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A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus

(A) AccR microcalorimetry measurements. The top panels show heat differences upon injection of ligand (L-arabinose-2-phosphate on the left and D-glucose-2-phosphate on the right) and lower panels show integrated heats of injection and the best fit (solid line) using Microcal Origin. Fitting values are indicated below. (B) Quantification of OC8HSL production and Ti plasmid conjugation induced by agrocinopine A, L-arabinose-2-phosphate and D-glucose-2-phosphate. Different A. tumefaciens C58 backgrounds were used as donor cells: C58 control, accF mutant, accF mutant complemented with p6000: accF wild type and accF mutant complemented with empty p6000). Results were obtained after 72 h of culture. SDs correspond to physical replicates. Experiments independently repeated between two and four times produce similar results.
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ppat.1005071.g006: (A) AccR microcalorimetry measurements. The top panels show heat differences upon injection of ligand (L-arabinose-2-phosphate on the left and D-glucose-2-phosphate on the right) and lower panels show integrated heats of injection and the best fit (solid line) using Microcal Origin. Fitting values are indicated below. (B) Quantification of OC8HSL production and Ti plasmid conjugation induced by agrocinopine A, L-arabinose-2-phosphate and D-glucose-2-phosphate. Different A. tumefaciens C58 backgrounds were used as donor cells: C58 control, accF mutant, accF mutant complemented with p6000: accF wild type and accF mutant complemented with empty p6000). Results were obtained after 72 h of culture. SDs correspond to physical replicates. Experiments independently repeated between two and four times produce similar results.

Mentions: In the A. tumefaciens C58 cytoplasm, the enzyme AccF releases the L-arabinose-2-phosphate and D-glucose-2-phosphate from agrocinopine A and agrocin 84. The question arose about their biological role in A. tumefaciens, especially in relation to regulation of the AccR/quorum-sensing pathway that controls dissemination of the Ti plasmid. We first measured the affinity of the synthetic agrocinopine A towards purified recombinant AccR by isothermal titration microcalorimetry. An unexpected outcome was that no affinity was detected. In contrast, the isothermal titration microcalorimetry data show that AccR displays affinity to L-arabinose-2-phosphate and D-glucose-2-phosphate in two steps with KD values for L-arabinose-2-phosphate of KD1 = 0.11 ± 0.06 (n1 = 0.4) μM for the first step and KD2 = 1.64 ± 0.8 (n2 = 0.9) μM for the second step and for D-glucose-2-phosphate KD1* = 0.43 ± 0.1 (n1* = 0.5) μM and KD2* = 1.64 ± 0.8 μM (n2* = 0.9) (Fig 6A). The binding stoichiometries of 2:1 (protein:ligand) in the first step and 1:1 in the second step are in line with the fact that AccR forms a dimer in solution as observed by native gel electrophoresis, suggesting that the binding of the effector to AccR may be described as following: first, a ligand binds to one molecule within the dimer before another binds to the second molecule. These results suggest L-arabinose-2-phosphate and D-glucose-2-phosphate are the effector molecules that regulate AccR and thus they should activate quorum-sensing synthesis in A. tumefaciens cells.


A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

(A) AccR microcalorimetry measurements. The top panels show heat differences upon injection of ligand (L-arabinose-2-phosphate on the left and D-glucose-2-phosphate on the right) and lower panels show integrated heats of injection and the best fit (solid line) using Microcal Origin. Fitting values are indicated below. (B) Quantification of OC8HSL production and Ti plasmid conjugation induced by agrocinopine A, L-arabinose-2-phosphate and D-glucose-2-phosphate. Different A. tumefaciens C58 backgrounds were used as donor cells: C58 control, accF mutant, accF mutant complemented with p6000: accF wild type and accF mutant complemented with empty p6000). Results were obtained after 72 h of culture. SDs correspond to physical replicates. Experiments independently repeated between two and four times produce similar results.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526662&req=5

ppat.1005071.g006: (A) AccR microcalorimetry measurements. The top panels show heat differences upon injection of ligand (L-arabinose-2-phosphate on the left and D-glucose-2-phosphate on the right) and lower panels show integrated heats of injection and the best fit (solid line) using Microcal Origin. Fitting values are indicated below. (B) Quantification of OC8HSL production and Ti plasmid conjugation induced by agrocinopine A, L-arabinose-2-phosphate and D-glucose-2-phosphate. Different A. tumefaciens C58 backgrounds were used as donor cells: C58 control, accF mutant, accF mutant complemented with p6000: accF wild type and accF mutant complemented with empty p6000). Results were obtained after 72 h of culture. SDs correspond to physical replicates. Experiments independently repeated between two and four times produce similar results.
Mentions: In the A. tumefaciens C58 cytoplasm, the enzyme AccF releases the L-arabinose-2-phosphate and D-glucose-2-phosphate from agrocinopine A and agrocin 84. The question arose about their biological role in A. tumefaciens, especially in relation to regulation of the AccR/quorum-sensing pathway that controls dissemination of the Ti plasmid. We first measured the affinity of the synthetic agrocinopine A towards purified recombinant AccR by isothermal titration microcalorimetry. An unexpected outcome was that no affinity was detected. In contrast, the isothermal titration microcalorimetry data show that AccR displays affinity to L-arabinose-2-phosphate and D-glucose-2-phosphate in two steps with KD values for L-arabinose-2-phosphate of KD1 = 0.11 ± 0.06 (n1 = 0.4) μM for the first step and KD2 = 1.64 ± 0.8 (n2 = 0.9) μM for the second step and for D-glucose-2-phosphate KD1* = 0.43 ± 0.1 (n1* = 0.5) μM and KD2* = 1.64 ± 0.8 μM (n2* = 0.9) (Fig 6A). The binding stoichiometries of 2:1 (protein:ligand) in the first step and 1:1 in the second step are in line with the fact that AccR forms a dimer in solution as observed by native gel electrophoresis, suggesting that the binding of the effector to AccR may be described as following: first, a ligand binds to one molecule within the dimer before another binds to the second molecule. These results suggest L-arabinose-2-phosphate and D-glucose-2-phosphate are the effector molecules that regulate AccR and thus they should activate quorum-sensing synthesis in A. tumefaciens cells.

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus