Abstract

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid-binding mode through three crystal structures at 1.4, 1.74 and 1.9 Å resolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Å resolution defines a different agropinic acid-binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.

Introduction

Agrobacteria are telluric and rhizosphere bacteria commonly isolated from roots of numerous plants as commensal bacteria. They can also be pathogenic when they harbor a tumor-inducing (Ti) plasmid [1,2]. Pathogenic agrobacteria create their own ecological niche (called tumor niche or opine niche) after plant cell transformation that leads to tumor formation in a wide range of plants [1]. Indeed, agrobacteria mediate the transfer of a portion of the pTi, namely the T-DNA, to the plant cell [3,4]. Once incorporated into the plant nuclear DNA, T-DNA gene expression drives the synthesis of (i) plant hormones leading to the tumor formation (crown-gall disease) and (ii) novel organic compounds called opines. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars [2,5,6]. They are specifically utilized as growth substrates by the inciting agrobacteria and confer upon these pathogens an advantage when in competition with other members of the soil microflora for the colonization of opine-rich environments [712]. Some opines, designated conjugative opines, such as octopine or agrocinopine (actually only the arabinose-2-phosphate moiety of the agrocinopine is recognized) [13], stimulate the horizontal transfer (conjugative transfer) of the Ti plasmid from pathogenic Agrobacterium to nonpathogenic Agrobacterium [6].

In Agrobacterium-induced plant tumors, over 20 opines are known. They fall into distinct families according to their precursors, which are sugars, amino acids and ketoacids. They are not all present at the same time in a tumor and some opines are specific for a given Ti plasmid/bacteria strains. For example, A. tumefaciens strains B6 and R10 possess Ti plasmid genes that code for enzymes responsible for the synthesis of the mannityl-opines family composed of four compounds containing mannose conjugated with either glutamate or glutamine. Mannopine (1-deoxymannosyl-glutamine) and mannopinic acid are unmodified conjugates, while agropine (1-deoxy-mannitosyl-glutamine,1′,2′-lactone) and agropinic acid are cyclized derivations of mannopine [14] (Figure 1). The ags gene product catalyzes the lactonization of mannopine to agropine [15,16] (Figure 1). Mannopine is formed from a precursor which is also an opine, but from the chrysopine family, named 1-deoxy-fructosyl-glutamine (DFG) [17]. Notably, mannopine, agropine and mannopinic acid can degrade into agropinic acid (mannityl-glutamic acid lactam) through a spontaneous lactamization [6,14].

Transport of agropine (gray and green) and agropinic acid (dark orange and green) from transformed plant cells to A. tumefaciens octopine/mannityl-type R10 strain.

Figure 1.
Transport of agropine (gray and green) and agropinic acid (dark orange and green) from transformed plant cells to A. tumefaciens octopine/mannityl-type R10 strain.

In plant tumor cells, the ags gene responsible for the biosynthesis of agropine from mannopine is located on the T-DNA transferred from the A. tumefaciens R10 Ti plasmid. All the genes allowing the synthesis of opines are located on the T-DNA incorporated into the plant genome. Agropinic acid results from the spontaneous lactamization of three mannityl opines: agropine, mannopine and mannopinic acid. In A. tumefaciens R10, the (agtABC) and (agaA-D) genes located on the Ti plasmid, proposed as coding for agropine and agropinic acid import, respectively, are shown in blue and magenta.

Figure 1.
Transport of agropine (gray and green) and agropinic acid (dark orange and green) from transformed plant cells to A. tumefaciens octopine/mannityl-type R10 strain.

In plant tumor cells, the ags gene responsible for the biosynthesis of agropine from mannopine is located on the T-DNA transferred from the A. tumefaciens R10 Ti plasmid. All the genes allowing the synthesis of opines are located on the T-DNA incorporated into the plant genome. Agropinic acid results from the spontaneous lactamization of three mannityl opines: agropine, mannopine and mannopinic acid. In A. tumefaciens R10, the (agtABC) and (agaA-D) genes located on the Ti plasmid, proposed as coding for agropine and agropinic acid import, respectively, are shown in blue and magenta.

Opine catabolic genes (import and degradation) are located in segments outside the T-DNA region on the pTi. They are generally clustered in operons and regulons, and their expression is inducible by the opine itself or a catabolized/degraded form of it [6,13,1820]. Two sets of genes are present in the catabolic region. The first one encodes the transport system that often consists of an ATP-binding cassette (ABC) transporter and its cognate periplasmic-binding protein (PBP). The second one encodes the enzymes involved in the degradation of the opines to molecules that belong to central bacterial metabolism. To date, we have characterized several opine-binding PBPs such as NocT (nopaline opine family import), AccA (agrocinopine opine family import), MotA (mannopine opine import) and OccJ (octopine opine family import) [12,13,21,22]. However, structural data on agropine and agropinic acid–opine–PBP complexes are still lacking.

In the present study, we investigated the structural and biochemical properties of two PBPs: AgtB, which stands for Agropine transport (Agt) protein B, and AgaA, which stands for agropinic acid (Aga) protein A. AgtB and AgaA have been predicted as an agropine-binding protein and an agropinic acid-binding protein, respectively. This prediction results from genetic investigations (mapping of gene regions and defective mutants), opine uptake/transport experiments [2327] and annotation of the agrobacterial genome (Figure 1). We first focused on the genetic and molecular role of the PBP AgtB through an integrative approach using a defective mutant in cellulo, crystallography and affinity measurements. We showed that AgtB was the PBP responsible for agropine import into A. tumefaciens R10. Nonetheless, AgtB can also bind agropinic acid with an affinity higher than that observed for agropine. We structurally characterized the binding mode of AgtB for agropinic acid in three different crystal forms at 1.4, 1.74 and 1.9 Å resolution. All attempts to obtain a structure of AgtB in complex with agropine failed mainly due to agropine instability. Nevertheless, a model of a bound agropine in AgtB was easily built based on the bound agropinic acid structure. In contrast, we demonstrated that the PBP AgaA is specific for agropinic acid and solved a liganded structure at 1.65 Å resolution. We identified two agropinic acid-binding signatures (a selective and a non-selective) allowing both PBPs to bind agropinic acid with a high affinity in the nanomolar range. This work provides new insights into the utilization of agropine and agropinic acid opines in host-interacting bacteria.

