Secreted Proteins and Several Polysaccharides Participate in Biofilm Development by Rhizobium leguminosarum bv. viciae

RUSSO, D. M.; WILLIAMS, A; KANNENBERG E; GIORDANO, W; BREWIN J A; DOWNIE, J. A; ZORREGUIETA, A

Atlanta, USA

Congreso; American Society for Microbiology, 105th ASM General Meetingr; 2005

American Society for Microbiology

Our previous results showed that Rhizobium leguminosarum bv. viciae 8401 pRL1JI develops on minimal medium a three-dimensional structure known as biofilm. The PrsDE secretion system in Rhizobium is responsible for secretion of several proteins including two â1-4-glycanases PlyA and PlyB and three adhesins called Rap. Previous observations indicate that the prsD and prsE secretion mutants are defective for biofilm formation on a glass surface. Our aim is to identify the genetic factors involved in cell-to-cell interactions and biofilm development. Confocal laser scanning microscopy (CLSM) of GFP-labelled bacteria cultured in chambered coverglass slides was used to visualise the different events of biofilm formation. Mutations in prsD, prsE and plyB severely affected biofilm maturation. A mutant in pssA, a crucial gene for EPS synthesis was unable to develop a biofilm although tight cell-to-cell interactions were observed in bacterial clumping. Congo red staining showed that cellulose biosynthesis is increased in pssA. We studied the nature of the molecules participating in bacterial clumping. A mutation in prsE destabilize clump formation while a mutation in celA, which encodes the cellulose synthase A abolished it, ndicating that cellulose synthesis is crucial in clumping in pssA genetic background. We also observed that cellulose synthesis is involved in bacterial clumping in an LPS mutant with an altered O-antigen. Another LPS mutant, which lacked the O antigen region, was unable to establish lateral cell-tocell interactions and does not induce bacterial clumping. Instead, pole-to-pole interactions were observed. Both LPS mutants were unable to develop a biofilm. Our results suggest that in R leguminosarum the LPS and the EPS are essential for biofilm formation and some PrsDE-secreted proteins are involved in biofilm maturation and cell-to-cell interactions. In addition, cellulose synthesis may have an impact in cell-to-cell interactions in certain genetic backgrounds.