Abstract
Poly-3-hydroxybutyrate (PHB), a biopolymer of important commercial applications, is found in a wide range of Gram-negative and Gram-positive bacteria and cyanobacteria. The present study has resulted in the identification of PHB in the luminescent marine bacteria, Vibrio harveyi, in spite of it being previously classified as PHB-negative. PHB granules with distinct membranes were detected by electron microscopy after fixation and staining of V. harveyi cells with malachite green. Analyses by gas chromatography, nuclear magnetic resonance, infrared, and ultraviolet spectroscopy clearly established the presence of PHB. The synthesis of PHB in V. harveyi was found to be under cell density regulation with the levels increasing from 0 (< 0.2) to 26 mg of PHB/g of dry cell weight during growth in a manner analogous to the induction of luminescence in this bacteria. Moreover, synthesis of PHB in V. harveyi was shown to be controlled by the lux autoinducer, N-(3-hydroxybutanoyl)homoserine lactone, providing not only a potential link between luminescence and PHB production but also showing that the lux autoinducer acts as a general signal transductant. These results have also extended the role of homoserine lactones in metabolic regulation to include the control of synthesis of potential energy reserves.
Highlights
Ner analogous to the induction of luminescencein this evidence is rapidly accumulating that homoserine bacteria
A constant amount of cells (OD, x volume = 10) were collectedand dried a t 110 "C to constant weight at different stages of cell growth (OD, = 1.5 to 5.5).An average of 5.1 mg of cells with a standard deviation of 0.4 mg was obtained with no dependence on cell density from OD, of 1.5-5.5, allowingdata to be presented per gram of matography (GC)analysis by followingthe procedure of Braunegg et al (1978).V. harueyi cells(200 ml) weresuspended in a mixture of 2 ml of acidic methanol (3% H,SO,v/v) and 2 mlofchloroform in a screw capped tube, and heated at 100 "C for 3% h
Peared tobe due to PHB, wiot uld be expected that the number Comparable results were obtained withanother autoinof sponge-like granules percell observed by electron microscopy ducer-deficient mutant, Dl,except PHB could not be detected should increase as the level of PHB rises with cell growth
Summary
Harueyi was recorded in CDC1, at room temperature and Polysciences Inc. Malachite green, poly-3-hydroxybutyrate, sodium compared with that of a reference. A constant amount of cells (OD,,, x volume = 10) were collectedand dried a t 110 "C to constant weight at different stages of cell growth (OD,,, = 1.5 to 5.5).An average of 5.1 mg of cells with a standard deviation of 0.4 mg was obtained with no dependence on cell density from OD,,, of 1.5-5.5, allowingdata to be presented per gram of matography (GC)analysis by followingthe procedure of Braunegg et al (1978).V. harueyi cells(200 ml) weresuspended in a mixture of 2 ml of acidic methanol (3% H,SO,,v/v) and 2 mlofchloroform in a screw capped tube, and heated at 100 "C for 3% h. Were harvested at different times of growth, prefured in situ at room temperature for 30 min by adding an equal volume of2% glutaralde-
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