Interaction of the RapA1 adhesin with the acidic exopolysaccharide in Rhizobium leguminosarum

VOZZA, N; ABDIAN, P; MORGIARDINI, E; RUSSO, D. M.; LODEIRO A; ZORREGUIETA, A

Villa Carlos Paz

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

SAMIGE

 INTERACTION OF THE RapA1 ADHESIN WITH THE ACIDIC EXOPOLYSACCHARIDE IN <i>Rhizobium leguminosarum</i>  Nicolás F Vozza; Patricia L Abdian; Elías Mongiardini, Daniela M. Russo, Anibal Lodeiro y Angeles Zorreguieta  We have shown that the acidic exopolysaccharide (EPS), the O-antigen of the LPS and a functional PrsDE type I secretion system are important for the formation of a structured and typical biofilm in <i>Rhizobium leguminosarum</i> bv <i>viciae</i> A34. The PrsDE system is responsible for the secretion of several proteins, such as two EPS-glycanases and the Rap (<i>Rhizobium</i> adhering proteins) family of proteins. It was previously shown that RapA1 adhesin from <i>R. leguminosarum</i> bv <i>trifolii</i> R200 localizes at one cell pole and that addition of recombinant RapA1 to a planktonic culture of R200 promote cell agglutination. In addition, it was suggested that attachment of RapA1 to the cell surface would be mediated by the EPS.  Although inactivation of the <i>rapA1</i> gene in the A34 background had not show a clear biofilm phenotype, overexpression of the <i>R. l</i>. bv <i>trifolii rapA1</i> in R200 and <i>R. l. </i> bv <i>viciae</i> A34 and 3841 wild type backgrounds resulted in disruption of tight and typical cell-cell interactions within the biofilm microcolonies as observed by Confocal Laser Scanning Microscope (CLSM). Using antiserum against R200-RapA1, we confirmed by Western blot that this phenotype was due to the secretion of RapA1 since in all cases, RapA1 was either associated to the cell surface or released to the extracellular milieu in a PrsDE-dependent manner. A faint band with a similar molecular weight to RapA1 was also observed in the surface protein fractions of RapA1-defective strains, suggesting that a RapA1 homologue is also located on the cell surface. By indirect inmunofluorescence and CLSM of live bacteria, we observed that in the A34 wild type background, RapA1 also localizes at one cell pole, as has been shown in R200.  However, overexpression of <i>rapA1</i> in A34 resulted in a localization of RapA1 in an extended region around one cell pole. Surprisingly, we observed by CLSM that overexpression of <i>rapA1</i> in an EPS-defective mutant caused unusual cell-cell interactions within biofilms, suggesting that even in the absence of EPS, RapA1 is able to interact with the bacterial surface.  In line with this observation, a polar localization of RapA1 was observed by inmunofluorescence in the <i>rapA1</i>-overexpressing cells of an EPS-defective mutant. Taken together, these observations suggest that in A34:  i) synthesis of EPS highly influences cell-cell interactions mediated by RapA1, ii) RapA1 localization at one cell pole is EPS-independent and iii) a RapA1 homologue also localizes at one bacterial pole.