Extracellular components that define Rhizobium leguminosarum biofilm matrix architecture

ABDIAN, P; VOZZA, N; CARAMELO, J.; VON BILDERLING C.; RUSSO, D. M.; PIETRASANTA, LI; ZORREGUIETA, A

Viena

Congreso; BIOFILMS 6. International Conference on Microbial Biofilms; 2014

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Rhizobia are soil bacteria that establish symbiotic interactions with leguminous plants inducing the formation of nitrogen-fixing root nodules. They are able to grow in varied environmental conditions producing different types of biofilms. We have studied the structure and the molecular determinants of biofilms formed by Rhizobium leguminosarum in vitro, a process probably more relevant to the attachment of bacterial cells to soil particles. Using confocal microscopy (CLSM) it was observed that the typical architecture of a mature R. leguminosarum biofilm consists of multiple layers of cells attached to each other mostly by lateral interactions, forming clusters that are interlaced by water channels. The main component of the extracellular matrix is an acidic exopolysaccharide or EPS. A flat lawn of bacteria without any architecture was observed in EPS-defective mutants, showing that EPS provides support to the biofilm structure. Moreover R. leguminosarum produces a capsular polysaccharide (CPS) structurally similar to EPS that seems to be involved in surface attachment during the first stages of biofilm development. Other components involved in R. leguminosarum biofilm are extracellular proteins, since mutations in the type I secretion system PrsDE reduced biofilm formation. Proteins secreted by PrsDE include several predicted calcium binding proteins and members of the Rap (Rhizobial adhering proteins) family, most of them of unknown function. Interestingly, the Raps possess one or more characteristic Ra/CHDL (cadherin like-) domains that confer carbohydrate binding ability. Among them, we have characterized RapA by biochemical and biophysical methods, and defined its role as a unipolar calcium binding lectin that specifically recognizes the EPS/CPS. Mutation of rapA in R. leguminosarum bv. viciae strain A34 produced no clear adhesion or biofilm phenotypes, but overexpression of RapA in different wild type backgrounds produced distinctive macroscopic phenotypes, suggesting that cellular interactions were altered. Observation of static biofilms by CLSM showed that RapA-overproducing strains had altered cell-to-cell interactions within the biofilms, losing intimate contact and the typical ordered structure. By Immunofluorescence (IF) we observed that RapA was re-distributed diffusely around RapA-overproducing cells. Moreover, RapA-overexpression had no effect on the adhesive phenotypes of an EPS/CPS-deficient strain, reinforcing the idea that the EPS/CPS is required for RapA to confer a particular adhesion phenotype. We have also studied other members of tha Rap family, the Ply glycanases that regulate the length of EPS chains in the extracellular medium. We have observed by atomic force microscopy (AFM) that the development of an ordered polysaccharide network is dependent on Ply secretion. Thus, Ply mutants are unable to form a typical biofilm. A model depicting the interaction of RapA, Ply glycanases and acidic polysaccharides (EPS and CPS) during the progress of biofilm matrix development is presented. Supported by CONICET and Agencia de Promoción Científica y Tecnológica-FONCYT