THE GLYCANASE PlyB FROM Rhizobium leguminosarum bv. viciae IS POLARLY LOCATED ON THE CELL SURFACE AND MODULATES THE LENGTH OF THE EXOPOLYSACCHARIDE AS SHOWN BY ATOMIC FORCE MICROSCOPY

PATRICIA L. ABDIAN; CATALINA VON BILDERLING; NICOLÁS VOZZA; DANIELA M. RUSSO; LÍA I. PIETRASANTA; ANGELES ZORREGUIETA

Villa Carlos Paz, Córdoba, Argentina

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiología General

The acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum is a key component for biofilm formation. Two glycanases identified in R. leguminosarum strain A34, named PlyA and PlyB are responsible for the EPS molecules length and seem to have synergistic effects. These enzymes are secreted by a type I secretion system (prsDE). PlyA remains attached to the cell surface, while PlyB is responsible for most of the diffusible activity. Surprisingly, both of them have been shown to be active only on the surface of EPS producing cells. The analysis of biofilm formation by a plyB mutant strain or a plyA double mutant, showed that an increase in EPS length affected normal biofilm development. We have raised a polyclonal antiserum to a truncated form of recombinant PlyB expressed in Escherichia coli. It was used for the location of PlyB in different strains of R. leguminosarum by western blot. The strains tested were wild type A34; the sequenced strain 3841; a mutant impaired in EPS production, A1077; and a mutant in type I secretion system, A412. PlyB was differentially located in the extracellular media or in a fraction of surface associated proteins, depending on the media in which cells were grown (rich or minimal medium, the last favouring EPS production). The antiserum was also employed in immunofluorescence assays with intact A34 cells which express the green fluorescent protein. Unexpectedly, PlyB was located at one pole of the cell.On the other hand, a direct comparison of the EPS produced by wild type A34 and mutant strain was performed by AFM single molecule measurements. We confirmed previous observations regarding an increased length of EPS molecules synthesized by the plyB mutant. We also observed a different association of the EPS molecules produced by the plyB mutant which result in the formation of an open and loose mesh as compared to the wild type. This could in part explain the deficient biofilm formation by the glycanase mutant previously reported.