Autoinducer-independent mutants of the LuxR transcriptional activator exhibit differential effects on the two lux promoters of Vibrio fischeri

Abstract

The LuxR protein is a transcriptional activator which, together with a diffusible small molecule termed the autoinducer [N-(3-oxohexanoyl)-L-homo-serine lactone], represents the primary level of regulation of the bioluminescence genes in Vibrio fischeri. LuxR, in the presence of autoinducer, activates transcription of the luxICDABEG gene cluster and both positively and negatively autoregulates transcription of the divergently oriented luxR gene, activating transcription at low levels of autoinducer, and repressing synthesis at high autoinducer concentration. Seven LuxR point mutants which activate V. fischeri lux transcription in the absence of autoinducer (LuxR*) have been characterized. The LuxR* proteins activated transcription of the bioluminescence genes to levels 1.5-40 times that achieved by wild-type LuxR without autoinducer. All of the LuxR* mutants retained responsiveness to autoinducer. However, in each case the degree of stimulation in response to autoinducer was lower than that observed for wild-type LuxR. The LuxR* proteins retained the requirement for autoinducer for autoregulation of the luxR gene. We propose that the LuxR protein exists in two conformations, an inactive form, and an active form which predominates in the presence of autoinducer. The LuxR* mutations appear to shift the equilibrium distribution of these two forms so as to increase the amount of the active form in the absence of autoinducer, while autoinducer can still convert inactive to active species. The differential effects of the LuxR* proteins at the two lux promoters suggest that LuxR stimulates PluxR transcription by a different mechanism to that used at the PluxI promoter, implying that binding of LuxR to its binding site, known to be necessary for transcriptional activation, may not be sufficient.