RapA1 secretion by the PrsDE type I system contributes to biofilm formation in a CPS/EPS dependent mode in Rhizobium leguminosarum

NICOLAS VOZZA; PATRICIA ABDIÁN; DANIELA RUSSO; SOREN MOLIN; ANGELES ZORREGUIETA

San Miguel de Tucumán

Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO; 2011

Sociedad Argentina de Microbiología General

Rhizobium leguminosarum is capable of forming biofilms in vitro that depend on the production of the acidic exopolysaccharide (EPS). The type-I secretion system PrsDE is also required for the development of a typical structured biofilm.  Among the proteins secreted by PrsDE are the nodulation protein NodO, the glycanases PlyA and PlyB that modulate the length of EPS chains and members of the Rap (Rhizobial adhering proteins) family.  The Raps are present only in R. leguminosarum and R. etli, and were identified by their ability to bind the bacterial cell surface at one pole.  It was originally proposed that RapA1, the only characterized member of the Rap family, has affinity for the EPS and would act as adhesin by bridging between bacterial EPS associated to the bacterial surface and other bacteria or a plant cell surface polysaccharide. Mutation of rapA1 in R. leguminosarum bv viciae strain A34 produced no clear adhesion or biofilm phenotypes.  In order to investigate if RapA1 is required for in vitro biofilm formation, we overexpressed RapA1 in different backgrounds and studied the bacterial surface properties and biofilm formation. Overexpression and secretion of RapA1 in three different wild type backgrounds produced distinctive macroscopic phenotypes; the colonies became dry and wrinkle and bacterial sedimentation was prevented, suggesting that cellular interactions were altered.  Observation of static biofilms by confocal microscopy showed that RapA1-overproducing strains had altered cell-to-cell interactions when compared to isogenic wild-type strains and cells within the biofilms have lost close contact and had more void space between them. By Immunofluorescence (IF) we observed that in the wild type strain, RapA1 localized to one bacterial pole as described previously. Intriguingly, IF showed that RapA1 localizes diffusely around the cell in RapA1-overexpressing strains, providing evidence of disruption of intimate contact between bacteria; however, microtiter plate biofilm assay showed that RapA1 overexpression enhanced attachment to polystyrene in comparison to wild type.  Interestingly, RapA1-overexpression had no effect on the adhesive phenotypes of an EPS-deficient strain, reinforcing the idea that the EPS is required for RapA1 to confer a particular adhesion phenotype. To determine if RapA1 overexpression was beneficial for biofilm formation under more stringent conditions, the development of a biofilm was assessed under continuous flow conditions.  We observed that RapA1-overexpressing strains showed increased adhesion and biofilm formation respect to wild type strains, and reached a ?mature biofilm? status in a shorter time. This effect was also dependent on EPS production. We propose that interaction of RapA1with the EPS influences attachment to abiotic surfaces and cell-cell interactions during biofilm formation