The glycanase PlyB from Rhizobium leguminosarum bv. viciae is polarly located on the cell surface and modulates the lenght of the exopolysaccharide as shown by Atomic Force Microscopy

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

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General. Sociedad Argentina de Microbiología General; 2009

SAMIGE

MM-O1THE 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. Abdian1,2 , Catalina von Bilderling3, Nicolás Vozza1, Daniela M. Russo1,2 , Lía I. Pietrasanta3, Angeles Zorreguieta1,2 1Fundación Instituto Leloir 2IIBBA, CONICET 3Centro de Microscopías Avanzadas, FCEyN, UBA (pabdian@leloir.org.ar)The acidic exopolysaccharide (EPS) is a key component of the biofilm matrix formed by Rhizobium leguminosarum bv. viciae. The PlyA and PlyB glycanases from R. leguminosarum strain A34 cleave the EPS molecules and seem to have synergistic effects. PlyA and PlyB 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 glycanases have been shown tobe active only on the surface of EPS producing cells. The analysis of biofilm formation in a plyB mutant strain or a plyAplyB double mutant showed that the 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. The anti- PlyB antiserum was used to analyze by Western blot the localization of PlyB in planktonic cultures of different strains of R. leguminosarum. The strains tested were wild type A34; the sequenced strain 3841; a mutant impaired in EPS production,  A1077 pssA; and a mutant in the type I secretion system, A412 prsD. PlyB was differentially located in the extracellular media or in the surface-associated protein fraction, depending on the medium in which cells were grown (rich or minimal medium, the last favoring EPS production). Surface localization of PlyB was analyzed by indirect immunofluorescence with intact A34 cells expressing the green fluorescent protein. Interestingly, PlyB was observed to localize at one pole of the cell. On the other hand, a direct comparison of the EPS produced by wild type A34 and plyBmutant strain was performed by atomic force microscopy (AFM) single molecule measurements. As expected, a significant increase in the length of EPS molecules synthesized by the plyB mutant compared with those produced by the A34 wild type strain was observed by AFM. We also observed a different association of the EPS molecules produced by the plyB mutant, which results in the formation of an open and loose mesh as compared to the wild type. This could in part explain the deficient biofilm formed by the glycanase mutant