Role of exopolysaccharides synthesis and biofilm formation during the establishment of the Sinorhizobium meliloti-alfalfa symbiosis.

RINAUDI, L., SORROCHE, F., ZORREGUIETA, A., GIORDANO, W.

Carlos Paz, Córdoba.

Congreso; VI congreso Argentino de Microbiología General (SAMIGE); 2009

Sinorhizobium meliloti is a nitrogen-fixing bacterium which establishes a symbiotic relationship with Medicago sativa (alfalfa). The synthesis by S. meliloti of at least one exopolysaccharides (EPSs), succinoglycan (EPS I) or galactoglucan (EPS II), is essential for a successful symbiosis. While exopolysaccharide-deficient mutants induce nodule formation, they fail to invade them, and as a result, no nitrogen fixation occurs. It has been shown that the ExpR/Sin quorum-sensing system controls biofilm formation in S. meliloti through the production of the EPS II, which provides the matrix for the development of biofilms, both in vitro and in vivo. Phosphate concentration constitutes an environmental signal that determines which EPS is produced by S. meliloti. The low-phosphate conditions normally found in the soil (typically 1 to 10 µM) stimulate EPS II production, while the high-phosphate concentrations inside the plant (10 to 100 mM)) block EPS II synthesis and induce the production of EPS I. Here we studied how phosphate availability regulates exopolysaccharides synthesis and the role of EPSs during invasion of the legume plant. We performed nodulation assays using wild-type strains of S. meliloti (Rm1021 and Rm8530) as well as mutants in EPS I (exoY) and EPS II (expA) production. Alfalfa plants were supplied with nitrogen-free Hoagland solution containing different phosphate concentrations (0.1 to 10 mM) as needed and scored for pink and white nodules after 30 days of growth at 25°C, 60% relative humidity, and a 16-h light cycle. Our results suggest that under phosphate limitation (0 and 0.001 mM), EPS II-producing strains are more efficient to nodulate alfalfa than succinoglycan-producing strains. However, when phosphate availability increases (0.1 and 10 mM), EPS I promotes a better nodulation than EPS II-producing strains. Our findings allow us to propose a model to explain the role of EPSs during the establishment of the symbiosis between S. meliloti and alfalfa. In this sense, low-phosphate conditions as the ones reported in soils would induce EPS II production which mediates attachment and root colonization. Once inside the plant, rhizobia would find higher phosphate concentrations that would induce EPS I production, which major role seems to be avoiding legume-defense responses. In light of the data presented here, it is tempting to speculate that the main role of EPS II is in positioning the bacteria appropriately on the host root by forming biofilms so that invasion can occur. The biofilm matrix could be providing S. meliloti with the suitable microenvironment necessary for colonization and eventual invasion of the root hairs, while the absence of exopolysaccharides appears to lead to a poor root association and host-invasion failure.