Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium 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.

FIG. 1.

Formation of a ring of biofilm in shaken cultures by the prsD secretion mutant is affected. R. leguminosarum strains A34 (WT) and A412 (prsD) were grown for 10 days in Y mannitol minimal medium. The prsE mutant (A755) produced a weak or greatly reduced ring of biofilm at the air-liquid interface similar to that seen with the prsD mutant (not shown).

FIG. 2.

Attachment to polystyrene is reduced by mutations affecting secreted proteins and the acidic EPS. Bacteria attached to the surfaces of wells in microtiter plates were stained with crystal violet, and the absorbance at 600 nm was measured. The strains used were A34 (WT), A412 (prsD), A755 (prsE), A600 (plyB), A638 (plyA), A640 (plyA/B), and A1077 (pssA). Data are presented as the mean absorbance values for four replicate wells plus standard errors from a representative experiment.

FIG. 3.

CMC degradation and protein secretion by the prsE secretion mutant. (A) CMC degradation was estimated by the lack of Congo red staining (seen as clear regions) in the agar directly below the colonies. The strains used were A34 (WT), A412 (prsD), and A755 (prsE) on CMC agar. (B) Culture supernatant proteins were precipitated from A34 (WT), A755 (prsE), and A412 (prsD); separated by SDS-PAGE; and stained with Coomassie blue. The migration positions of molecular mass markers (94, 67, 43, and 30 kDa) are shown, and the arrows indicate the positions of the proteins absent from the supernatants of prsE and prsD mutants.

FIG. 4.

Developing stages of biofilm formation in R. leguminosarum bv. viciae. A34 (WT), A412 (prsD), and A1077 (pssA) containing the GFP-expressing plasmid pRU1319 were grown for 4 days in chambered cover slides and observed daily by CLSM. The panels (left to right) show horizontal projected images (x-y axis) from each day. (A34, day 1) The arrow indicates cell attachment to the glass surface by one pole in vertical projected images (z axis). (A34, day 3) The inset shows a detail of a typical wild-type microcolony. The prsD mutant, A412, developed an immature biofilm after 4 days and was restored to wild-type phenotype with the prsD-prsE genes cloned in pIJ7349 (A412 prsD + pIJ7349). The pssA mutant, A1077, did not form a typical biofilm after 4 days. CLSM images were also acquired from mixed (1:1) cultures of A34/pRU1319 GFPuv-labeled (green) and A34/pMP4518 EYFP-labeled (red) (A34g/A34r). At early stages of biofilm development (A34g/A34r, days 1 and 2), dual-color A34 cultures showed mixed-color bacteria interacting (arrow) and developing microcolonies; at later stages, the typical microcolonies progressed to a honeycomb structure (A34g/A34r, day 4). Magnifications, ×1,000; zooms (insets), ×3,000. The size bars in panels and in the inset of the first panel indicate 2 μm.

FIG. 5.

Effects of different mutations on biofilm formation. (A) Altered biofilm phenotypes of the plyB (A600) and plyA plyB (A640) mutants observed by CLSM. The panels show horizontal projected images (x-y axis) from 4-day static cultures in chambered cover slides of A34/pRU1319, A600 plyB/pRU1319, A638 plyA/pRU1319, and A640 plyA plyB/pRU1319. (B) The panels (left to right) show horizontal projected images (x-y axis) of A34/pRU1319, the prsE mutant A755/pRU1319, and the EPS mutant A550/pRU1319. Magnifications, ×1,000. Size bars indicate 2 μm.

FIG. 6.

Incorporation of protein secretion and EPS-defective mutants into mixed biofilms. Dual-color mixed cultures were all inoculated at a 1:1 ratio of bacteria. (A) Mixed cultures of A34/pMP4518 EYFP-labeled (red) and A1077(pssA)/pRU1319 GFPuv-labeled (green) were followed during a 4-day experiment. (B) Biofilms formed by mixed cultures after 4 days are shown for the following mixes: A34/pRU1319 (green) and A412(prsD)/pMP4518 (red), A1077(pssA)/pMP4518 (red) and A412(prsD)/pRU1319 (green), and A550 (pssC-V)/pRU1319 (green) and A34/pMP4518 (red). The panels show horizontal projected images (x-y axis), and zoom images (insets) illustrate tight interactions between dual-color bacteria inside the cluster.

FIG. 7.

Analysis of extracellular proteins from the prsD mutant and the wild type. Cultures of A34 (WT) or A412 (prsD) were grown for 24 h to an OD₆₀₀ of 0.6, and proteins from the culture supernatants were precipitated and analyzed by SDS-12% PAGE. The proteins were stained with Coomassie brilliant blue R-250 (A) or silver (B). The arrows indicate extracellular proteins that were absent in the secretion mutant A412. The numbers indicate the molecular weights of protein standards (st).