates the locus of the first amino acid residue changed by an addition of adenine in the nucleotide sequence of _ccrB1_∗ as described in the text. (b) Phylogenetic relationships among ccrA genes, ccrB genes, and four site-specific recombinases. The four site-specific recombinases that show high similarity to ccr genes and that were selected to investigate phylogenetic relationships are the integrase (int) of bacteriophage phi-FC1 found in E. faecalis (1,216 bp; DDBJ/EMBL/GenBank accession no. AF124258), the integrase of bacteriophage TP901-1 found in L. lactis (1,458 bp; DDBJ/EMBL/GenBank accession no. X85213), the site-specific recombinase found in C. acetobutylicum ATCC 824 (1,635 bp; DDBJ/EMBL/GenBank accession no. AE007636), and the site-specific recombinase found in S. pneumoniae R6 (1,680 bp; DDBJ/EMBL/GenBank accession no. AE008466). The nucleotide sequences of six ccrA genes, six ccrB genes, and four site-specific recombinases were aligned by using the PileUp program with a Genetics Computer Group default scoring matrix. Phylogenetic relationships were examined with the Paupsearch program by the neighbor-joining method by creating 1,000 bootstrap replicates and by selecting 70% of consensus. The tree was visualized with Tree View software, which was obtained from the Tree View 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. (c) The alignment of deduced amino acid sequences between OrfXaur representative from NCTC 10442 MRSA strain and OrfXhom in GIFU12263.

FIG. 1.

FIG. 1.

(a) The deduced amino acid sequences of CcrB1 of GIFU 12263 S. hominis were aligned with those of NCTC 10442 MRSA. A black arrowhead indicates the locus of the first amino acid residue changed by an addition of adenine in the nucleotide sequence of _ccrB1_∗ as described in the text. (b) Phylogenetic relationships among ccrA genes, ccrB genes, and four site-specific recombinases. The four site-specific recombinases that show high similarity to ccr genes and that were selected to investigate phylogenetic relationships are the integrase (int) of bacteriophage phi-FC1 found in E. faecalis (1,216 bp; DDBJ/EMBL/GenBank accession no. AF124258), the integrase of bacteriophage TP901-1 found in L. lactis (1,458 bp; DDBJ/EMBL/GenBank accession no. X85213), the site-specific recombinase found in C. acetobutylicum ATCC 824 (1,635 bp; DDBJ/EMBL/GenBank accession no. AE007636), and the site-specific recombinase found in S. pneumoniae R6 (1,680 bp; DDBJ/EMBL/GenBank accession no. AE008466). The nucleotide sequences of six ccrA genes, six ccrB genes, and four site-specific recombinases were aligned by using the PileUp program with a Genetics Computer Group default scoring matrix. Phylogenetic relationships were examined with the Paupsearch program by the neighbor-joining method by creating 1,000 bootstrap replicates and by selecting 70% of consensus. The tree was visualized with Tree View software, which was obtained from the Tree View 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. (c) The alignment of deduced amino acid sequences between OrfXaur representative from NCTC 10442 MRSA strain and OrfXhom in GIFU12263.

FIG. 2.

FIG. 2.

Genomic structure of SCC12263. (a) ORFs in and around SCC12263. The ORFs corresponding to sequences more than 90 amino acids in size and twenty-six possible reading frames are indicated by squares. Those above and those below the line are the ORFs whose transcription is directed to the right and those whose transcription is directed to the left, respectively. The white squares indicate the ORFs that were conserved among three SCC_mec_ elements. The black squares indicate the ORFs that showed no identity to the four SCC_mec_ elements. The gray square indicates the ORF with identity to type II SCC_mec_. (b) The essential structure of SCC12263. Locations of the essential genes are illustrated. The only restriction sites indicated are those of of _Hin_dIII and _Eco_RI. _orfX_hom is shown in orange, the ccr1 genes are shown in red, and the arrow indicates the direction of transcription. Nucleotide sequences of the left and right boundaries of attL and attR in SCC12263 were aligned with those of attL in NCTC10442 and attR in N315, respectively. The orange arrows indicate inverted complementary repeats, IR-L and IR-R, at both extremities of SCC12263, and the blue arrows indicate those of MRSA. The direct repeats in SCC12263 are indicated by magenta arrows, and those of MRSA are indicated by green arrows. The sequences that are colored are identical to each other. (c) Boundary of SCC12263. Nucleotide sequences around the attL and attR, respectively, are aligned with the sequence of the presumptive integration site, _att_B, on the GIFU12263 SCC12263(−) chromosome. The orange arrows indicate inverted complementary repeats, IR-L and IR-R, at both extremities of SCC12263. The red arrows indicate direct repeats. The orange and red sequences are identical to each other.

FIG.3.

FIG.3.

Homologous regions of nucleotide sequences of SCC12263 and SCC_mec_ type I, II, III, IVa, and IVb and their identities. The nucleotide positions are relative to the SCCs. The SCC region of SCC12263 corresponds to the region from nucleotide 770 to 22358 in GIFU12263, the type I SCC_mec_ corresponds to the region from nucleotide 4504 to 38867 in AB033763, the type II SCC_mec_ corresponds to the region from nucleotide 4687 to 57653 in D86934, the type III SCC_mec_ corresponds to the region from nucleotide 899 to 67794 in AB037671, the type IVa SCC_mec_ corresponds to the region from nucleotide 975 to 25222 in AB063172, and type IVb SCC_mec_ corresponds to the region from nucleotide 301 to 21219 in AB063173. The regions A through E (see text for description of location) and how they correspond to the structure presented in Fig. 2a are shown at the bottom.

FIG. 4.

FIG. 4.

PCR for detection of attB_scc region on the chromosome. Amplification was carried out with a set of primers cL3 and cR2 specific for the regions to the left and right of the chromosome-SCC_mec junction point. Lanes: 1, 1-kb ladder as a molecular weight marker; 2, N315; 3, N315(pSR2); 4, N315(pSR1hom); 5, N315(pSR1aur); 6, 85/1940; 7, 85/1940(pSR2); 8, 85/1940(pSR1hom); 9, 85/1940(pSR1aur).

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