Recombination at the coagulase locus in Staphylococcus aureus: plasmid integration and amplification

The integrating plasmid pCOA18, comprising pUC18 linked to a mutated coagulase (coa) gene from Staphylococcus aureus, and constructed by substituting coa sequences with a tetracycline (Tc)-resistance marker (delta coa::Tcr), was transformed into Staphylococcus aureus RN4220, where it underwent recombination with the chromosomal coa locus. Allele-replacement mutants were recovered at a low frequency directly after transformation. The majority of transformants carried pCOA18 integrated in the chromosome by a single Campbell-type recombination event. The majority of integrants contained tandem repeats of pCOA18 and expressed high levels of resistance to Tc (> 30 micrograms ml-1) compared to the single-copy integrants and allele-replacement mutants (15 micrograms ml-1). Transduction of a single-copy integrant to a Coa+ recipient allowed the cointegrant to be resolved and allele-replacement recombinants to be selected. In addition, growth of a single-copy integrant on high concentrations of Tc (> 30 micrograms ml-1) selected for amplified derivatives at a frequency of 10(-5). It was estimated that up to 19 copies of pCOA18 could occur in a tandem array in the chromosome.