Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi

LuxO is the central regulator integrating the quorum sensing signals contro lling autoinduction of luminescence in Vibrio harveyi. We have previously p urified to homogeneity a new lux regulator, LuxT, that binds to the luxO pr omoter. Based on the sequence of the tryptic peptides of LuxT, degenerate o ligonucleotides were designed for PCR of the genomic DNA. A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 a mino acids which shares homology with the AcrR/TetR family of transcription al regulators. The recombinant and native LuxT gave the same footprint bind ing between 117 and 149 bp upstream from the luxO initiation codon. Gene di sruption of luxT in V. harveyi increased luxO expression and affected the c ell density dependent induction of luminescence showing that LuxT was a rep ressor of luxO. As LuxT also affected the survival of the V. hm harveyi cel ls at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regula tory protein appears to be a general rather than a lux-specific regulator. (C) 2000 Elsevier Science B.V. All rights reserved.