Experimental

Synthesis and purification of opines

Agropine and agropinic acid were synthesized from mannopine as previously described [14]. Because agropine can spontaneously lactamize into agropinic acid, we purified agropine stocks on a Waters 2695 Alliance HPLC System equipped with a Waters 2420 ELSD and a Waters 996 HPLC Photodiode Array Detector. Agropine was injected on a Waters Atlantis Hilic silica column washed with 95% of 10 mM ammonium formate, pH 3 (A) and 5% acetonitrile (B) during 5 min, and eluted by gradient until a ratio A : B of 75 : 25 at a 4 ml/min flow. Acetonitrile was then evaporated and residual agropine was frozen into liquid nitrogen. Solutions of agropine and agropinic acid were checked by mass spectrometry (Supplementary Figure S3). The used mannopine was the same as in Marty et al. [21], and mannopinic acid was a laboratory product also checked by mass spectrometry.

Construction of the non-polar AgtB-defective mutant in A. tumefaciens R10

The octopine–mannityl-type R10ΔagtB-defective mutant was constructed according to a strategy recently described [28]. Briefly, approximately 1000 pb of the recombinant region, containing the upstream and downstream region flanking the agtB gene, was generated by PCR using primers listed in Supplementary Table S1. These fragments were designed to delete the coding sequence of agtB without affecting neighboring genes. The recombinant fragment was inserted into a SmaI digested suicide vector (pJQ200sk [29]) by infusion technology (Takara Clonetech, Kyoto, Japan). The resulting pJQ200sk-ΔagtB plasmid was introduced into A. tumefaciens R10 by electroporation. Colonies with plasmid integration were selected on YPG medium plates supplemented with gentamicin (20 µg/ml) and by PCR with primers UpagtBF-DwagtBR. Double-crossover events were searched by culturing gentamicin-resistant colonies into YPG medium without antibiotic overnight before plating to YPG plates containing 5% of sucrose. The loss of the suicide vector and the deletion of agtB in the sucrose-resistant colonies were verified by PCR (primers UpagtBF-DwagtBR) and sequencing (GenoScreen, Lille, France).

Bacterial culture conditions

The octopine–mannityl-type A. tumefaciens R10 and R10ΔagtB strains were cultivated overnight at 28°C in AB media supplemented with mannitol (2 g/l). After washing the pellet with 0.8% NaCl, strains were inoculated in AB minimal medium supplemented with 2 mM of carbon/nitrogen sources (agropine or agropinic acid) or in the absence of any carbon/nitrogen source, in 24-well sterile plates incubated at 28°C during 4 days. Growth of cells was measured every 8 h by optical density at 600 nm. Analyses were performed in three replicates.

Cloning, expression and purification of mature AgtB and AgaA

The synthetic genes (Genscript) coding for the mature AgtB and AgaA were inserted into pET-28b and pET-29b, respectively. Escherichia coli BL21 pLysS was transformed by pET-28b-agtB and E. coli Rosetta pLysS by pET-29b-agaA. Cells were grown at 37°C in LB broth supplemented with 0.5 mM isopropyl β-d-1-thiogalactopyranoside to induce protein production. Cells were centrifuged, resuspended in a buffer of 50 mM Tris–HCl, pH 8.0, 20 mM imidazole and 500 mM NaCl, and disrupted by sonication. After centrifugation at 25 000 g for 30 min at 4°C, the supernatant was loaded onto a 5 ml His-Trap column (GE Healthcare). Protein elution was performed with 50 mM Tris–HCl pH 8.0, 300 mM imidazole and 500 mM NaCl. Protein fractions were loaded onto a gel filtration column (HiLoad 26/60 Superdex 200 prep grade, GE Healthcare) equilibrated with 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl. The protein fractions were pooled, concentrated at 30 mg/ml and 15 mg/ml for AgtB and AgaA, respectively, and stored at −80°C.

Crystallization and structure determination

Crystallization conditions (Table 1) for liganded AgtB and AgaA (molar ratio of protein : ligand used was 1 : 5) were screened using QIAGEN kits (Valencia, CA) with a Cartesian nanodrop robot (Genomic solutions) and were manually optimized. Crystals were transferred to a cryo-protectant solution (mother liquor supplemented with 25% PEG 400 or/and 25% glycerol) and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K on the PROXIMA 1 and 2 beamlines equipped with a PILATUS 6M and an EIGER 9M as a detector, respectively, at SOLEIL synchrotron (Saint-Aubin, France). Data processing was performed using the XDS package [30] (Table 1). Structure determinations were performed by molecular replacement with PHASER [31]. The coordinates of the N- and C-terminal domains of the PBP MotA (PDB: 5L9G) [21] were used as search models for the liganded AgtB structures. For the AgaA–agropinic acid structure, the coordinates of the N-terminal and the C-terminal domains of an AgaA partial model built using the server @TOME-2 [32], which performs fold-recognition, were used. The partial AgaA model from the best matching template based on the structure of the E. coli putative-binding protein Ylib (PDB: 1UQW) was built using SCWRL 3.0 [33]. Refinement of each structure was performed with BUSTER-2.10 [34] and TLS group. NCS restraints were applied when more than one molecule was present in the asymmetric unit. Inspection of the density maps and manual rebuilding was performed using COOT [35]. The three-dimensional models of agropinic acid and agropine were generated with the ProDRG webserver [36]. Refinement details of each structure are shown in Table 1. Molecular graphics images were generated using PyMOL (http://www.pymol.org).

