Purification and Structural Identification of an Autoinducer for the Luminescence System of Vibrio harveyi

• Published in: The Journal of biological chemistry 1989-12, Vol.264 (36), p.21670-21676
• Main Authors: Cao, J G, Meighen, E A
• Format: Article
• Language: English
• Subjects: Acetates - metabolism; Biological and medical sciences; Cell physiology; Chromatography, Thin Layer; Fundamental and applied biological sciences. Psychology; Homoserine - analogs & derivatives; Homoserine - chemical synthesis; Homoserine - isolation & purification; Homoserine - metabolism; Kinetics; Luminescent Measurements; Magnetic Resonance Spectroscopy; Miscellaneous; Molecular and cellular biology; Mutation; Vibrio - genetics; Vibrio - growth & development; Vibrio - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(20)88238-6

An autoinducer required for the growth-dependent development of luminescence in Vibrio harveyi has been purified, structurally identified, and chemically synthesized. The autoinducer, which is excreted by the cells, was extracted with chloroform from conditioned media in which V. harveyi cells had been grown. The concentrated extract was separated on a silica gel column and the autoinducer activity further purified by thin layer, paper, and high performance liquid chromatography. The structure of the partially purified autoinducer was identified by 1H NMR and mass spectrometry as N-(β-hydroxybutyryl)homoserine lactone. This compound was chemically synthesized by condensation of β-hydroxybutyrate with α-amino-γ-butyrolactone hydrobromide using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a carboxyl group activator. The pure synthetic autoinducer gave the characteristic NMR and mass spectra, co-migrated with the natural autoinducer on thin layer plates, and specifically stimulated induction of luminescence of V. harveyi. Light emission of a regulatory dark mutant of V. harveyi could be stimulated over 1000-fold by the addition of N-(β-hydroxybutyryl)homoserine lactone, reaching intensities comparable to that of the native strain. The similarity in structure of the autoinducer of V. harveyi to that of Vibrio fischeri suggests that the regulation of luminescence induction in these bacteria may be related in spite of their differences in lux gene organization.