title: A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.
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.
authors.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.
authors.forename: Abbas
authors.initials: A
authors.lastname: El Sahili
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, ICBMS, Université de Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, INSA Lyon, CPE-Lyon, Bât J. Verne, Villeurbanne, France.
authors.forename: Si-Zhe
authors.initials: SZ
authors.lastname: Li
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lang
authors.affiliation: Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Virus
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.
authors.forename: Sara
authors.initials: S
authors.lastname: Planamente
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, ICBMS, Université de Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, INSA Lyon, CPE-Lyon, Bât J. Verne, Villeurbanne, France.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Ahmar
authors.affiliation: Synchrotron SOLEIL, Gif sur Yvette, France.
authors.forename: Beatriz G
authors.initials: BG
authors.lastname: Guimaraes
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Protein-Protein Interaction Platform, CNRS CEA University Paris-Sud, Orsay, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Aumont-Nicaise
authors.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.
authors.forename: Armelle
authors.initials: A
authors.lastname: Vigouroux
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, ICBMS, Université de Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, INSA Lyon, CPE-Lyon, Bât J. Verne, Villeurbanne, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Soulère
authors.affiliation: Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Reader
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, ICBMS, Université de Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, INSA Lyon, CPE-Lyon, Bât J. Verne, Villeurbanne, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Queneau
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Faure
authors.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.
authors.forename: Solange
authors.initials: S
authors.lastname: Moréra
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D011487:Protein Conformation; D053038:Quorum Sensing / Q000502:physiology*; D013403:Sugar Phosphates / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; C082936:AccA protein, Agrobacterium tumefaciens; D000227:Adenine Nucleotides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D013403:Sugar Phosphates; C013799:agrocin 84; C038030:agrocinopine