Table 1
Crystallographic data and refinement parameters

Values for the highest resolution shell are in parentheses.

Protein AgtB–agropinic acid AgtB–agropinic acid AgtB–agropinic acid AgaA–agropinic acid 
Crystallization conditions 15% PEG 4K, 0.1 M Tris (pH 8.5), 0.2 M sodium acetate 30% PEG 8K, 0.1 M Tris (pH 8.5) 28% PEG 8K, 0.1 M Tris (pH 8.5), 0.2 M CaCl2 1.8 M AS, 0.1 M Mes (pH 6.5), 0.2 M Zn acetate 
PDB code 6HLY 6HLZ 6HM2 6HLX 
Space group P212121 CPP6522 
Cell parameters (Å,°) a = 40.8
b = 108.8
c = 64.1 
a = 170.8
b = 40.9
c = 117.4
β = 115.1 
a = 40.6
b = 86.3
c = 106.5
α = 71.1
β = 81
γ = 76.3 
a = 89.3
b = 89.3
c = 285.8 
Number of molecules in the au 
Resolution (Å) 50–1.4 (1.48–1.4) 50–1.9 (2–1.9) 50–1.74 (1.85–1.74) 50–1.65 (1.74–1.65) 
No. of observed reflections 294 363 (35 081) 316 647 (46 781) 451 101 (64 995) 2 274 669 (341 154) 
No. of unique reflections 57 167 (8745) 58 982 (8971) 129 664 (19 223) 82 437 (12 777) 
Rsym (%)1 5 (95.1) 14.9 (93.3) 11.6 (89.7) 13.2 (205.2) 
Rpim (%) 3.4 (90.1) 10.5 (70.4) 10.2 (73.8) 3.8 (76.7) 
Completeness (%) 99.2 (95.7) 98.8 (93.9) 95.6 (87.3) 99.6 (97.6) 
I/σ 15.3 (1.1) 7 (1.2) 7.58 (1.3) 19.2 (1.73) 
CC1/2 99.9 (48.9) 99.2 (68.8) 99.4 (52.8) 99.9 (60.5) 
Rcryst (%)2 18.2 (24.8) 20.5 (29.2) 17.3 (24.6) 15.6 (25.1) 
Rfree (%)3 21.3 (23.6) 25.4 (32.4) 20.1 (24.7) 17.4 (27.7) 
No. of atoms 
 Protein 2488 4858 12 075 3865 
 Ligand 20 40 80 20 
 Water 291 620 999 470 
 r.m.s. bond deviation (Å) 0.01 0.01 0.01 0.01 
 r.m.s. angle deviation (°) 0.98 0.97 
Average B2
 Protein 26.9 34.7 37.0 27.6 
 Ligand 15.5 29.3 21.8 20 
 Water 38.1 41.9 31.9 39.3 
 Clashscore4 0.99 2.09 1.7 2.63 
 MolProbity score4 0.94 1.21 1.05 1.11 
Ramachandran plot (%) 
 Favored 98 98 98 99 
 Allowed 
 Outliers 
Protein AgtB–agropinic acid AgtB–agropinic acid AgtB–agropinic acid AgaA–agropinic acid 
Crystallization conditions 15% PEG 4K, 0.1 M Tris (pH 8.5), 0.2 M sodium acetate 30% PEG 8K, 0.1 M Tris (pH 8.5) 28% PEG 8K, 0.1 M Tris (pH 8.5), 0.2 M CaCl2 1.8 M AS, 0.1 M Mes (pH 6.5), 0.2 M Zn acetate 
PDB code 6HLY 6HLZ 6HM2 6HLX 
Space group P212121 CPP6522 
Cell parameters (Å,°) a = 40.8
b = 108.8
c = 64.1 
a = 170.8
b = 40.9
c = 117.4
β = 115.1 
a = 40.6
b = 86.3
c = 106.5
α = 71.1
β = 81
γ = 76.3 
a = 89.3
b = 89.3
c = 285.8 
Number of molecules in the au 
Resolution (Å) 50–1.4 (1.48–1.4) 50–1.9 (2–1.9) 50–1.74 (1.85–1.74) 50–1.65 (1.74–1.65) 
No. of observed reflections 294 363 (35 081) 316 647 (46 781) 451 101 (64 995) 2 274 669 (341 154) 
No. of unique reflections 57 167 (8745) 58 982 (8971) 129 664 (19 223) 82 437 (12 777) 
Rsym (%)1 5 (95.1) 14.9 (93.3) 11.6 (89.7) 13.2 (205.2) 
Rpim (%) 3.4 (90.1) 10.5 (70.4) 10.2 (73.8) 3.8 (76.7) 
Completeness (%) 99.2 (95.7) 98.8 (93.9) 95.6 (87.3) 99.6 (97.6) 
I/σ 15.3 (1.1) 7 (1.2) 7.58 (1.3) 19.2 (1.73) 
CC1/2 99.9 (48.9) 99.2 (68.8) 99.4 (52.8) 99.9 (60.5) 
Rcryst (%)2 18.2 (24.8) 20.5 (29.2) 17.3 (24.6) 15.6 (25.1) 
Rfree (%)3 21.3 (23.6) 25.4 (32.4) 20.1 (24.7) 17.4 (27.7) 
No. of atoms 
 Protein 2488 4858 12 075 3865 
 Ligand 20 40 80 20 
 Water 291 620 999 470 
 r.m.s. bond deviation (Å) 0.01 0.01 0.01 0.01 
 r.m.s. angle deviation (°) 0.98 0.97 
Average B2
 Protein 26.9 34.7 37.0 27.6 
 Ligand 15.5 29.3 21.8 20 
 Water 38.1 41.9 31.9 39.3 
 Clashscore4 0.99 2.09 1.7 2.63 
 MolProbity score4 0.94 1.21 1.05 1.11 
Ramachandran plot (%) 
 Favored 98 98 98 99 
 Allowed 
 Outliers 
1

