Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

  Characterising quorum-sensing antiactivation in Mesorhizobium loti R7A   (#48)

Drew A Hall 1 , Joshua P Ramsay 1 , Gavin J Knott 2 , Tom Caradoc-Davies 3 , Elizabeth L Watkin 1 , Clive W Ronson 4 , Charles S Bond 2
  1. Biomedical Science, Curtin University, Perth, WA, Australia
  2. Chemistry and Biochemistry, University of Western Australia, Perth, WA, Australia
  3. the Australian Synchrotron, Melbourne, Victoria, Australia
  4. Microbiology and Immunology, University of Otago , Dunedin, New Zealand

Mesorhizobium loti R7A is a legume symbiont that fixes atmospheric nitrogen. The ability to fix atmospheric nitrogen requires genes located on a mobile genetic element called the symbiosis island. This symbiosis island can excise, circularise and transfer via conjugation to non-symbiotic mesorhizobia in the environment, converting them into symbionts. Transfer of the symbiosis island occurs rarely, but the rate is increased at high cell-densities. Population density is controlled by a LuxR/I-family quorum-sensing system encoded on the symbiosis-island, however, even in high population density, island excision is only activated in ~ 5% of cells . The restriction of quorum-sensing activated transfer is accomplished by an antiactivator protein called QseM. QseM binds and inhibits both the quorum-sensing activator TraR and the symbiosis island excision activator FseA. QseM binds to both these proteins in vivo and inhibits their function through an unknown mechanism. To understand how QseM binds and inhibits TraR and FseA, we seek to determine the structure of QseM and of QseM complexed with FseA and TraR, using X-ray crystallography.

We have successfully expressed, purified and crystallised QseM with and without the incorporation of seleno-methionine. Native and seleno-methionine containing QseM crystals shared similar morphology, space-group and unit cell dimensions. Native diffraction data was collected to 2.8 Å, however, seleno-methionine data was limited to ~3.5 Å and is not sufficient for structure determination. By improving crystallisation of QseM with heavy atoms we can determine its structure and begin targeted mutagenesis of QseM and its partner proteins to isolate residues required for interaction via in vivo assays. We will also attempt to elucidate the structure of QseM bound to FseA and TraR by co-crystallisation X-ray crystallography.

Structural information gained from this work will guide future protein interaction assays and identify key residues or secondary structure features required for binding. This study will broaden our understanding of quorum-sensing regulation and horizontal gene transfer.

 

  1. Sullivan, J. T., Patrick, H. N., Lowther, W. L., Scott, D. B., & Ronson, C. W. (1995). Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment. Proceedings of the National Academy of Sciences of the United States of America, 92(19), 8985-8989.
  2. Sullivan, J. T., & Ronson, C. W. (1998). Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene. Proceedings of the National Academy of Sciences, 95(9), 5145-5149.
  3. Ramsay, J. P., Tester, L. G. L., Major, A. S., Sullivan, J. T., Edgar, C. D., Kleffmann, T., . . . Ronson, C. W.(2015). Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element. Proceedings of the National Academy of Sciences, 112(13), 4104-4109.