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

Abstract

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. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for 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 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 termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles 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.

The ExpR/Sin Quorum-Sensing System Controls Succinoglycan Production in Sinorhizobium meliloti

“…It is interesting, however, that similar phenotypes have been linked to biofilm production (22,26,73). Biofilms have recently been shown to occur in Rhizobia (23,62,65). In Rhizobium leguminosarum, the PrsD-PrsE secretion system was shown to be involved in biofilm production (65).…”

Aggregation by depletion attraction in cultures of bacteria producing exopolysaccharide

“…We used chambered coverslides, whereby the base of the culture chamber forms a coverslip, to visualize biofilm formation, as this allowed us to visualize aggregates as they sedimented. Chambered coverslides have also been used in studies of R. leguminosarum [11], S. meliloti [46] and X. campestris [47] biofilms. We found that the exoS mutant forms an inhomogeneous structure, consisting of interlinked aggregates separated by gaps that are characteristic of the 'water channels' described in many biofilms.…”

“…In studies on rhizobial biofilms, many have used chambered coverslides, which consist of a coverslip with culture chambers mounted on top, that are imaged using an inverted confocal microscope [11,46,47]. We used the same arrangement in our work as it allowed the visualization of the sediment that formed as bacteria aggregate and sediment.…”

“…Bacteria also colonize surfaces, forming microcolonies and aggregative structures known as biofilms. Integral to aggregation is the production of exopolysaccharides by the bacteria; for example, observations of bacterial aggregates from wastewater treatment by electron microscopy showed the presence of extracellular fibrils that were later confirmed to be polysaccharides [1][2][3][4][5][6], and, more recently, studies of Agrobacterium species [7][8][9][10], Rhizobium leguminosarum [11][12][13], Xanthomonas campestris [14] and Sinorhizobium meliloti [15,16] have shown that mutants affected in exopolysaccharide production have altered aggregation and biofilm formation.…”

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…Also, the degree of EPS polymerization is important for proper development of biofilms in rhizobia. Mutant strains of R. leguminosarum, having mutations in genes involved in EPS processing (prsD and prsE encoding components of the type I protein secretion system and plyB encoding glycanase), are only able to form biofilms of immature structure (Russo et al 2006).…”