Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.     

Iron control of the Vibrio fischeri luminescence system in Escherichia coli

Iron influences liminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB ⁺ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICD ABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di (o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB ⁺ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of -galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8–9 fold each for tonB, 2–3 fold each for tonB ⁺) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.     

Biosynthesis and stereochemistry of the autoinducer controlling luminescence in Vibrio harveyi.

Knowledge of the pathway for synthesis of the autoinducer, N-(beta-hydroxybutyryl)-homoserine lactone (HBHL), controlling luminescence in Vibrio harveyi can provide important information concerning the relationship between the nutrition and physiology of the bacteria and the phenomenon of light emission. In this study, the D and L isomers of the autoinducer containing the stereoisomers of beta-hydroxybutyric acid were synthesized and characterized by proton nuclear magnetic resonance in the presence of a chiral shift reagent, a europium(III) derivative of Tris[3-(heptafluoropropyl-hydroxymethylene)-(+)-camphorato]. By using a newly isolated autoinducer mutant which responds to low physiological concentrations of the autoinducer, it could be shown that autoinducer activity was associated with D-HBHL and not L-HBHL. Blockage of fatty acid biosynthesis by the addition of fatty acids and/or the antibiotic cerulenin to the cells prevented synthesis of the autoinducer as measured by the loss of autoinducer activity and a decrease in the incorporation of labelled acetate into the partially purified autoinducer. These results indicate that fatty acid biosynthesis is necessary for light emission in luminescent bacteria because it controls formation of the lux autoinducer.     

Synthesis of the lux gene autoinducer in Vibrio fischeri is positively autoregulated

The enzymes for luminescence in Vibrio fischeri are induced only after the accumulation of a sufficient concentration of a metabolic product (the autoinducer) generated by the bacteria themselves. Genetic analyses by others have previously suggested that biosynthesis of the autoinducer is catalyzed by a single gene product (autoinducer synthetase) presumably from precursors typically present in the bacterial cell. Also, the biosynthesis was predicted to be autocatalytic such that in the presence of autoinducer, more autoinducer synthetase should be produced. We have directly tested these predictions and found that autoinducer synthesis is indeed positively autoregulated. In addition, we have demonstrated autoinducer synthesis in vitro and have tentatively identified the substrates of autoinducer synthetase as S-adenosylmethionine and 3-oxohexanoyl coenzyme A.Abbreviations AdoMet S-adenosylmethionine - AI autoinducer, i.e. 3-oxohexsanoyl homoserine lactone - C-10 decanoyl homoserine lactone - HPLC high performance liquid chromatography - LM luminescence medium - LM-BT luminescence medium without tryptone - LU light units - 3-oxo 3-oxohexanoyl-coenzyme A - SWC sea water complete medium     

Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi

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-(beta-hydroxybutyryl)homoserine lactone. This compound was chemically synthesized by condensation of beta-hydroxybutyrate with alpha-amino-gamma-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-(beta-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.     

黄曲霉毒素对费氏弧菌发光的影响

[目的]探讨黄曲霉毒素对一种发光细菌——费氏弧菌发光的抑制效应.[方法]黄曲霉毒素或产黄曲霉毒素的菌株培养液对费氏弧菌进行处理后,利用多功能酶标仪检测费氏弧菌的发光强度,研究黄曲霉毒素对费氏弧菌发光的影响.[结果]黄曲霉毒素浓度的对数值与费氏弧菌发光的抑制率呈线性关系,依据所得的回归方程可以快速准确地检测不同微生物产毒素的情况:6株不同来源的黄曲霉菌株均能够产毒素,以黄曲霉毒素含量表示的毒素量在14.94 - 46.45mg/L之间,1株米曲霉不产毒素.[结论]费氏弧菌发光强度的改变可以较准确地反映微生物产毒素的能力,尤其是微生物产黄曲霉毒素的能力,为在工农业生产中快速检测黄曲霉毒素提供了新的线索,有望发展成为一种检测黄曲霉毒素的新技术.