Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi

Bioluminescence in the marine bacterium Vibrio harveyi is cell density depe ndent and is regulated by small molecules (autoinducers) excreted by the ba cteria. The autoinducer signals are relayed to a central regulator, LuxO, w hich acts in its phosphorylated form as a repressor of the lux operon at th e 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 f ive major chromatography steps, including a highly effective DNA affinity c hromatography step and reverse-phase HPLC. Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guan idine 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 th at LuxT is a new V. harveyi lux regulatory protein, (C) 2000 Academic Press .