Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis

Abstract

Bacterial responses to phosphorus limitation, commonly inorganic phosphate (P(i)), are important survival mechanisms in a variety of environments. The two-component sensor kinase PhoR and its cognate response regulator PhoB are central to the P(i) limitation response of many bacteria and control the large Pho regulon. Limitation for P(i) significantly increased attachment and biofilm formation by the plant pathogen Agrobacterium tumefaciens, and this was driven by PhoB. Surprisingly, it was also found that both phoR and phoB were essential in A. tumefaciens. Expression of a plasmid-borne copy of the low affinity P(i) transporter (pit) from Sinorhizobium meliloti in A. tumefaciens abolished the phoB and phoR essentiality in A. tumefaciens and allowed direct demonstration of the requirement for this regulatory system in the biofilm response. Increased attachment under P(i) limitation required a unipolar polysaccharide (UPP) adhesin. Mutation of a polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) called uppE abolished UPP production and prevented surface attachment under P(i)-replete conditions, but this was rescued under P(i) limitation, and this rescue required phoB. In low P(i) conditions, either uppE or a paralogous gene Atu0102 is functionally redundant, but only uppE functions in UPP synthesis and attachment when P(i) is replete. This conditional functional redundancy illustrates the influence of phosphorus availability on A. tumefaciens surface colonization.

Fig. 1. Biofilm formation in flow cells is stimulated by phoB expression under P_i-replete conditions, and P_i-limitation-enhanced biofilm formation requires phoR

(A) SDCM images of A. tumefaciens TD5 (phoB::pTD105; pTD115, P_lac-traR, P_traI-phoB) mutant derivatives expressing GFP and cultivated in once-through flow cells for 96 h in ATMN medium. Side and bottom panels are orthogonal views of the biofilms. (B) Static biofilm assays of A. tumefaciens WT and JW6 (P_lac-pit_Sm, phoR::Ω Km) with 72 h cultures in ATGN medium (79 mM P_i) and P_i-limited medium (50 μM). The A₆₀₀ of solubilized CV stain from adherent biomass was normalized by planktonic growth (OD₆₀₀) of the culture. Values are averages of triplicate assays and error bars are standard deviation.

Fig. 2. A. tumefaciens C58 and S. meliloti orfA-pit loci and gene products

Diagram shows the genetic context of the orfA-pit genes in A. tumefaciens C58 and S. meliloti 1021. Pit and Pit* gene products are also shown, with gray ovals indicating transmembrane domains.

Fig. 3. Biofilm formation and the P_i limitation effect require the UPP but not other polysaccharides

(A) CV-stained coverslip biofilms (48 h) of A. tumefaciens polysaccharide mutant derivatives grown in ATGN (high P_i, 79 mM) and P_i-limiting medium (low P_i, 50 μM). (B) Measurement of A₆₀₀/OD₆₀₀ values for acetic-acid-solubilized CV-stained 72-h biofilms under high and low P_i. Values are averages of triplicate assays and error bars are standard deviation. (C) Lectin labeling of A. tumefaciens C58 in short-term binding assays on PVC coverslip inoculated from planktonic cultures grown in ATGN and P_i-limiting medium. Cells were mixed with fl-DBA just prior to inoculation and, after 1 h, viewed at 100X magnification on a Nikon E800 epifluorescence microscope (excitation, 460-500 nm; emission, 510-560 nm) with an overlay of phase contrast and fluorescence images.

Fig. 4. Paralogues of UppE- and PhoB-dependent functional redundancy under P_i limitation

(A) CV-stained coverslip biofilms (72 h) of A. tumefaciens mutant derivatives grown in ATGN (high P_i, 79 mM) and P_i-limiting medium (low P_i, 50 μM). (B) Measurement of A₆₀₀/OD₆₀₀ values for acetic-acid-solubilized CV-stained 72 h biofilms under high and low P_i. Values are averages of triplicate assays and error bars are standard deviation. (C) Diagram of domain structure for UppE and Atu0102 and ExoY paralogues. Gray ovals indicate transmembrane domains, the large dark gray bar is the WbaP domain and the white rectangles indicate the PHPT I, II and III motifs. (D) Neighbor-joining tree of UppE paralogues. The optimal tree with the sum of branch length = 1.22142857 is shown. The tree is drawn to scale, with branch lengths reflecting evolutionary distances. The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) and are in the units of the number of amino acid differences per site. All positions containing gaps and missing data were eliminated, and a total of 210 positions were in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).

Fig. 5. Lectin labeling of UPP in the phoB mutant and PHPT paralogue mutants under high and low P_i

Short-term binding assays were performed with A. tumefaciens derivatives grown in high P_i (79 mM) and low P_i (50 μM) cultures and incubated in suspension with PVC coverslips after 2 h incubation with af-WGA at room temperature. After washing, coverslips were viewed at 100X magnification on a Nikon E800 epifluorescence microscope (excitation, 510-560 nm; emission, >610 nm) with an overlay of phase contrast and fluorescence images.