Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

RUSSO, D. M.; WILLIAMS, A; EDWARDS, A; POSADAS, D. M.; FINNIE, C; DANKERT, M; DOWNIE, J. A; ZORREGUIETA, A

JOURNAL OF BACTERIOLOGY

American Society for Microbiology

Año: 2006 vol. 188 p. 4474 - 4486

0021-9193

The Type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. Both the prsD and prsE secretion mutants were greatly affected in the formation of a ring of biofilm on the surface of the glass in shaken cultures. Confocal laser scanning microscopy (CLSM) analysis of GFP-labelled bacteria showed that during growth in minimal medium R. leguminosarum wild type develops microcolonies, which progress to a characteristic three-dimensional biofilm structure.  However, the prsD and prsE secretion mutants were only able to form an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed an altered timing of biofilm formation and its structure was atypical. A mutation in an essential gene for the EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the essential role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system and  N-terminal sequencing revealed peptides homologous to the N-terminal of proteins from the Rap family (Rhizobium adhering proteins), which could have a role in cellular adhesion in R. leguminosarum.  We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.