Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription

Abstract

The LuxR protein is a transcriptional activator involved in regulation of the genes required for bioluminescence (lux) in the marine bacterium Vibrio fischeri. Transcription of the two divergently oriented lux operons (luxR and luxICDABEG) is activated by LuxR in the presence of a diffusible inducer (autoinducer). Transcription of the luxR gene is subject to both positive and negative autoregulation as well as activation by the cAMP-catabolite gene activator protein complex (cAMP-CAP). Transcription of luxR was studied using both luminescence in vivo as a reporter and primer extension analysis of mRNA synthesized in vivo. Mutation of the lux CAP-binding site resulted in a reduction in luminescence from the reporter and the complete loss of luxR positive autoregulation. Positive autoregulation was restored if luxR was provided in trans, demonstrating that LuxR and autoinducer activate luxR transcription in the absence of cAMP-CAP. By means of primer extension analysis, three sites of initiation of luxR transcription were demonstrated; initiation at two of these sites required cAMP-CAP. The quantity of all three transcripts was increased in the presence of LuxR and autoinducer when a plasmid with a wild-type CAP-binding site was used. Initiation at the cAMP-CAP-dependent sites was not observed from a plasmid with a mutated CAP-binding site in the presence or absence of autoinducer even with luxR supplied in trans. Instead, with luxR supplied in trans, initiation at the cAMP-CAP-independent initiation site was specifically stimulated by LuxR and autoinducer. Thus, in the course of positive autoregulation, the LuxR protein activates transcription from two luxR promoters by a cAMP-CAP-dependent mechanism and a third promoter by a cAMP-CAP-independent mechanism.