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Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens.

ABSTRACT: A signal turnover system is an essential component of many genetic regulatory mechanisms. The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms. We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode. A. tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL), also known as Agrobacterium autoinducer. By using Tn5 mutagenesis and a functional cloning approach, we identified two genes that are involved in switching from a conjugal quorum-sensing mode to a nonconjugal mode at the onset of stationary phase. First, we located attJ, which codes for an IclR-type suppressor that regulates the second gene attM. The latter encodes a homologue of N-acylhomoserine lactone (AHL)-lactonase. Mass spectrometry analysis shows that the enzyme encoded by attM is an AHL-lactonase that hydrolyzes the lactone ring of 3OC8HSL. In wild-type A. tumefaciens, attM expression is initially suppressed by AttJ but significantly elevated at the stationary phase accompanied a sharp decline in 3OC8HSL. DNA gel retardation analysis shows that AttJ specifically binds to the promoter that controls AHL-lactonase expression. Mutation of attJ resulted in constitutive production of AHL-lactonase that abolishes 3OC8HSL accumulation and Ti plasmid transfer. These data suggest that A. tumefaciens has a sophisticated multicomponent quorum-sensing signal turnover system, allowing the cell to sense a change in growth and adjust cellular activities accordingly.

SUBMITTER: Zhang HB

PROVIDER: S-EPMC123700 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens.

Zhang Hai-Bao HB Wang Lian-Hui LH Zhang Lian-Hui LH

Proceedings of the National Academy of Sciences of the United States of America 20020401 7

A signal turnover system is an essential component of many genetic regulatory mechanisms. The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms. We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode. A. tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL), also known as Agrobacterium autoinducer. By usi ...[more]

PMID: 11930013

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Project description: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.

Project description:Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. The At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologs of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR is preferentially biased toward its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

Dataset Information

Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens.

Publications

Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens.

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