Amino acid residues in LuxR critical for its mechanism of transcriptional activation during quorum sensing in Vibrio fischeri

Abstract

PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the Vibrio fischeri lux operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the lux operon was examined by using in vivo assays in recombinant Escherichia coli. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant E. coli was subsequently used to measure the ability of the variant forms of LuxR to bind to the lux box, the binding site of LuxR at the lux operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the lux box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the lux operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.

FIG. 1

Effects of alanine substitutions on LuxR-dependent cellular luciferase levels in recombinant E. coli. The value for each alanine-substitution mutant represents the average of two independent experiments with individual luciferase assays performed in quadruplicate. The error bars represent the range of each experiment from the mean. The wild-type strain (pSC300) value was set at 100% for each experiment. The negative-control strain (pKK223-3; Amersham-Pharmacia Biotech, Piscataway, N.J.) exhibited less than 0.3% of wild-type levels of luciferase (data not shown). The letter preceding each alanine-substitution position indicates an abbreviation for the amino acid residue at that position in the wild-type sequence. An asterisk highlights those LuxR variants exhibiting a “dark” phenotype (less than 50% of the wild-type levels of luminescence and luciferase).

FIG. 2

Western immunoblot of cell extracts from strains exhibiting the “dark” phenotype. The LuxR band is highlighted with an asterisk on the right. The mobility of molecular size standards is indicated by arrows. Residue numbers for the position of the alanine substitutions in LuxR are given at the top. Lanes + and − illustrate the levels of wild-type LuxR expressed from pSC300 and from the vector control pKK223-3, respectively.

FIG. 3

Effects of alanine substitutions on the ability of LuxR to bind to the lux box in recombinant E. coli. The value for each alanine-substitution mutant represents the average of two independent experiments each performed in triplicate. The error bars represent the range of each experiment from the mean. The wild-type value (pSC300) was set at 100% for each experiment, with the actual average value being equivalent to 7.48 ± 0.75-fold repression in the presence of 3-oxo-C6-HSL over all experiments. The negative-vector control (pKK223-3) value shown in the graph is the average value from all experiments. The letter preceding each alanine-substitution position indicates an abbreviation for the amino acid residue at that position in the wild-type sequence. An asterisk highlights those LuxR variants exhibiting a “dark” phenotype, and arrowheads highlight variants with wild-type levels of luminescence and luciferase but unable to bind to the lux box in the presence of 3-oxo-C6-HSL and to repress transcription in the assay.