Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome mec , a Staphylococcus aureus Genomic Island

The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci,mecA , is carried on a genomic island, SCCmec (for staphylococcal cassette chromosomemec ). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec . Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB ) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed byEscherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducibleccrA ,ccrB , or both genes was assessed by PCR inStaphylococcus aureus . CcrB alone could mediate excision but excision was at an alternateatt site (attR2 ) within the right extremity of SCCmec . In contrast, both CcrB and CcrA were required to mediate excision at the chromosomalattB site (calledattR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS ) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required forattB site-specific excision and SCCmec chromosomal insertion.