Environmental pH sensing: resolving the VirA/VirG two-component system inputs for Agrobacterium pathogenesis

Abstract

Agrobacterium tumefaciens stands as one of biotechnology's greatest successes, with all plant genetic engineering building on the strategies of this pathogen. By integrating responses to external pHs, phenols, and monosaccharides, this organism mobilizes oncogenic elements to efficiently transform most dicotyledonous plants. We now show that the complex signaling network used to regulate lateral gene transfer can be resolved as individual signaling modules. While pH and sugar perception are coupled through a common pathway, requiring both low pH and sugar for maximal virulence gene expression, various VirA and ChvE alleles can decouple pH and monosaccharide perception. This VirA and ChvE system may represent a common mechanism that underpins external pH perception in prokaryotes, and the use of these simple genetic elements may now be extended to research on specific responses to changes in environmental pH.

FIG. 1.

Protein expression of VirA truncation mutants. (A) Schematic representation of VirA constructs. The plasmids expressing VirA constructs are listed on the left; in parentheses are the corresponding plasmids of VirA constructs with a G665D mutation in the kinase domain. Open arrow, virA promoter; solid arrow, P_N25 promoter; white box, His₆ tag. (B) Western blot analysis of VirA constructs. Clarified lysates from bacteria carrying indicated plasmids were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% Bis-Tris NuPage (Invitrogen) gels followed by Western blot analysis using anti-RGS-His monoclonal antibody.

FIG. 2.

pH responses of VirA constructs. A. tumefaciens A136 carrying the indicated VirA constructs together with pRG109 containing P_virB-lacZ and P_N25-virG was cultured in induction medium supplemented with 14 mM glucose and assayed for β-galactosidase activity. (A) Expression of P_virB-lacZ at pH 5.5 with 0 μM AS, pH 5.5 with 200 μM AS, pH 7.0 with 0 μM AS, and pH 7.0 with 200 μM AS. (B) Ratio of the β-galactosidase activity, pH 5.5/pH 7.0.

FIG. 3.

pH response of VirA(G665D) constructs. A. tumefaciens A136 carrying the indicated VirA constructs and pRG109 was cultured as described above in the presence of 14 mM glucose. (A) Expression of P_virB-lacZ at pH 5.5 with 0 μM AS, pH 5.5 with 200 μM AS, pH 7.0 with 0 μM AS, and pH 7.0 with 200 μM AS. (B) Ratio of the β-galactosidase activity at pH 5.5 to that at pH 7.0 with 0 and 200 μM AS.

FIG. 4.

P_virB-lacZ expression by wild-type VirA as a function of AS concentrations. A. tumefaciens A136/pRG109 containing pVRA8 was cultured for 16 h in media supplemented with 1% glycerol at pH 5.5 (Δ), pH 5.5 with 14 mM glucose (▴), pH 7.0 (□), and pH 7.0 with 14 mM glucose (▪). β-Galactosidase activity calculated as Miller units (A) and expressed as percentages of the maximal activity (B).

FIG. 5.

P_virB-lacZ expression by wild-type VirA as a function of AS concentrations in a background in which chvE was disrupted. A. tumefaciens DL8/pRG109 containing pVRA8 (A and B) or pRG168 (C and D) was cultured as described at pH 5.5 (Δ), pH 5.5 with 14 mM glucose (▴), pH 7.0 (□), and pH 7.0 with 14 mM glucose (▪). β-Galactosidase activity calculated as Miller units (A and C) and expressed as percentages of the maximal activity (B and D).

FIG. 6.

pH and sugar responses of VirA and ChvE allele products. P_virB-lacZ expression by DL8/pRG109 containing pRG169 with wild-type VirA and P_N25-chvE(T211M) at pH 5.5 (Δ), pH 5.5 with 14 mM glucose (▴), pH 7.0 ([squo]), and pH 7.0 with 14 mM glucose (▪), in terms of β-galactosidase activity calculated as Miller units (A) and expressed as percentages of the maximal activity (B). (C) P_virB-lacZ expression by wild-type VirA and VirA with aa 242 through 257 deleted (Δ242-257) with 200 μM AS.

FIG. 7.

Simulated structure of ChvE. The structural data from GBP and RBP were used as templates to generate the coordinates for ChvE by use of Swiss-Prot. The resulting structure of ChvE contains two domains joined by three hinges with the sugar binding site located within the domain cleft. Based on the RBP and GBP models, the indicated side chains are involved in protein-protein interactions.