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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 simplified schematic representing the agrocinopine A and agrocin 84 roles in A. tumefaciens C58.Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain A. radiobacter K84 (which colonizes the same plant environment as the A. tumefaciens C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of A. tumefaciens C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In A. tumefaciens C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing acc and traR genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the tra, trb and rep genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.
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ppat.1005071.g001: A simplified schematic representing the agrocinopine A and agrocin 84 roles in A. tumefaciens C58.Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain A. radiobacter K84 (which colonizes the same plant environment as the A. tumefaciens C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of A. tumefaciens C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In A. tumefaciens C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing acc and traR genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the tra, trb and rep genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.

Mentions: AccA also plays a key-role in the importation of the characteristic plant tumour-derived compounds such as the opines agrocinopines A and B discovered in 1981 in crown gall tumour tissue [8]. Agrocinopine A is composed of a sucrose linked to a L-arabinose via a phosphodiester bond. Agrocinopine B results from the cleavage of the sucrose moiety of agrocinopine A. Hence agrocinopine B is composed of a fructose linked to a L-arabinose via a phosphodiester bond. The agrocinopines A and B were purified from tumours induced by A. tumefaciens strain C58. Upon infection, A. tumefaciens genetically engineers the plant host by transferring a piece of DNA (the T-DNA) from its tumour inducing (Ti) plasmid to the nuclear genome of plants. Proliferation of the transformed plant cells results in the formation of tumours colonized by the bacteria. In plant tumours, T-DNA genes expression redirects the metabolism towards the production of several opines which are used by the pathogen as nutrients (C, N and P sources) and signals to control the quorum-sensing signal expression [9–11]. Indeed, agrocinopines A and B play a crucial role in the A. tumefaciens C58 infection process by inducing the synthesis of the quorum-sensing signal 3-oxo-octanoylhomoserine lactone (OC8HSL) which increases aggressiveness of agrobacterium and activates the dissemination of the Ti plasmid by horizontal transfer (by conjugation) [12,13] (Fig 1). Binding of agrocinopine to the transcriptional repressor AccR, which belongs to the DeoR transcriptional factors family, is proposed to inhibit its repression [14], releasing the expression of a second transcriptional factor TraR, which binds the quorum-sensing signal OC8HSL. In turn, the complex TraR-OC8HSL activates the expression of quorum-sensing regulated genes such as the tra and trb operons, encoding for horizontal transfer of the Ti plasmid by conjugation and the rep operon which controls the replication of the Ti plasmid. In addition to quorum-sensing, AccR also regulates the transcription of acc (agrocinopine catabolism) genes [15]. These genes encode an agrocinopine ABC transporter system including the PBP AccA and two putative enzymes required for catabolism of agrocinopine, the phosphodiesterase AccF and the phosphatase AccG.


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 simplified schematic representing the agrocinopine A and agrocin 84 roles in A. tumefaciens C58.Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain A. radiobacter K84 (which colonizes the same plant environment as the A. tumefaciens C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of A. tumefaciens C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In A. tumefaciens C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing acc and traR genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the tra, trb and rep genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526662&req=5

ppat.1005071.g001: A simplified schematic representing the agrocinopine A and agrocin 84 roles in A. tumefaciens C58.Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain A. radiobacter K84 (which colonizes the same plant environment as the A. tumefaciens C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of A. tumefaciens C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In A. tumefaciens C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing acc and traR genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the tra, trb and rep genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.
Mentions: AccA also plays a key-role in the importation of the characteristic plant tumour-derived compounds such as the opines agrocinopines A and B discovered in 1981 in crown gall tumour tissue [8]. Agrocinopine A is composed of a sucrose linked to a L-arabinose via a phosphodiester bond. Agrocinopine B results from the cleavage of the sucrose moiety of agrocinopine A. Hence agrocinopine B is composed of a fructose linked to a L-arabinose via a phosphodiester bond. The agrocinopines A and B were purified from tumours induced by A. tumefaciens strain C58. Upon infection, A. tumefaciens genetically engineers the plant host by transferring a piece of DNA (the T-DNA) from its tumour inducing (Ti) plasmid to the nuclear genome of plants. Proliferation of the transformed plant cells results in the formation of tumours colonized by the bacteria. In plant tumours, T-DNA genes expression redirects the metabolism towards the production of several opines which are used by the pathogen as nutrients (C, N and P sources) and signals to control the quorum-sensing signal expression [9–11]. Indeed, agrocinopines A and B play a crucial role in the A. tumefaciens C58 infection process by inducing the synthesis of the quorum-sensing signal 3-oxo-octanoylhomoserine lactone (OC8HSL) which increases aggressiveness of agrobacterium and activates the dissemination of the Ti plasmid by horizontal transfer (by conjugation) [12,13] (Fig 1). Binding of agrocinopine to the transcriptional repressor AccR, which belongs to the DeoR transcriptional factors family, is proposed to inhibit its repression [14], releasing the expression of a second transcriptional factor TraR, which binds the quorum-sensing signal OC8HSL. In turn, the complex TraR-OC8HSL activates the expression of quorum-sensing regulated genes such as the tra and trb operons, encoding for horizontal transfer of the Ti plasmid by conjugation and the rep operon which controls the replication of the Ti plasmid. In addition to quorum-sensing, AccR also regulates the transcription of acc (agrocinopine catabolism) genes [15]. These genes encode an agrocinopine ABC transporter system including the PBP AccA and two putative enzymes required for catabolism of agrocinopine, the phosphodiesterase AccF and the phosphatase AccG.

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