Replication of staphylococcal multiresistance plasmids — ASN Events
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Replication of staphylococcal multiresistance plasmids (#146)

Stephen M Kwong 1 , Michael A Liu 2 , Maria A Schumacher 3 , Neville Firth 1
  1. School of Biological Sciences, University Of Sydney, NSW, Australia
  2. School of Molecular Bioscience, University Of Sydney, Sydney, NSW, Australia
  3. Department of Biochemistry, Duke University, Durham, NC, USA

Theta-replicating, low copy number plasmids have contributed significantly to the evolution of antibiotic resistance in Staphylococcus aureus. These plasmids act as an assembly point for the rise of large antimicrobial resistance gene clusters, which are then capable of spreading to other bacteria as a single entity by horizontal DNA transfer mechanisms.

Most S. aureus multiresistance plasmids have been found to carry an evolutionarily related replication region that encodes a replication initiation protein (Rep) belonging to the RepA_N family. The Rep protein from conjugative multiresistance plasmid pSK41 initiates plasmid replication by binding to four directly repeated sequences in the origin of replication (oriV) found at the centre of the rep gene. The DNA-binding domain is located in the N-terminal 120 residues of Rep. The role of the Rep C-terminal region has yet to be determined; however, phylogenetic analyses indicated a higher level of sequence conservation in plasmids from the same genera suggesting that the C-terminal region could interact with host-encoded protein(s). The Rep-oriV nucleoprotein complex presumably causes strand melting at oriV, followed by the assembly of host-encoded proteins to form the replisome, although the intricacies of these molecular processes in pSK41 replication are unknown. 

In this study, we utilised the yeast two-hybrid system to investigate potential protein-protein interactions that occur between the Rep protein and host-encoded replication proteins. An S. aureus genomic DNA library was constructed and screened using pSK41 Rep as the bait. We detected a direct interaction between pSK41 Rep and DnaG primase, a primosomal protein essential for DNA synthesis. As a component of the primosome, DnaG continually synthesises the primers for Okazaki fragments during lagging-strand replication. In pSK41 replication, DnaG may also play a role in priming of the leading strand. DnaG is also known to interact directly with DnaB, and thus, could assist in the recruitment of other replisome components to the pSK41 oriV.

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