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 the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, 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 microM, 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 ligand causes the protein to multimerize. Chemical cross-linking analysis supported 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.
Welch
M
M
Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW.
Todd
D E
DE
Whitehead
N A
NA
McGowan
S J
SJ
Bycroft
B W
BW
Salmond
G P
GP
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015780:Carbapenems / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D006714:Homoserine / Q000031:analogs & derivatives* / Q000378:metabolism; D007783:Lactones / Q000378:metabolism*; D008024:Ligands; D016973:Pectobacterium carotovorum / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C091096:CarR protein, Azospirillum brasilense; D015780:Carbapenems; D017931:DNA Primers; D004269:DNA, Bacterial; D007783:Lactones; D008024:Ligands; D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; D006714:Homoserine