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.

Quorum‐dependent transfer of the opine‐catabolic plasmid pAoF64/95 is regulated by a novel mechanism involving inhibition of the TraR antiactivator TraM

“…For example, allolactose, a metabolic product of lactose is the true ligand for LacI (Burstein, Cohn, Kepes, & Monod, ; Jobe & Bourgeois, ), while tyramine, a catabolic intermediate in the conversion of phenylethylamine to phenylacetate, is the ligand recognized by FeaR, the AraC‐like activator that regulates the pathway in Escherichia coli (Zeng & Spiro, ). More relevant to this system, arabinose‐2‐phosphate, the first intermediate in catabolism of agrocinopines A and B, is the ligand recognized and bound by AccR, the FucR‐like repressor that controls expression of the acc operon as well as traR in the arc operon of pTiC58 (El Sahili et al., ).…”

“…Both operons are controlled by AccR, a FucR-like repressor (Beck von Bodman, Hayman, & Farrand, 1992;Piper et al, 1999). This regulator loses affinity for its operator sequences in the acc and arc promoter regions when it binds arabinose-2-phosphate, the first intermediate in catabolism of the two agrocinopine opines (El Sahili et al, 2015). In both Ti plasmids, the two signals thus form a regulatory cascade with the conjugative opine controlling expression of the quorum-sensing system which, in turn, is responsible for inducing transcription of the tra regulon ( Figure S1).…”

Can Plant Microbiome Studies Lead to Effective Biocontrol of Plant Diseases?

“…In the case of agrocin 84 its N 6 -D-glucofuranosyloxyphosphoramidate substituent 'tricks' a high-efficiency transport system for its uptake into target cells. This substituent mimics the region of agrocinopine A, arabinose-2-phosphate, responsible for its specific interaction with a Ti plasmid-encoded periplasmic binding protein, AccA, the first component of an ABC transporterbased agrocinopine A permease (El Sahili et al 2015). Interestingly agrocin 84 itself is not toxic until processed inside the target cell by the AccF enzyme, a phosphodiesterase involved in degradation of agrocinopine A (Kim and Farrand 1997).…”

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders

“…The same Ti-plasmid also codes for QS-signalling, which controls the dissemination of the Ti-plasmid by bacterial conjugation between a virulent Ti-plasmid donor and an avirulent recipient individual, which thereby becomes virulent through Ti-plasmid acquisition2021. Ti-plasmid conjugation occurs in the induced plant-tumour tissues, as some opines are required for increasing the transcription of Ti-plasmid genes responsible for the production ( traI ) and sensing ( traR ) of the QS-signal2122. Virulent Ti-plasmids thus modify the host-plant habitat of A. tumefaciens cells by inducing opine production in infected plant cells and favour their own spread through a QS-controlled horizontal transfer.…”