Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter

Abstract

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding.

Fig. 1.

Genetic organization of the choXWV locus. The cho genes are flanked by two ORFs: atu2282, encoding a putative thymidine kinase, and atu2278, a NolR-like regulator. choX encodes a periplasmic choline-binding protein, choW encodes a permease, and choV encodes an ATPase. The positions of the gentamicin (Gm) cassette insertion and corresponding mutants (choX::Gm> and choX::Gm<) are indicated. The upper part shows the constructed lacZ fusions.

Fig. 2.

β-Galactosidase activities of plasmid-encoded transcriptional choX-lacZ, choW-lacZ, and atu2278-lacZ fusions in A. tumefaciens C58. Cells were grown in AB minimal medium in the absence or presence of 1 mM choline at 30°C. The plasmid pAC01 containing the promoterless lacZ gene was used as a negative control. Values are the mean of three independent experiments ± standard deviations.

Fig. 3.

PC formation in A. tumefaciens wild-type (wt) and mutant strains. Cells were grown in M9 minimal medium in the absence (−) or presence (+) of 1 mM choline for 24 h. Lipids were extracted, separated by one-dimensional TLC, and visualized by Molybdenum Blue staining. PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin; MMPE, monomethyl-PE; DMPE, dimethyl-PE; PC, phosphatidylcholine.

Fig. 4.

[¹⁴C]choline uptake analyses of the A. tumefaciens wild type (wt) and choX mutants. (A) Initial choline uptake of the A. tumefaciens wild type and choX mutants with a final choline concentration of 0.1 mM [methyl-¹⁴C]choline chloride (55 mCi/mmol) at the time indicated. The points shown are the mean values of three individual experiments. (B) Complementation of the choX mutants by plasmid (pVSBAD)-encoded choX expression. Uptake was assayed over 10 min with a final [¹⁴C]choline concentration of 5 μM. Total [¹⁴C]choline uptake of the A. tumefaciens wild type and choX mutants (C) and the Δpcs choX::[Gm<] double mutant (D) was assayed using a final choline concentration of 10 μM [¹⁴C]choline. The accumulation of radioactivity was quantified after a 24-h incubation. The values shown are the means of two individual experiments.

Fig. 5.

Purification and in vitro choline-binding properties of recombinant ChoX. (A) Size exclusion chromatography of Ni-IDA-purified PmtA. The peak fractions 1, 2, and 3 were analyzed by SDS-PAGE as depicted on the right. AU, absorbance units. Lane M (inset), BenchMark protein standard (Invitrogen). (B) Radioactive choline-binding activity of recombinant ChoX. Choline-binding activity was analyzed with 5 μM ChoX and 5 μM [¹⁴C]choline over 5 min. The displacement assay (+unlabeled choline excess) contained, in addition, 10-fold unlabeled choline. (C) Competitive displacement of radioactively labeled choline by unlabeled choline (0 to 50 μM). The assay mixture contained 5 μM recombinant ChoX and 5 μM [¹⁴C]choline. (D) Plots of choline-binding data for wild-type ChoX. A reaction volume of 50 μl contained 5 μM recombinant ChoX and 0 to 50 μM [¹⁴C]choline. Changes in bound [¹⁴C]choline (y axis) were plotted against choline concentration (x axis). All data sets were fitted to the equation for one-site binding (see Materials and Methods) by nonlinear regression using SigmaPlot, version 9.0. The points shown are the mean values of three individual experiments.

Fig. 6.

Fluorescence-based ligand-binding analysis of ChoX. (A) Emission spectra of ChoX (10 μM) in the absence or presence of 100 μM choline. au, arbitrary units. (B) Binding affinities (K_Ds) of ChoX for choline, acetylcholine, and betaine were determined via intrinsic tryptophan quenching after addition of the ligands (0 to 100 μM) to ChoX protein (10 μM). Changes in the emission maximum (Δλ in nm) were plotted against the ligand concentration (data not shown), and the Michaelis-Menten equation was used for calculation of ligand-binding affinities. The data were fitted by nonlinear regression using SigmaPlot, version 9.0. Values are the means of three independent experiments ± standard deviations.

Fig. 7.

Homology model of A. tumefaciens ChoX. (A) ChoX model was generated using the online server I-TASSER (41) and visualized by PyMOL (http://www.pymol.org). The fold is characterized by a bilobal organization into two lobes connected via a linker region. The proposed ligand-binding site is located within the two lobes. Proposed choline-binding residues are shown in red, and two aromatic residues (W283 and W294) outside the binding pocket, depicted as negative controls, are shown in blue. (B) Detailed view of the ligand-binding site of A. tumefaciens ChoX. Proposed residues participating in ligand binding and the ligand choline are shown in stick and in ball-and-stick representation, respectively.

Fig. 8.

In vitro choline-binding properties of point-mutated ChoX proteins. (A) [¹⁴C]choline binding was assayed with 5 μM ChoX and 5 μM [¹⁴C]choline. (B) Choline affinity constants (K_Ds) of ChoX variants. To calculate affinity constants, 5 μM ChoX protein was titrated with 0 to 50 μM [¹⁴C]choline, and bound [¹⁴C]choline (in disintegrations per minute) was plotted against the [¹⁴C]choline concentration (data not shown). The Michaelis-Menten equation was used for calculation of ligand-binding affinities. The data were fitted by nonlinear regression using SigmaPlot, version 9.0. Values are the means of three independent experiments ± standard deviations. (C) Circular dichroism analysis of the wild type and inactive ChoX variants. The spectrum of each protein with a concentration of 10 μM is plotted as ellipticity in millidegrees (mdeg) versus wavelength.