PURIFICATION AND CHARACTERIZATION OF A POLY(DA-DT) LUX-SPECIFIC DNA-BINDING PROTEIN FROM VIBRIO-HARVEYI AND IDENTIFICATION AS LUXR

A lux-specific DNA-binding protein was purified to homogeneity from Vi brio harveyi by chromatography on DEAE-Sepharose, DNA-cellulose, Super ose 12, and Mono Q. A single polypeptide of M(r) = 23,000 was found on SDS-polyacrylamide gel electrophoresis with an amino-terminal sequenc e corresponding to that predicted for luxR, a gene that causes a shift in the transcriptional start site from position -123 to -26 base pair s upstream of the initiation codon of luxC in the V. harveyi lux opero n and is required for high expression of lux mRNA in recombinant Esche richia coli. Identification of the DNA-binding protein as LuxR was con firmed by showing its absence in V. harveyi luxR-mutants and its synth esis in recombinant E. coli containing V. harveyi luxR. The LuxR prote in was shown to bind to two specific (A + T)-rich regions of DNA upstr eam of the V. harveyi luxC gene: region A, -290 to -253 base pairs, an d region B, -170 to -116 base pairs. Synthetic poly(dA-dT) but not pol y(dA)-poly(dT) competed with the lux DNA for binding to LuxR suggestin g that this protein may be a novel poly(dA-dT)-binding protein in prok aryotes. The LuxR protein inhibited transcription from the -123 promot er in vitro; however, transcription from the -26 promoter was not reco nstituted suggesting the possible requirement for other factors in lux gene regulation. LuxR shared sequence identity with two proteins link ed to the regulation of enzymes involved in electron transport indicat ing that it may be a member of a family of regulators of metabolic fun ctions responsible for diverting electrons from the respiratory chain.