Amino acid residues in LuxR critical for its mechanism of transcriptional activation during quorum sensing in Vibrio fischeri.
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.
Trott
A E
AE
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Stevens
A M
AM
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014733:Vibrio / Q000235:genetics* / Q000378:metabolism*
D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; D000409:Alanine