Fig. 5: Effect of chromosomal integration on the bactericidal activity of ePICI.
A Schematic representation of the oligonucleotide design used to detect the linear and circular forms of ePICIrsaE and its integrase-deficient mutant (ePICIrsaE ∆int). Oligonucleotides E18 and E19 amplify a fragment of ePICI (PCRC) regardless of whether it is integrated into the genome (linear form) or excised (circular form). Oligonucleotides OL17 and OL18 produce an amplification product (PCRD) only when ePICI is excised from the chromosome and forms the circular intermediate. B Agarose gel electrophoresis showing PCR amplification products obtained using oligonucleotides E18 and E19 (top panel) and the divergent primers OL17 and OL18 (bottom panel). PCR was conducted on bacteria with either ePICIrsaE (Int ePICI +) or ePICIrsaE ∆int (Int ePICI −), along with an empty plasmid or one carrying the ePICI integrase, both before and after mitomycin C treatment. Lanes 1–8 from the top gel show a 4 kb band corresponding to ePICIrsaE integrated into the chromosome or its excised form following mitomycin treatment. Lanes 1–8 from the bottom gel show an amplification product only in mitomycin-treated samples, confirming the circular form of ePICIrsaE after excision from the bacterial chromosome. C Growth curves of the S. aureus HG001 strain and HG001 complemented with a plasmid expressing the ePICI integrase, in the presence of ePICIrsaE or ePICIrsaE ∆int purified from bacteria complemented with an empty plasmid or the plasmid producing ePICI integrase in trans. Data are representative of three independent biological replicates.
