Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi.
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.
Lin
Y H
YH
Department of Biochemistry, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada.
Miyamoto
C
C
Meighen
E A
EA
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*
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