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

Abstract

ABSTRACT The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA , is carried on a genomic island, SCC mec (for staphylococcal cassette chromosome mec ). The chromosomal excision and integration of types I to IV SCC mec 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 SCC mec . 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 by Escherichia coli two-hybrid analysis. SCC mec excision mediated by plasmid-encoded and inducible ccrA , ccrB , or both genes was assessed by PCR in Staphylococcus aureus . CcrB alone could mediate excision but excision was at an alternate att site ( attR2 ) within the right extremity of SCC mec . In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCC mec is integrated). Insertion of a plasmid containing the SCC mec 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 for attB site-specific excision and SCC mec chromosomal insertion.