A plasmid-borne system to assess the excision and integration of staphylococcal cassette chromosome mec mediated by CcrA and CcrB

Abstract

Resistance to methicillin and other ß-lactam antibiotics in staphylococci is due to mecA, which is carried on a genomic island, staphylococcal cassette chromosome mec (SCCmec). The chromosomal excision and integration of SCCmec are mediated by the site-specific recombinase CcrAB or CcrC, encoded within this element. A plasmid-borne system was constructed to assess the activities of CcrA and CcrB in the excision and integration of SCCmec in Escherichia coli and Staphylococcus aureus. The excision frequency in E. coli mediated by CcrAB from methicillin-resistant S. aureus (MRSA) strain N315 was only 9.2%, while the integration frequency was 31.4%. In S. aureus the excision and integration frequencies were 11.0% and 18.7%, respectively. Truncated mutants identified the N-terminal domain of either CcrB or CcrA to be necessary for both integration and excision, while the C-terminal domain was important for recombination efficiency. Site-directed mutagenesis of the N-terminal domain identified S11 and R79 of CcrA and S16, R89, T149, and R151 of CcrB to be residues essential for catalytic activities, and the critical location of these residues was consistent with a model of the tertiary structure of the N terminus of CcrA and CcrB. Furthermore, CcrAB and CcrC, cloned from a panel of 6 methicillin-resistant S. aureus strains and 2 methicillin-resistant Staphylococcus epidermidis strains carrying SCCmec types II, IV, and V, also catalyzed integration at rates 1.3 to 10 times higher than the rates at which they catalyzed excision, similar to the results from N315. The tendency of SCCmec integration to be favored over excision may explain the low spontaneous excision frequency seen among MRSA strains.