title: Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi.
abstract: Bioluminescence in the marine bacterium Vibrio harveyi is cell density dependent and is regulated by small molecules (autoinducers) excreted by the bacteria. The autoinducer signals are relayed to a central regulator, LuxO, which acts in its phosphorylated form as a repressor of the lux operon at the early stages of cell growth. We report in these studies the purification to homogeneity of a luxO DNA binding protein (LuxT) from V. harveyi after five major chromatography steps, including a highly effective DNA affinity chromatography step and reverse-phase HPLC. Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guanidine hydrochloride. It was also demonstrated that the functional LuxT was a dimer of 17 kDa that bound tightly (K(d) = 2 nM) to the luxO promoter. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the Protein Data Bank, indicating that LuxT is a new V. harveyi lux regulatory protein.
authors.affiliation: Department of Biochemistry, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada.
authors.forename: Y H
authors.initials: YH
authors.lastname: Lin
authors.forename: C
authors.initials: C
authors.lastname: Miyamoto
authors.forename: E A
authors.initials: EA
authors.lastname: Meighen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D018983:DNA Footprinting; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors; D014357:Trypsin / Q000378:metabolism; D014733:Vibrio / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C088907:LuxO protein, Vibrio harveyi; C416202:LuxT protein, Vibrio harveyi; D012097:Repressor Proteins; D014157:Transcription Factors; D014357:Trypsin