Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

Antimicrobial resistance inStaphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES proteinin vitro . Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureus plasmids that contain sequences similar to theoriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriT sequences of these nonconjugative plasmidsin vitro . Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT , it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT . These data indicate that the conjugative relaxase intrans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.IMPORTANCE Understanding the mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT -like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intrans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.