Role of the exopolysaccharides in the early steps of Rhizobium-legume symbiosis

Principal Investigator: Leonilde Morais Moreira

Contract: PTDC/AGR-AAM/66977/2006

Start date: 01/03/2007

Duration: 36 months


The soil bacterium Sinorhizobium meliloti fixes atmospheric nitrogen in a symbiosis with leguminous plant Medicago sativa (alfalfa) through the formation of root nodules. This implies a complex chemical dialogue between partners and drastic changes on both plant roots and bacteria. Several reports pointed out the importance of rhizobial surface polysaccharides in the establishing of the highly specific symbiosis between symbionts. These polysaccharides appear to be essential for the early infection process but the exact mechanisms of symbiotic development are still unclear. S. meliloti 1021 produces two acidic exopolysaccharides (EPS), succinoglycan (EPS I) and galactoglucan (EPS II) and at least one of them is required to properly nodulate alfalfa. Nodules induced by EPS mutant strains are small, contain few or no bacteroids, and cannot fix nitrogen. Both biosynthetic gene clusters directing the biosynthesis of EPS I (exo and exs genes) and EPS II (exp genes) are known. Most of the proteins involved in the biosynthesis of the nucleotide sugars as well as repeat-unit assembly and decoration are well known. Contrastingly, the later steps of polysaccharide biosynthesis such as polymerization and secretion remain totally unknown.
Our previous work on protein secretion in S. meliloti demonstrated the involvement of the outer membrane TolC in the secretion of ExpE1 and ExsH proteins, important for EPS II and LMW-EPS I biosynthesis, respectively. The lack of EPS I from the tolC deletion mutant was unexpected and impose important questions about the mechanism of EPS polymerization and secretion. Beside that, it was never described before the involvement of a protein such as TolC in polysaccharide biosynthesis. It was also observed that the tolC deletion mutant is able to induce nodules in Medicago that remained empty and do not fix nitrogen. The aim of this project is to understand the role of TolC in EPS I biosynthesis and symbiosis and also evaluate if the EPS I is a determinant of host-plant specificity in nodulation. More specifically we propose to: 1) assess whether the involvement of TolC protein in EPS I biosynthesis is due to secretion of proteins required for EPS I biosynthesis or whether is required to interact with other Exo ptoteins responsible for EPS I polymerization/secretion. To test the first hypothesis the extracellular proteome of the wild-type and tolC mutant strains will be compared by 2D-gel electrophoresis followed by protein identification. For the second hypothesis we will test possible interactions between TolC and proteins involved in polymerization/secretion of EPS I (Exo P, Exo T, Exo Q and Exo F), using in vivo bacterial two-hybrid system and surface plasmon resonance; 2) determine which step of the symbiotic development is impaired in the tolC mutant, by labelling it with the green fluorescent protein and follow entry in the roots of Medicago by confocal microscopy; 3) measure global gene expression of Medicago roots in the presence of S. meliloti EPS defective mutants or in the presence of EPS oligosaccharides only, by using Affymetrix Medicago truncatula GeneChip arrays; 4) analyse microarray data using web resources like MapMan in order to evaluate if there are genes or pathways that are specifically activated or repressed by exopolysaccharides; 5) perform a more detailed characterization of the genes/proteins of interest by evaluating the symbiotic properties of the mutant plants and perform protein localization in the roots of transgenic plants expressing promoter- reporter gene fusions. With this set of proposed experiments, new insights on the role of the exopolysaccharides in legumerhizobia symbiosis will be provided. Since this symbiosis is of agronomic importance, the exploration of new genes and gene networks can create better legumes and sustainable agriculture practices with benefit to environmental and human health. This proposal includes researchers from the Center for Biological and Chemical Engineering/IST, from the Instituto Gulbenkian de Ciência, Oeiras and from the Biology Department of Bielefeld University, Germany.