Conversion of the Vibrio fischeri transcriptional activator, LuxR, to a repressor

The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sens ing system that involves the transcriptional activator (LuxR) and an acyl-h omoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has pr oven difficult to study in vitro. A truncated form of LuxR has been purifie d, and together with sigma(70) RNA polymerase it can activate transcription of the lux: operon. Both the truncated LuxR and RNA polymerase are require d for binding to lux regulatory DNA in vitro. We have constructed an artifi cial lacZ promoter with the lax box positioned between and partially overla pping the consensus -35 and -10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiple hu: boxes o n an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase bin ding to the promoter region. A variety of LuxR mutant proteins were studied , and with one exception there was a correlation between function as a repr essor of the artificial promoter and activation of a native lax operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.