Influence of exopolysaccharides in biofilm formation in Mesorhizobium loti.

SUPANITSKY A.; RUSSO D.M.; ZORREGUIETA A.; LEPEK V.

Córdoba

Congreso; XI Congreso Argentino de Microbiología General. Asociación Civil de Microbiología General- SAMIGE 2015; 2015

Asociación Civil de Microbiología General

Biofilm lifestyle plays an important role in survival of bacteria under different environmental stresses. The interaction between symbiotic microorganisms and the environment, in particular with the host plant, depend on superficial and extracellular molecules. Mesorhizobium loti produces several exopolysaccharides that participate in the symbiotic process with the host plant. In previous works,our results suggested that EPS production is crucial for biofilm formation in M.loti Ayac 1 BII.         Analysis of the M. loti MAFF303099 sequenced strain revealed the presence of a cluster of exo genes involved in EPS biosynthesis, polymerization, transport, and post-production processing, and a distant locus putatively related  to EPS biosynthesis. By directed mutagenesis,we generated two EPS mutants of M. loti MAFF303099, interrupting the mll5252exo Y and mlr6756 genes. mll5252 gene encodes a protein homologue to the galactosyltransferase ExoY, which is involved in exopolysaccharide I(EPS I) production in S. meliloti. The mlr6756 gene encodes aputative exopolysaccharide biosynthetic protein. Biofilm formation was assayed in polystyrene multiwell plates using crystal violet staining. EPS levels were determined by Anthrone reaction, and cellulose production was evaluated by Congo red staining. Biofilm formation was reduced in mll5252 mutant incomparison with WT whereas the mlr6756 mutant showed no significant differences compared with the WT. mll5252 mutant formed non-mucoidcolonies that  were strongly stained with Congo red in comparison with the WT strain, while mlr6756 formed mucoid colonies that overproduced EPS.  Finally,nodulation experiments were carried out on Lotus tenuis plants inoculated with WT strain or mutants. mll5252 mutant strain showed a significant reduction in the total number of nodules in comparison with the WT. Plants inoculated with mlr6756 mutant developed an increased number of nodules that seemed to develop earlier than the WT. Our results suggest that in M. loti MAFF303099, the production of EPS(s) is required for biofilm development and nodulation.