Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids inStaphylococcus aureus

Abstract

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

“…However, the analogous relaxase in trans mobilization phenomenon recently described for the distinct pWBG749-like conjugative plasmids provides a precedent that likely explains this apparent paradox. Namely, pWBG749 oriT-like sequences exist as subtypes differentiated by sequence divergence in an inverted repeat (IR2) located adjacent to the nic site-containing core sequence (18). This results in specificity between various mobilizable plasmids and particular pWBG749-like conjugative plasmids.…”

“…This results in specificity between various mobilizable plasmids and particular pWBG749-like conjugative plasmids. Thus, pWBG749 can mobilize plasmids with a pWBG749-like oriT of subtype OT49 but not those carrying an OT45 subtype, which instead can be mobilized by pWBG749-like conjugative plasmids that possess a cognate OT45 subtype oriT (18). Despite this, pWBG749 was able to stimulate recombination between OT49-and OT45-type oriT sequences carried on the same mobilizable plasmid, indicating that the pWBG749 relaxosome could recognize the OT45-type oriT even though it cannot mediate transfer of that subtype (18).…”

“…The plasmid database compiled and analyzed for pWBG749 family oriT sequences by O'Brien et al was analyzed for oriT sequences similar to that of pSK41 (18). The online interface of BLASTN was used to search these plasmids for the sequence ATAAGTG CGCCCTTACGGGATTTAAC from the pSK41 oriT, and each sequence with a match was manually inspected for an adjacent DNA hairpin sequence (23).…”

“…pSK41 was introduced into each resulting strain by conjugation with strain SK5428, and resulting Cm r Gm r transconjugants were used as donors in mobilization experiments. Mobilization assays were conducted in brain heart infusion (BHI) liquid medium (Sigma-Aldrich) containing 40% (final concentration) polyethylene glycol (PEG) as described previously (18). The WBG4515 strain was used as a recipient, and Sm and Nb were used to select against donors.…”

“…Recently, O'Brien et al showed that another staphylococcal conjugative plasmid, pWBG749, which is unrelated to pSK41, can facilitate the mobilization of other plasmids that lack mob genes (17). They demonstrated that this transfer is facilitated by origin-oftransfer sequences on the mobilizable plasmids that mimic the pWBG749 origin-of-transfer sequence, suggesting a conjugative relaxase in trans mechanism (18). We identified sequences similar to the pSK41 origin of transfer on numerous nonconjugative staphylococcal resistance plasmids (see Data Sets S1 and S2 in the supplemental material), raising the possibility that pSK41 family plasmids likewise facilitate mobilization of other plasmids by an analogous relaxase in trans mechanism mediated by NES.…”