Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system

Abstract

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells. Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected. Functions encoded on the two large plasmids of A. tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect. The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB. The A. tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential. Expression of the A. tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A. tumefaciens surface interactions.

FIG. 1.

Phosphate concentration influences A. tumefaciens C58 adhesion in static culture. (A) Light micrographs (×100 objective) of A. tumefaciens C58 cells adhering to PVC coverslips and grown for 48 h in 50 or 500 μM phosphate, as indicated. (B) CV staining of adherent A. tumefaciens biofilms on PVC coverslips grown for 48 h in 150 or 500 μM, as indicated. (C) Different cultures of A. tumefaciens C58 grown over a range of phosphate concentrations for 48 h with monitoring of the OD₆₂₀ of planktonic-phase cultures (filled circles), the AP activity of planktonic cells (open circles), the A₆₂₀ values of DMSO-solubilized CV from adherent cells (squares), and the CV A₆₂₀/OD₆₂₀ ratio (triangles).

FIG. 2.

Flow cell biofilms in low and high phosphate concentrations. CSLM images from 62-h A. tumefaciens C58 (pJZ383, P_tac::gfpmut3) biofilms grown on PVC at 50 and 500 μM phosphate. The right side and bottom of each panel are reconstructed vertical cross sections of the biofilm. Images were obtained with a Zeiss LSM 510 microscope and processed with Imaris 3.3 software. See Table 2 for COMSTAT analysis of the biofilms.

FIG. 3.

Mutagenesis constructs for phoR and phoB. (A) Allelic replacement mutagenesis of A. tumefaciens phoR with derivative pTD102 to generate strains TD1 (single recombinant) and TD2 (double recombinant). (B and C) Campbell integration mutagenesis of phoB with pTD104 to retain a functional copy of phoB in TD3 (B) and pTD105 to disrupt phoB in TD5 (C). Single quotes indicate truncation of phoB at either the 5′ end (‘phoB), the 3′ end (phoB'), or both ends (‘phoB').

FIG. 4.

AAI-controlled expression vector system. (A) Schematic representation of pTD115 organization and derivation of pTD115 (P_traI::phoB) and pTD116 (P_traI::lacZ-Km) indicating unique restriction sites (N, NheI; S, SmaI/XmaI; X, XbaI; B, BamHI). (B) β-Galactosidase dose-response curve of A. tumefaciens harboring pTD116 (P_traI::lacZ-Km) as a function of the crude AAI preparation in the medium.

FIG. 5.

Controlled expression of phoB influences biofilm formation at high and low phosphate concentrations. Dose-response curves of A. tumefaciens TD5 (phoB::pTD105) harboring pTD115 (P_traI::phoB) over a range of AAI concentrations after 48 h of growth at 50 (A) or 500 (B) μM phosphate are shown. In panels A and B, the OD₆₀₀ of planktonic cells (filled circles), the A₆₀₀ values of DMSO-solubilized CV from adherent cells (filled squares), and the CV A₆₀₀/OD₆₀₀ ratio (filled triangles) are shown. Dashed lines with open symbols correspond to filled symbols but are for A. tumefaciens NTL4 (wild-type phoB) without pTD115. (C) AP activities in response to AAI dose at 50 (inverted triangles) and 500 (upright triangles) μM phosphate in either A. tumefaciens TD5(pTD115) (filled symbols) or A. tumefaciens NTL4 without pTD115 (open symbols).