Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Finding a target: how a single protein regulates transcription, excision and transfer in a bacterial integrative and conjugative element. (93851)

Callum J Verdonk 1 2 , Josh P Ramsay 2 , Charlie S Bond 1 3
  1. School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia
  2. Curtin Health Innovation Research Institute and Curtin Medical School, Curtin University, Perth, WA, Australia
  3. Marshall Centre for Infectious Disease Research and Training, The University of Western Australia, Perth, WA, Australia

Integrative & Conjugative Elements (ICEs) are mobile genetic elements that reside within the chromosomes of their hosts but can excise and horizontally transfer by conjugation. Integration of ICEs within the host chromosome is catalysed by a tyrosine recombinase enzyme but excision of the ICE usually also requires a recombination directionality factor protein. Here we present the 2.4 ångström X-ray crystal structure of RdfS, the recombination directionality factor that stimulates excision and conjugative transfer of ICEMlSymR7A, a 502-kb ICE carrying genes for nitrogen-fixing symbiosis in members of the genus Mesorhizobium. RdfS stimulates excision by binding DNA targets within recombinase attachment sites and through transcriptional activation of the recombinase gene. Promoter-lacZ fusion assays reveal RdfS also negatively autoregulates rdfS transcription to ensure stable levels of protein expression, as artificial overexpression of RdfS is lethal. Gel-filtration and band shift assays with purified RdfS suggest formation of variably sized oligomers that specifically bind in a concentration-dependent manner to distinct DNA sequences that share poor sequence similarity. Analysis of the X-ray crystal structure of RdfS reveals it is a winged helix-turn-helix (wHTH) protein that forms head-to-tail left-handed helices. This quaternary structure is partially mediated by a novel N-terminal α-helix not present in other wHTH domains. We propose that the ability of RdfS to specifically bind several DNA sites with distinct sequences arises from an ability to form variable-length helical filaments that recognise DNA structure through an indirect readout mechanism. This likely explains how a single protein, through binding diverse DNA targets at different concentrations, can coordinate regulation of ICEMlSymR7A transcriptional regulation, excision and conjugation. 

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  2. Verdonk, C. J., Sullivan, J. T., Williman, K. M., Nicholson, L., Bastholm, T. R., Hynes, M. F., Ronson, C. W., Bond, C. S. & Ramsay, J. P. (2019). Plasmid, 104, 102416. https://doi.org/10.1016/j.plasmid.2019.102416
  3. Verdonk, C. J., Marshall, A. C., Ramsay, J. P. & Bond, C. S. (2022). Acta Cryst. D 78(10). https://doi.org/10.1107/S2059798322008579