Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens

Abstract

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.

FIG. 1.

P_virB-lacZ expression (A) and in vivo phosphorylation (B) of VirG mutants. A. tumefaciens A136 strains carrying pYW48 (wild type [WT]), pAM13 (N54D), or pRG112b (I77V/D52E) together with a P_virB-lacZ reporter plasmid were assayed in parallel with in vivo phosphorylation. Aliquots (1 ml) of phosphate-starved cultures were taken out, supplemented with phosphates, and incubated for 10 h prior to the β-galactosidase assay. The rest of the phosphate-starved cultures were used for in vivo phosphorylation. The phosphorimaging and Western blotting detected by anti-His are shown in the upper and lower lanes of panel B, respectively. Arrows mark the positions of VirG proteins.

FIG. 2.

P_virB-lacZ expression by VirG alleles with an additional D52E or D9A mutation. β-Galactosidase activity was assayed for A136(pRG129) strains containing a plasmid-borne virA and the following plasmids: (from left to right) pRG80, pRG112b, pYW47, pAM18, pAM19, pA1, pB1, and pC1. WT, wild type.

FIG. 3.

Role of the conserved phosphorylation center in VirG phosphorylation. In vivo phosphorylation patterns of VirG alleles are shown in the upper panels, while protein expression profiles are shown in the lower panels. (A) Phosphorylation of VirGs with the D52E mutation. A136(pSW209Ω) strains carrying the following plasmids were used: lanes 1 and 2, pYW48; lane 3, pAM13; lane 4, pAM19; lane 5, pAM21; lane 6, pAM20 (lanes 1 and 2 were from Fig. 1 for comparative purposes). (B) Phosphorylation of VirGs with the D9A mutation. A136(pRG129) strains containing a plasmid-borne virA and the indicated plasmids were labeled with H₃³²PO₄ in the presence of 200 μM AS. Lane 1, pC1; lane 2, pA1; lane 3, pYW48; lane 4, pAM13. (C) Chemical stability of the phosphate on VirGN54D. Three identical membranes from ³²P-labeled A136(pRG129, pRG80) samples were incubated with Tris-buffered saline at pH 7.0 (Neutral), 1 M HCl (Acid), or 3 M NaOH (Base) for 2 h at room temperature prior to the phosphorimaging and Western blotting. WT, wild type.

FIG. 4.

P_virB-lacZ expression by VirG alleles in E. coli strains with mutations in the acetyl phosphate synthesis pathway. (A) Diagram of the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway. acCoA, acetyl-CoA; acP, acetyl phosphate. (B) P_virB-lacZ expression in E. coli strains carrying pRG145/pFQ95 (VirGN54D) or pRG146/pRG149 (VirGI77V/D52E).

FIG. 5.

Gel retardation assay of the binding of virB promoter with (A) VirG, (B) VirGN54D, and (C) VirGI77V/D52E. VirG proteins were added to a final concentration of 0 μM (lane 1), 0.1 μM (lane 2), 0.2 μM (lane 3), 0.6 μM (lane 4), 2 μM (lane 5), and 6 μM (lane 6).

FIG. 6.

Model structures of VirG. The structures of VirG before and after the phosphorylation were modeled by SWISS-PROT as described in Material and Methods. Residues involved in the conserved Asp-Ser/Thr-Tyr/Phe (D52-S79-F99) “aromatic switch” (3, 15, 22) were highlighted together with N54 and I77: white, C; red, O; blue, N; orange, P. The N54 residue is shown as sticks for a clear view of the phosphorylation site.

FIG. 7.

P_virB-lacZ expression of VirG mutants. β-Galactosidase activity was assayed for A136(pRG129) strains carrying the following plasmids: (from left to right) pRG111, pRG112b, pB9, pB7, and pB8.