Rsym = Σhkl Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣIi(hkl), where Ii(hkl) is the ith observed amplitude of reflection hkl and 〈I(hkl)〉 is the mean amplitude for all observations i of reflection hkl.

2

Rcryst = Σ||Fobs| − |Fcalc||/Σ|Fobs|.

3

5% of the data were set aside for free R-factor calculation. CC1/2 = percentage of correlation between intensities from random half-dataset [45].

4

Calculated with MolProbity [46].

Fluorescence titration measurements

Each ligand bound to AgtB or AgaA was monitored by autofluorescence by excitating the protein at a wavelength of 295 nm and monitoring the quenching of fluorescence emission of tryptophans at 335 nm. All experiments were performed at 22°C in 96-well plates (1/2 Area Plate-96F, PerkinElmer) using Tecan Infinite M1000 (Tecan, Männedorf, Switzerland) in 25 mM Tris–HCl (pH 8.0) and 150 mM NaCl with a fixed amount of proteins (1 µM) and increasing concentrations of ligand. Each ligand has no emission signal at 335 nm. The data were analyzed using Origin® 7 software and fitted to the following equation:  
formula

Isothermal titration microcalorimetry measurements

Isothermal titration microcalorimetry experiments were performed with an ITC200 isothermal titration calorimeter from MicroCal (Malvern). The experiments were carried out at 20°C. Protein concentration in the microcalorimeter cell (0.2 ml) varied from 30 to 75 µM. Nineteen injections of 2 µl ligand solution (agropine, agropinic acid, mannopine and mannopinic acid) at concentrations ranging from 400 to 675 μM were performed at intervals of 180 s while stirring at 500 rpm. The experimental data were fitted to theoretical titration curves with the software supplied by MicroCal (ORIGIN®). This software uses the relationship between the heat generated by each injection and ΔH (enthalpy change in kcal mol−1), Ka (the association-binding constant in M−1), n (the number of binding sites), total protein concentration, and free and total ligand concentrations [37].

Distance tree

Sequences were analyzed using BlastP from NCBI (https://blast.ncbi.nlm.nih.gov/) and MicrosScope (https://www.genoscope.cns.fr/). Alignments of AgtB/AgaA and related sequences were conducted using MUSCLE software. The distance tree was built using Mega software, version 7. Topology was inferred using the neighbor-joining method [38]. The bootstrap consensus tree inferred from 1000 replicates was generated to represent the sequence relatedness of the proteins analyzed. The sequence distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.

Results

Agtb and AgaA exhibit a high affinity for agropinic acid but only AgtB binds agropine

Binding of agropinic acid and freshly purified agropine to the purified recombinant mature proteins AgtB and AgaA were explored using tryptophan fluorescence spectroscopy (AgtB and AgaA possess 16 and 5 tryptophans, respectively) and isothermal titration microcalorimetry. Intrinsic protein fluorescence titration experiments for AgtB yielded apparent dissociation constant KD values of 1.4 ± 0.2 μM with agropine. No interaction could be measured between AgaA and agropine. In contrast, AgaA and AgtB share a similar affinity in the nanomolar range for agropinic acid (47 ± 5.6 and 67 ± 10.6 nM, respectively), showing that both PBPs are efficient for agropinic acid binding (Figure 2). Using isothermal titration microcalorimetry, very similar KD values were obtained (Figure 2). The microcalorimetry data confirmed the 1 : 1 binding stoichiometry for both AgtB and AgaA, and revealed a negative enthalpy change for all ligands, meaning that the binding was enthalpy-driven, involving polar interactions mainly. No interaction between each protein and mannopine or mannopinic acid could be measured.

ITC and fluorescence KD measurements of AgtB and AgaA towards agropine/agropinic acid.

Figure 2.
ITC and fluorescence KD measurements of AgtB and AgaA towards agropine/agropinic acid.

The top graphs show fluorescence monitoring of each protein upon titration with each ligand and fit (solid line) to a single-binding model using Origin 7. Measures were done in triplicate. The lower graphs correspond to microcalorimetry experiments. The top panel shows heat differences upon injection of ligand and the low panel shows integrated heats of injection with the best fit (solid line) to a single-binding model using Microcal ORIGIN. ITC experiments were performed twice. Calculated parameters for each experiment are indicated in a table.

Figure 2.
ITC and fluorescence KD measurements of AgtB and AgaA towards agropine/agropinic acid.

The top graphs show fluorescence monitoring of each protein upon titration with each ligand and fit (solid line) to a single-binding model using Origin 7. Measures were done in triplicate. The lower graphs correspond to microcalorimetry experiments. The top panel shows heat differences upon injection of ligand and the low panel shows integrated heats of injection with the best fit (solid line) to a single-binding model using Microcal ORIGIN. ITC experiments were performed twice. Calculated parameters for each experiment are indicated in a table.

