This study examined recombination events when the integrating plasmid pCOA18 was introduced into Staphylococcus aureus. The plasmid carried a deletion-substitution mutation in the coagulase gene (Δcoa::Tc') and lacked a functional origin of replication in S. aureus. When transformed by electroporation into S. aureus strain RN4220, the plasmid underwent recombination at the chromosomal coa locus. While allele-replacement mutants were recovered at low frequency, the majority of transformants contained pCOA18 integrated by single Campbell-type recombination events. Unexpectedly, most integrants carried tandem multimers of the plasmid and exhibited high tetracycline resistance (>30 μg/ml) compared to single-copy integrants (15 μg/ml). Southern hybridization analysis revealed complex integration patterns. Single-copy integrants could be selected and subsequently amplified to higher copy numbers through growth on high tetracycline concentrations, with up to 19 copies detected in tandem arrays. Transduction experiments demonstrated that single-copy integrants could be resolved to generate allele-replacement recombinants, providing a method to isolate targeted mutations in S. aureus.

Key findings

• Most pCOA18 transformants carried tandem multimers of the plasmid integrated at the coa locus rather than single-copy integrations
• Plasmid amplification occurred through unequal crossing-over during chromosome replication, enabling selection of derivatives carrying up to 19 copies of pCOA18
• Single-copy integrants expressed lower tetracycline resistance (15 μg/ml) compared to amplified derivatives (>30 μg/ml)
• Transduction of single-copy integrants to recipient strains allowed resolution of cointegrants and selection of allele-replacement mutants
• High-frequency electroporation transformation (10³/μg DNA) successfully delivered the integrating plasmid, enabling direct isolation of both plasmid integrants and allele-replacement mutants