Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

Deciphering the molecular recognition of origin-of-transfer sequences by the pWBG749 family of Staphylococcus aureus conjugative plasmids (#356)

Karina Yui Eto 1 2 , Geoffrey W. Coombs 2 3 , Charlie S. Bond 1 , Joshua P. Ramsay 2 3
  1. University of Western Australia, Crawley, WA, Australia
  2. School of Biomedical Sciences, Curtin University, Bentley, WA, Australia
  3. The Australian Collaborating Centre for Enterococcus and Staphylococcus (ACCESS) Typing and Research, School of Veterinary and Life Sciences, Murdoch University and School of Biomedical Sciences, Curtin University., Perth, WA, Australia

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. In S.aureus, a large proportion of antimicrobial-resistance and virulence genes are carried on plasmids that lack classical mobilisation loci. The pWBG749-family is a recently identified class of conjugative plasmid that is capable of mobilising non-conjugative plasmids that carry mimics of the pWBG749 origin-of-transfer (oriT) sequence. These oriT mimics have been identified on over 50% of plasmids. The oriTs can be grouped into 5 distinct sequence families, each differing in an inverted-repeat sequence IR2. The two most common oriT sites, OT49 and OT45, with IR2 half-sites CTATC and CCCCC, are only recognised by pWBG749-family plasmids carrying the same oriT, i.e. pWBG749 and pWBG745, but this limitation is overcome if the cognate conjugative-plasmid’s SmpO gene (smpO49 or smpO45) is provided in trans. Thus the predicted DNA-binding ribbon-helix-helix protein SmpO, appears to be the specificity factor that distinguishes between oriT sites, rather than the relaxase SmpP. The pWBG749/pWBG745 SmpO sequences differ considerably in their N-terminal predicted DNA-binding β-sheet regions. Directed mutagenesis of the SmpO49 β-sheet region was performed in attempt to make SmpO49 recognise OT45 sites in conjugative mobilisation experiments. Twenty smpO49 alleles were constructed, each carrying substitutions with 2 to 4 amino-acids found on SmpO45 in the same positions. Half of the smpO49 mutants enabled mobilisation of OT45 sites. A single F7K substitution was conserved in these mutants, suggesting this sole residue distinguished between the two oriT sites. This was confirmed by construction of an SmpO­49 variant with a single F7K substitution, implying that small changes in SmpO sequence can change DNA-binding specificity. This discovery provides insights into the coevolution between DNA-binding proteins and their cognate DNA sites and highlights how rapidly horizontal-gene-transfer mechanisms may evolve to mobilise new sequences. Our current investigations are focused on dissecting the molecular mechanisms of oriT recognition by the pWBG749 relaxase protein SmpP and the oriT specificity protein SmpO.