PBP AgtB is responsible for agropine uptake

The octopine–mannityl-type A. tumefaciens R10 strain was from our laboratory collection. The non-polar R10ΔagtB mutant corresponds to a mutant defective for AgtB, i.e. a strain in which the sole agtB gene is deleted with no effect on the expression of the downstream genes, when compared with the wild-type (WT) R10 strain. The growth profiles of WT and R10ΔagtB were compared in minimal medium containing agropinic acid or freshly purified agropine as the sole source of carbon/nitrogen or no carbon/nitrogen source (Figure 3). Under these conditions, both the R10ΔagtB mutant and the WT strains grew on agropinic acid, suggesting that another transport system can uptake agropinic acid in line with the presence of the PBP AgaA. In contrast with what was observed for the WT strain, the R10ΔagtB mutant did not grow on agropine during the first 50 h. After 50 h, growth was observed for the mutant due to the appearance of agropinic acid in the media, a feature that we verified by mass spectrometry. This presence of agropinic acid originated from spontaneous lactamization of agropine in line with what has been previously observed and reported [6,14]. Therefore, AgtB associated with its ABC transporter is the sole transport system responsible for the uptake and assimilation of agropine in pure culture.

AgtB involvement in agropine consumption.

Figure 3.
AgtB involvement in agropine consumption.

Four days’ growth (OD at 600 nm) of A. tumefaciens R10 WT strain (top panel) and the R10ΔagtB mutant (bottom panel) in AB minimal medium supplemented with agropine (▴) or agropinic acid (⬤) as carbon/nitrogen sources and in the absence of any carbon/nitrogen source (▪). Analyses were performed in three replicates.

Figure 3.
AgtB involvement in agropine consumption.

Four days’ growth (OD at 600 nm) of A. tumefaciens R10 WT strain (top panel) and the R10ΔagtB mutant (bottom panel) in AB minimal medium supplemented with agropine (▴) or agropinic acid (⬤) as carbon/nitrogen sources and in the absence of any carbon/nitrogen source (▪). Analyses were performed in three replicates.

Ligand-binding site of AgtB: a PBP from cluster D

The mature AgtB expression plasmid was a synthetic gene lacking the first 29 signal sequence residues that serve for the localization to the bacterial periplasm. Nonetheless, the expressed protein contains an N-terminal sequence of 20 residues corresponding to cloning artifact including a His-tag. The first X-ray structure of the liganded AgtB was determined at 1.74 Å resolution in P1 space group (Table 1) by molecular replacement using the coordinates of the N- and C-terminal domains of the PBP MotA (PDB: 5L9G) [21] as search models. The liganded crystal contains five very similar molecules in the asymmetric unit as indicates the average root-mean-square deviation (RMSD) of 0.39 Å for all Cα atoms. Because electron density maps were of high resolution, they revealed the structure of a ligand bound between the two closed lobes of AgtB. It was an agropinic acid molecule with a very well-defined oxoproline ring, no agropine as expected. The mass spectrometry analysis of the agropine solution used for the first AgtB–agropine co-crystallization trials revealed the presence of both agropine and agropinic acid. Using freshly purified agropine, we screened additional crystallization conditions to obtain an AgtB–agropine complex. New crystals appeared within 2 weeks. A second structure in a different space group (C2) at 1.9 Å resolution also revealed the presence of a bound agropinic acid. The asymmetric unit contains two similar AgtB–agropinic acid molecules. We determined at 1.4 Å resolution in P212121 the structure of AgtB co-crystallized with agropinic acid. One molecule is present in the asymmetric unit. The monomeric AgtB of 343 amino acids possesses a typical fold of cluster D within the PBP structural classification [39] (Figure 4A). The N-terminal lobe consists of residues (30–127 and 253–342), and the C-terminal lobe comprises the residues 133–247. Two short segments define the hinge region connecting the two lobes.

Ribbon representation of AgtB and AgaA structures and their ligand-binding sites.

Figure 4.
Ribbon representation of AgtB and AgaA structures and their ligand-binding sites.

(A) Agropinic acid in green is located in the cleft of AgtB between the lobes 1 and 2 shown in cyan and in blue, respectively, and the hinge region is in red. (B) Agropinic acid bound to the binding site of AgtB is shown in the same code color as in (A). Hydrogen bonds between AgtB and its ligand are shown as dashed lines in black (distances are up to 3.2 Å). The ligand is shown in its annealing FoFc omit map contoured at 4σ. (C) Agropinic acid in green is located in the cleft of AgaA between the lobes 1 and 2 shown in purple and in pink, respectively, and the hinge region is in red. (D) Agropinic acid bound to the binding site of AgaA is shown in the same code color as in (C). Hydrogen bonds between AgaA and its ligand are shown as dashed lines in black (distances are up to 3.2 Å). The ligand is shown in its annealing FoFc omit map contoured at 4σ. (E) Superposition of the bound agropinic acid in AgtB (cyan) and in AgaA (red) showing one functionally conserved residue. A rotation of ∼80° around the carbon-bond CAH–CAI between both sugar moieties of agropinic acid is observed.

Figure 4.
Ribbon representation of AgtB and AgaA structures and their ligand-binding sites.

