Quorum sensing in Vibrio fischeri: Elements of the luxl promoter

Abstract

Although cell density-dependent regulation of the luminescence genes in Vibrio fischeri is a model for quorum sensing in Gram-negative bacteria, relatively little is known about the promoter of the luminescence operon. The luminescence operon is activated by the LuxR protein, which requires a diffusible acyl-homoserine lactone signal. The lux box, a 20 bp inverted repeat, is located in the luxl promoter region and is required for LuxR-dependent induction of the luminescence genes. Using primer extension, we mapped the LuxR-dependent transcriptional start site of the lux operon to 19 bp upstream of the luxl start codon. This indicates that the lux box is centred at -42.5 bp from the start of transcription. To gain evidence about the location of the -10 sequence, we placed a consensus -35 hexamer at different locations relative to the luxl transcriptional start site and measured constitutive levels of luminescence in recombinant Escherichia coli. The strongest constitutive promoter contained a TATAGT hexamer 17 bp from the -35 consensus sequence and 6 bp from the transcriptional start site. We propose that this is the -10 hexamer. Also in recombinant E. coli, both half-sites of the lux box were required for LuxR-dependent gene activation and for activation by an autoinducer-independent, monomeric LuxR deletion protein. LuxR-dependent activation of luminescence was eliminated when the lux box was centred at -47.5, -52.5 and -62.5 with respect to the luxl transcriptional start site. Our evidence, taken together with other information, points to a model in which a LuxR dimer overlaps the -35 region of the luxl promoter and functions as an ambidextrous activator with each LuxR subunit interacting with a different region of RNA polymerase.