Research objectives and content
The Gram-negative soil bacterium R. meliloti is able two produce two exopolysaccharides, EPS I (succinoglycan) and EPS II (galactoglucan). At least one of these is required to establish an effective symbiosis with alfalfa. Two gene clusters directing the biosynthesis of EPS I or EPS II (exp genes) were studied and functions were proposed fo of the exp gene products. These data suggested that expDI and expD2 encode components of bacterial ABC (ATP binding cassette) exporter complexes involved in the secretion of proteins containing a C-terminal secretion signal. The expEl gene located just downstream of these two exporter genes might encode a protein secreted by this complex, since ExpEI contains a putative C-terminal secretion signal. Furthermore, ExpE1 displayed homologies to rhizobial NodO proteins. These proteins have pore-forming activity in lipid bilayers and bind Ca-ions. The functions of the three exp gene products in EPSII biosynthesis is unknown, but mutants in expDl and expDI were blocked in EPSII production. In this project biochemical aspects of three exp gene products of R. meliloti will be studied. Analyses of ExpEI will be carried out to demonstrate if it constitutes a secreted protein and if its secretion is mediated by the ExpDI and ExpD2 complex ExpE1 is secreted further analysis concerning the secretion signal and the Ca-ion-binding activity are carried out. We also intend to further determine the specific functions of these proteins in EPS II biosynthesis.
Training content (objective, benefit and expected impact)
This study will be based on the application of modern methods of bacterial genetics in conjunction with the appropriate use of techniques in protein biochemistry. All genetic and biochemical techniques to be used are well-established in the host group and have already been successfully applied to elucidate the genetics of R. meliloti EPS I and EPS II biosynthesis. Therefore, the research project proposed will greatly benefit from the experience of the EPS research group at the Lehrstuhl fuer Genetik of the University of Bielefeld. The same applies to me because I will obtain an in-depth training in the application of both genetic and biochemical techniques. This will enable me to carry out comparable research projects independently, when I return to my home country. The expected impact of this project is the analysis of the involvement of protein exporters and exported proteins in EPS II biosynthesis.