(A) Agropinic acid in green is located in the cleft of AgtB between the lobes 1 and 2 shown in cyan and in blue, respectively, and the hinge region is in red. (B) Agropinic acid bound to the binding site of AgtB is shown in the same code color as in (A). Hydrogen bonds between AgtB and its ligand are shown as dashed lines in black (distances are up to 3.2 Å). The ligand is shown in its annealing FoFc omit map contoured at 4σ. (C) Agropinic acid in green is located in the cleft of AgaA between the lobes 1 and 2 shown in purple and in pink, respectively, and the hinge region is in red. (D) Agropinic acid bound to the binding site of AgaA is shown in the same code color as in (C). Hydrogen bonds between AgaA and its ligand are shown as dashed lines in black (distances are up to 3.2 Å). The ligand is shown in its annealing FoFc omit map contoured at 4σ. (E) Superposition of the bound agropinic acid in AgtB (cyan) and in AgaA (red) showing one functionally conserved residue. A rotation of ∼80° around the carbon-bond CAH–CAI between both sugar moieties of agropinic acid is observed.

All the liganded AgtB structures are very similar to the bound agropinic acid making the same numerous direct interactions with AgtB or via water molecules. The oxoproline ring of the agropinic acid is surrounded by two aromatic residues (Tyr37 and Phe130), and forms six H-bonds involving its carbonyl group and the side chains of Tyr37, Glu252 and Glu89 as well as its carboxylate group and the side chains of Arg229 and Tyr288 (Figure 4B). Glu89 appears to be a critical amino acid as its side chain maintains both the oxoproline ring and the deoxymannosyl (sugar) part of the ligand through three H-bonds. The deoxymannosyl part of agropinic acid interacts with the main chain amino group of Ala208 and with five water molecules. Only one of these is shown in Figure 4B, which establishes the most numerous polar interactions, involving the main chain of Asp164 as well as the carbonyl main chain and the side chain of Thr166. Although the oxoproline part of agropinic acid is well maintained in the binding site, no interaction was measured by fluorescence between AgaA and a single oxoproline residue, suggesting the requirement of the sugar part for the ligand binding.

SSM-EBI (http://www.ebi.ac.uk/msd-srv/ssm) reports the PBPs MotA (PDB: 5L9G) and A. tumefaciens C58 Atu4243 with GABA (PDB: 4EUO) as structurally closest to AgtB with RMSD lower than 2 : 1.56 Å over 283 Cα atoms with 31% sequence identity and 1.65 Å over 301 Cα atoms with 33% sequence identity, respectively. Although AgtB and MotA bind opines from the same opine family, their ligand-binding sites have one residue in common corresponding to Arg229 in AgtB and Arg238 in MotA, which locks the carboxylate group of the ligand. Upon superposition of both liganded MotA and AgtB structures, the presence of the tryptophans cluster at positions 165, 214 and 235 in MotA would prevent the binding of agropinic acid to MotA due to steric hindrance with the deoxymannosyl moiety (Supplementary Figure S1). In the same way, AgtB could not bind mannopine as observed in MotA because the glutamine part of mannopine would make steric hindrance with Met128 and Tyr288 (Ser128 and Thr297 in MotA, respectively), and the presence of Tyr37 in AgtB (Ser37 in MotA) would also create a steric clash with the deoxymannosyl moiety of mannopine (Supplementary Figure S1). The ligand-binding sites between AgtB and the GABA-binding Atu4243 share three residues: Glu89, Arg229 and Tyr288 in AgtB, which are Glu82, Arg225 and Tyr284 in Atu4243. Atu4243 presents a narrow pocket adapted for a selective GABA-binding due to the presence of several aromatic residues making van der Waals contacts and restricting the size of the ligand-binding site to accommodate GABA. As observed for MotA, AgtB possesses a larger binding site compared with Atu4243 preventing GABA binding.

Ligand-binding site of AgaA: a PBP from cluster C

The mature AgaA expression plasmid was obtained by cloning the agaA gene lacking the nucleotides corresponding to the first 24 signal sequence residues that serve for localization to bacterial periplasm. The X-ray structure of the liganded AgaA with agropinic acid was determined at 1.65 Å resolution (Table 1) by molecular replacement using the coordinates of the N- and C-terminal domains of a partial model of AgaA built from the E. coli putative-binding protein Ylib (PDB: 1UQW) as search models. The liganded crystal contained one molecule in the asymmetric unit. AgaA is a monomer of 493 residues composed of two lobes, each formed by a central β-sheet flanked by α-helices (Figure 4C). The biggest lobe (lobe 1) consists of residues 25–258 and 479–509, and the smallest (lobe 2) comprises the residues 264–473. Two short segments define the hinge region connecting the two lobes. AgaA fold belongs to the cluster C within the PBP structural classification [39] as SSM-EBI (http://www.ebi.ac.uk/msd-srv/ssm) reports: RMSD between AgaA and similar PBP structures binding oligopeptide range from 1.85 to 2.6 Å over 423–456 residues. A detailed structural comparison is irrelevant because AgaA presents a distinct ligand-binding site.

The agropinic acid bound between the two lobes of AgaA is well defined in the electron density maps and is surrounded by Gln43, Thr46, Tyr239, Gln267, Tyr355, Gln358, Trp383, Arg386, Gly401, Thr425, Asn426 and Arg475 residues (Figure 4D). The oxoproline part of the ligand, wedged between the aromatic residues Tyr239, Tyr355 and Trp383, interacts via its carboxylate group with the guanidinium group of Arg475 and the side chains of Gln358 and Tyr239, and via its carbonyl group with Thr46. Each hydroxyl group of the ligand deoxymannosyl moiety makes at least one H-bond with AgaA involving five side chains (Gln43, Gln267, Arg386, Thr425 and Asn426) and the main chain carbonyl of Gly401. As observed for AgtB, although the oxoproline part of agropinic acid is well held in the binding site, no interaction was measured by fluorescence between AgaA and a single oxoproline residue, suggesting that the sugar part is strictly needed for ligand binding.

