INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
congresos y reuniones científicas
EXOPOLYSACCHARIDE CHARACTERIZATION OF Rhizobium favelukesii LPU83 AND ITS ROLE IN THE SYMBIOSIS WITH MEDICAGO SATIVA
LUCHETTI, A.; PÉREZ-GIMÉNEZ, J.; NILSSON, J; TORRES TEJERIZO, G. A.; CASTELLANI, L; PISTORIO, M.
Congreso; REUNIÓN CONJUNTA SAIB-SAMIGE 2020; 2020
Leguminous plants can develop a symbiotic interaction with rhizobia, allowing the biological reduction of N2. It was shown thatrhizobial surface polysaccharides play an important role in the establishment of the symbiosis between bacteria and leguminous plants. Rhizobia can produce different types of surface polysaccharides relevant for the symbiosis establishment,such as exopolysaccharides (EPS). Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia that is capable of nodulating alfalfa (Medicago sativa) but inefficient in nitrogen fixation. This symbiosis has other particular features, such as not requiring sulfated forms of the Nod factors (NF), a fact that does not occur during the symbiosis between alfalfa and Ensifer meliloti, the model of efficient rhizobia that needs sulfated NFs to nodulate. We knew from previous work, that LPU83 showed mucoid macrocolonies, which predict the production of polysaccharides. Aiming to identify the molecular determinants that allow R. favelukesii to infect, its polysaccharide production was studied. First, we searched for genes needed for the biosynthesis of surface polysaccharides by BLAST. E. meliloti has a main cluster (exoBZQFYXUVWTIHKLAMONP) needed for the synthesis of succinoglycan (EPS I). We found that in R. favelukesii LPU83 the cluster is split into three different sections: one clustered in the chromosome, one in the pLPU83a plasmid and the exoB gene not clustered in the chromosome. We performed phylogenetic analysis and found that these sections may have arose by an event of horizontal gene transfer. Moreover, the structural analysis of the EPS I from R. favelukesii LPU83 showed that this exopolysaccharide is identical to the one produced byE. meliloti. To answer the question of whether these three sections involved in the synthesis of EPS I were relevant for the biosynthesis, we decided to delete both clusters and the exoB gene to generate mutant strains. Then we combined the mutations producing double and triple mutants. We found that none of the mutants produced EPSI and neither showed fluorescence in medium containing calcofluor. Only the wild type strains were able to produce EPS I. Therefore, both clusters and the exoB gene are needed for the EPS I production. We evaluated the nodulation of alfalfa using all the constructed strains. Previously, it was shown that when E. meliloti only produces EPS I, this EPS I is essential for the symbiosis. In contrast, our results show that LPU83 devoid of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa. By means of confocal microscopy, we observed a similar distribution pattern of the bacteria inside de nodules produced by the different strains of LPU83, were the bacteria showed lack of differentiation. Overall, our results show that, unlike E. meliloti, R. favelukesii is a particular species that is able to infect alfalfa when is devoid of all the genes needed for the synthesis of EPS I.