Journal of Bacteriology, February 2000, p. 805-811, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Department of Microbiology and Graduate Program in Molecular Biology, University of Iowa, Iowa City, Iowa 52242
Received 18 August 1999/Accepted 8 November 1999
The Vibrio fischeri luminescence (lux)
operon is regulated by a quorum-sensing system that involves the
transcriptional activator (LuxR) and an acyl-homoserine 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 proven difficult to study in vitro. A truncated form of LuxR
has been purified, and together with
70 RNA polymerase
it can activate transcription of the lux operon. Both the
truncated LuxR and RNA polymerase are required for binding to
lux regulatory DNA in vitro. We have constructed an
artificial lacZ promoter with the lux box
positioned between and partially overlapping 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
lux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to
lux boxes independently of RNA polymerase binding to the
promoter region. A variety of LuxR mutant proteins were studied, and
with one exception there was a correlation between function as a
repressor of the artificial promoter and activation of a native
lux 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.
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