Comparison between AgtB and AgaA structures

AgtB and AgaA overall structures show no similarity (RMSD value of 19.55 Å), as both PBPs belong to two different clusters. Hence, the ligands bound in the binding site exhibit different conformations and opposite orientations.

Superimposed AgtB–agropinic acid and AgaA–agropinic acid structures according to their ligand show no structurally common residue in the binding site. However, Arg229 residue in AgtB and Arg475 in AgaA conserved the same function in both proteins: their guanidinium group makes two interactions with the carboxylate group of the agropinic acid (Figure 4E). The curvature of the sugar moiety of agropinic acid in AgtB differs in AgaA by a rotation of ∼80° around the carbon-bond CAH–CAI, while the oxoproline moiety of the ligand adopts a similar conformation in both PBPs.

Agtb is present in a few bacteria only

Searching for AgtB conservation in the bacterial kingdom (protein database at NCBI) and subsequent topology analysis revealed the existence of 18 PBPs with sequences at least 65% identical with that of AgtB from strain R10 and the occurrence of clades (Figure 5). One of these, termed the AgtB clade, contains AgtB R10 and 9 highly similar orthologues sharing 93% sequence identity. It encompasses octopine/mannityl-opine-type (R10, B6, Ach5 and TT111), agrocinopine/mannityl-opine-type (Bo542, ATAM) A. tumefaciens strains and other plant-associated bacteria such as Rhizobium sp. and R. rhizogenes (formerly A. rhizogenes). More distantly related (73% sequence identity), AgtB proteins from the Agrobacterium larrymoorei strain ATCC 51759 and β-proteobacteria Burkholderia sp. strain PAMC 26561 seem capable of binding agropine and agropinic acid. All the above-mentioned bacteria possess the agropinic acid-binding signature of seven amino acids: Tyr37, Glu89, Phe130, Ala208, Arg229, Glu252 and Tyr288. Outside the AgtB clade, AgtB orthologues were detected in the Burkholderia, Pseudomonas, Polaromonas, Ensifer and Sinorhizobium genera. In these bacteria, the signature slightly degenerates. Modeling indicates that the binding may not be affected. The binding signature, however, appears strongly degenerated in MotA, a PBP that displayed only 31% sequence identity with AgtB.

Distance tree and AgtB-binding signature.

Figure 5.
Distance tree and AgtB-binding signature.

For each protein clade, the residues, which are identical with (black) and different from (red) those involved in the agropinic acid binding of A. tumefaciens R10 AgtB, are indicated. The sequence distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.

Figure 5.
Distance tree and AgtB-binding signature.

For each protein clade, the residues, which are identical with (black) and different from (red) those involved in the agropinic acid binding of A. tumefaciens R10 AgtB, are indicated. The sequence distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.

AgaA is conserved among rhizobiales

Searching for AgaA conservation leads to the identification of 72 PBPs with sequences at least 40% identical with that of AgaA from strain R10 and to the delineation of the AgaA clade with a solid bootstrap value of 100% containing the highly AgaA-similar PBPs (14 in total) that share more than 91% sequence identity (Figure 6). All of them are described as agropinic acid-binding proteins and display an agropinic acid-binding signature composed of 12 amino acids: Gln43, Thr46, Tyr239, Gln267, Tyr355, Gln/His358, Trp383, Arg386, Gly401, Thr425, Asn426 and Arg475. Residue 358 can be a glutamine (five PBPs) or histidine (nine PBPs); these two residues assume the same role toward the ligand. They all belong to Agrobacterium and Rhizobium genera. In the nearest subgroup (8 PBPs) that gathers sequences from the Rhizobium and Neorhizobium genera, the only changes observed in PBPs were the replacement of Thr425 by Leu425 and that of Gln43 by Asn43 in the binding signature. These two changes should not alter the binding of agropinic acid, as modeling revealed. Outside the AgaA clade and its closest group, the signature degenerates with changes that should alter the agropinic acid binding.

Distance tree and AgaA-binding signature.

Figure 6.
Distance tree and AgaA-binding signature.

For each protein clade, the residues, which are identical with (black) and different from (red) those involved in the agropinic acid binding of A. tumefaciens R10 AgaA, are indicated. Number in bracket represents the number of AgaA-relative PBPs per clade. The sequence distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.

Figure 6.
Distance tree and AgaA-binding signature.

For each protein clade, the residues, which are identical with (black) and different from (red) those involved in the agropinic acid binding of A. tumefaciens R10 AgaA, are indicated. Number in bracket represents the number of AgaA-relative PBPs per clade. The sequence distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.

Discussion

Ti plasmid genes drive the synthesis (in the plant cells) and assimilation (in the bacterial cells) of opines that are instrumental in the construction of the ecological niche of Agrobacterium. The mannityl-opine family gathers four opines: mannopine, mannopinic acid, agropine and agropinic acid. Mannopine and mannopinic acid are imine conjugates of mannose and glutamine or glutamate, respectively. Agropine is a cyclized derivative (a lactone) of mannopine and agropinic acid (a lactam) is generated non-enzymatically from the three other mannityl opines by spontaneous rearrangements [6,14]. We focused on these two latter opines because molecular and structural data on their transporter in pathogenic agrobacteria were missing, in contrast with the characterized PBP MotA involved in mannopine transport [21].

