Characterization of DNA sequences required for the CcrAB-mediated integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island.

Abstract

The mobile element staphylococcal cassette chromosome mec (SCCmec), which carries mecA, the gene responsible for methicillin resistance in staphylococci, inserts into the chromosome at a specific site, attB, mediated by serine recombinases, CcrAB and CcrC, encoded on the element. This study sought to determine the sequence specificity for CcrB DNA binding in vitro and for CcrAB-mediated SCCmec insertion in vivo. CcrB DNA binding, as assessed in vitro by electrophoretic mobility shift assay (EMSA), revealed that a 14-bp sequence (CGTATCATAAGTAA; the terminal sequence of the orfX gene) was the minimal requirement for binding, containing an invariant sequence (TATCATAA) found in all chromosomal (attB) and SCCmec (attS) integration sites. The sequences flanking the minimal attB and attS binding sites required for insertion in vivo were next determined. A plasmid containing only 37 bp of attS and flanking sequences was required for integration into the attB site at 92% efficiency. In contrast, at least 200 bp of sequence within orfX, 5' to the attB core, and 120 bp of specific sequence 3' to the orfX stop site and attB core were required for the highest insertion frequency. Finally, an attS-containing plasmid was inserted into wild-type Staphylococcus aureus strains without integrated SCCmec (methicillin susceptible) at various frequencies which were determined both by sequences flanking the att site and by the presence of more than one att site on either the chromosome or the integration plasmid. This sequence specificity may play a role in the epidemiology of SCCmec acquisition.