Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

A single amino-acid substitution switches DNA-binding and mobilisation specificity of antimicrobial-resistance plasmids (#264)

Karina Yui Eto 1 2 , Joshua P Ramsay 2 , Charles S Bond 1
  1. School of Chemistry and Biochemistry, University of Western Australia, Crawley, WA, Australia
  2. School of Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia

The diverse ribbon-helix-helix (RHH) DNA-binding proteins family contain an N-terminal anti-parallel β-sheet that binds DNA in the major groove, directly decoding the DNA site. Recently the SmpO RHH-protein was discovered to be critical for recognition of the pWBG749-family of bacterial conjugative ‘origin-of-transfer’ DNA sequences (oriT), carried by 53% Staphylococcus aureus antimicrobial-resistance plasmids. These oriTs contain inverted-repeat sequences likely required for recognition and processing by the pWBG749-encoded DNA endonuclease/relaxase SmpP. The two most prevalent pWBG749-family conjugative plasmids pWBG749 and pWBG745 share an almost identical nucleotide sequence, but carry distinct oriT sequences, named OT49 and OT45. pWBG749 and pWBG745 only mobilise plasmids carrying the same oriT, but this limitation can be overcome by additionally providing the cognate pWBG749 or pWBG745-encoded SmpO gene, smpO45 or smpO49, in trans. This suggests that the predicted ribbon-helix-helix DNA-binding proteins SmpO49 and SmpO45, rather than the relaxase SmpP, distinguish between OT49 and OT45 sites. In this current work we used directed mutagenesis of the predicted DNA-binding β-sheet region of SmpO49, in an attempt to force SmpO49 to recognise non-cognate OT45 sites in conjugative mobilisation experiments. Twenty mutant smpO49 alleles were constructed, each of which changed 2-4 amino acids of SmpO49 to match those on SmpO45. Surprisingly, a single F7K substitution switched the mobilisation specificity of pWBG749 from OT49 oriT sites to OT45 sites, indicating this sole residue controls the ability of SmpO to discriminate between the two oriTs. Conversely, a K7F substitution in SmpO45 switched mobilisation specificity of pWBG745 from OT45 oriT sites to OT49 sites. Preliminary analysis of purified SmpO proteins indicates they exist as tetramers in solution. These discoveries provide insight into the coevolution between DNA-binding proteins and their cognate DNA sites and highlight how rapidly horizontal-gene-transfer mechanisms may evolve to facilitate mobilisation of new DNA targets.

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