Previous genetic experiments have shown that agropine and agropinic acid should be imported into agrobacteria via the AgtB- and AgaA-mediated transport system, respectively [23,26,27,40,41]. This work reveals the structural basis of the PBPs AgtB and AgaA, which belong to two different structural clusters [39] and are determined by genes located in two different regions of Ti-plasmids of octopine/mannityl-opine-type and agrocinopine/mannityl-opines-type Agrobacterium strains. Using a genetic approach and growth assays, we showed that the presence of AgtB is compulsory for the binding and assimilation of the opine agropine. Using two different biophysical methods, we demonstrated that AgtB can bind agropine with an affinity in the micromolar range. An unexpected outcome of our study was that AgtB can also bind agropinic acid. Remarkably indeed, AgtB that so far appears to be the sole agrobacterial agropine transporter is not selective for agropine since it displays an affinity for agropinic acid 30-fold higher than that for agropine. This feature does not impair Agrobacterium ability to take up and utilize agropine because agropine is the precursor of agropinic acid and because the uptake of agropine is a process faster than the lactamization. In agreement, in young tumors induced by octopine/mannityl-opine-type Agrobacterium strains (R10/B6 strains), the main mannityl opines are agropine, mannopine and mannopinic acid, agropinic acid being barely detectable ([42] and Dessaux, unpublished results). The main role of AgtB would therefore be the transport of the abundant agropine in the first stage of tumor formation, i.e. as soon as this opine is produced.

But, the PBP AgaA recognizes agropinic acid as a unique ligand and displays a high affinity for it. Thus, the plant pathogen A. tumefaciens strains R10/B6 possess two Ti plasmid regions for agropinic acid import/degradation and exhibit two agropinic acid-binding PBPs, each with its own molecular signature characterized from the high-resolution crystal structures. Similarly, two pathways/regions (suggesting two PBPs) were reported for mannopinic acid assimilation using genetic approaches: one specific and the other unspecific [23,27,43]. Therefore, two uptake systems coexist for each agropinic and mannopinic acid. Nonetheless, they are differently regulated. Indeed, genes of the specific region containing the determinants of the PBPs AgaA and MoaA for agropinic and mannopinic acids uptake, respectively, are inducible by these two opines, while the nonspecific ones containing the genes coding the PBPs AgtB and likely MotA for agropinic and mannopinic acids uptake, respectively, are inducible by mannopine and agropine. Though this global scheme seems to be well established, the fine regulation of mannityl-opines utilization is still unclear, partly due to the complexity in the organization of the genes, the imbrication of the transport and catabolic pathways, and the lack of molecular and structural data on the numerous transcriptional regulators controlling these functions.

It was tempting to speculate that the occurrence of two PBPs (AgtB and AgaA) capable of transporting efficiently agropinic acid might correlate with an abundance of agropinic acid in the tumor resulting from the spontaneous degradation of the other mannityl opines. As indicated above, however, agropinic acid is not the most abundant opine in young tumors, but it is eventually present in concentrations equivalent to those of the other mannityl opines in aging tumors ([42] and Dessaux, unpublished results). A dual uptake system for agropinic acid that involves high-affinity transporters may therefore provide a selective advantage for pathogenic agrobacteria in this environment, mostly considering that bacterial competitors of Agrobacterium are more frequently prone to use agropinic acid than agropine (Dessaux, unpublished results). Agropinic acid uptake therefore remain essential for agrobacteria to assimilate this opine and maintain their competitivity in the tumor environment where the substantial production of mannityl opines [14] contributes to the establishment of the ecological niche of agrobacteria [44].

On the structural point of view, all attempts to obtain agropine bound to AgtB failed due to (i) the instability of agropine in the time course of crystallization process, which lasts for 10–15 days and to (ii) a better affinity of AgtB for agropinic acid. Co-crystallization with agropine led to different crystal forms with agropinic acid bound to AgtB. Nevertheless, it was straightforward to model manually an agropine at the position of the observed bound agropinic acid as both ligands share a common sugar moiety, which has served as a guide to anchor the ligand in the ligand-binding site (Supplementary Figure S2). This revealed that the ligand-binding site of AgtB can accommodate agropine. If the co-crystals of AgtB–agropine could appear in a short time (e.g. few hours), the structure of AgtB with agropine could be determined.

Database Depositions

The atomic coordinates and structure factors have been deposited at the Protein Data Bank under PDB: 6HLY, 6HLZ and 6HM2 for AgtB with agropinic acid in P212121, C2 and P1, respectively, and PDB: 6HLX for AgaA with agropinic acid.

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • PBP

    periplasmic-binding protein

  •  
  • RMSD

    root-mean-square deviation

  •  
  • WT

    wild-type

Author Contribution

Y.D. provided the agropine, agropinic acid, mannopine and mannopinic acid compounds and information on their metabolism and related determinants. F.P. purified agropine. T.M. and C.L. performed the non-polar AgtB mutant. A.V. performed the growth assays. L.M., A.V. and S.M. performed all the crystallography work. L.M. and A.V. performed the fluorescence assays. M.A.-N. performed the microcalorimetry experiments. A.V. and S.M. performed the phylogenetic analysis. S.M. wrote the manuscript. All the authors discussed the results and contributed to the writing of the manuscript.

Funding

T.M. received a doctoral grant from the French Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. L.M., A.V. and S.M. were supported by CNRS and ANR-Blanc SENSOR [ANR-12-BSV8-0003-01/02/03]. This work benefited from the I2BC crystallization and microcalorimetry platforms and from the server @TOME-2, all supported by FRISBI ANR-10-INSB-05-01 and the I2BC mass spectrometry SICaPS platform.

Acknowledgements

We acknowledge SOLEIL for the provision of synchrotron radiation facilities (proposal ID: 20130869, 20140774 and 20160782) in using Proxima beamlines. We deeply thank Ricardo Rodriguez de la Vega (ESE, Orsay) for his helpful discussion on tree construction.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.