ANALOGS OF THE AUTOINDUCER 3-OXOOCTANOYL-HOMOSERINE LACTONE STRONGLY INHIBIT ACTIVITY OF THE TRAR PROTEIN OF AGROBACTERIUM-TUMEFACIENS

The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-d ensity-dependent expression of the Ti plasmid tra regulon. TraI synthe sizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C-8-HSL), while TraR is an 3-oxo-C-8-HSL-responsive transcript ional activator. We have compared the abilities of 3-oxo-C-8-HSL and 3 2 related compounds to activate expression of a TraR-regulated promote r. In a strain that expresses wild-type levels of TraR, only 3-oxo-C-8 -HSL was strongly stimulatory, four compounds were detectably active o nly at high concentrations, and the remaining 28 compounds were inacti ve. Furthermore, many of these compounds were potent antagonists. In c ontrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist . We propose a model in which autoinducers enhance the affinity of Tra R either for other TraR monomers or for DNA binding sites and that ove rexpression of TraR potentiates this interaction by mass action. Wild- type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. How ever, under all conditions tested, 3-oxo-C-8-HSL was more abundant tha n any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C-8-HSL significantly stimulates tra gene expressi on, while many autoinducer analogs are potent antagonists; (ii) TraR o verexpression increases agonistic activity of autoinducer analogs, all owing sensitive biodetection of many antoinducers; and (iii) autoinduc er stimulatory activity is potentiated by TraR overproduction, suggest ing that autoinducers may shift an equilibrium between TraR monomers a nd dimers or oligomers. When autoinducer specificities of other quorum -sensing proteins are tested, care should be taken not to overexpress those proteins.