N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls th e biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcript ion of the carbapenem biosynthetic genes is dependent on the LuxR-type acti vator protein, CarR, Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was exami ned using fluorescence quenching, DNA bandshift analysis, limited proteolys is and reporter gene assays, CarR bound the most physiologically relevant l igand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 mu M, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the liga nd causes the protein to multimerize. Chemical cross-linking analysis suppo rted this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.