A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,llaS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCI2-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonasphaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucwmerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol.Phytoalexins are low mol wt antimicrobial compounds produced by plants in response to challenge by microorganisms. These compounds accumulate at the site of infection and are involved in the plant's defense response to potential pathogens (3,8,14).The accumulation of the pterocarpan phytoalexins, glyceollin4 (Fig. 1), by soybean (Glycine max) in response to fungal attack has been well documented (12). In vitro studies have shown glyceollin to be inhibitory to the growth of both bacteria and fungi (1,11,12 for which we propose the trivial name glycinol. This pterocarpan has been previously observed as a quantitatively minor constituent in CuCl2-treated soybean cotyledons, although no antifungal activity was recognized to be associated with it (18). The present study describes an improved isolation procedure for the pterocarpan leading to mg quantities of the crystalline compound. We present also the results of our studies of the effect of glycinol on growth of a variety of microorganisms. MATERIALS AND METHODSPlant Material. Certified quality soybean seed (Glycine max [L.] Merr. cv. Wayne) was obtained from Wilkens Seed Co.Pontiac, IL. The seed was stored in closed containers at 4 C and hand sorted for soundness before planting. The sorted seeds were planted at a density of about 75 g seed/tray (40 x 30 x 9 cm) on a 2-cm layer of potting soil (Bacto Potting Medium, Michigan Peat Co., Houston, TX) overlaying a water-soaked 4-cm layer of vermiculite (W. R. Grace and Co., Cambridge, MA). The seed was then overlayed with a 2 cm layer of vermiculite, and watered with 400 ml deionized H20/...
The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits -lactate-dependent transport of I14CIglycine but not vectorial phosphorylation-mediated transport of 114CIa-methyl glucoside. Thus, capsidiors mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.Phytoalexins are low-mol-wt antibiotics produced by plants in response to various biotic and abiotic elicitors (1, 2,20,27,31,39 glycinol may inhibit the growth of widely diverse microorganisms by a similar mechanism of action (45).Previous studies on the mechanism of action of structurally diverse phytoalexins have implicated the membrane as the site of antimicrobial action (19,23,29,30,34,42). The research detailed in this paper was undertaken to determine the locus of action of glycinol. A comparison ofglycinol's effect on bacterial membranes with the effect on the membranes of other phytoalexins has allowed us to examine the question of whether the mode of action of glycinol is representative of other phytoalexins. (i-z-y+a ), a gift of Dr. R. Kaback, Roche Institute of Molecular Biology, Nutley, NJ, was maintained on nutrient agar (Difco) slants and plates. The E. coli used in the preparation of membrane vesicles was grown on Escherichia medium 63 (9). MATERIALS AND METHODS Materials. L-General. Liquid culture assays of the ability of glycinol to inhibit bacterial growth were performed as described (45). Glycinol concentrations were determined by A at 287 nm in absolute ethanol (Es7 = 5800). Membrane vesicles were prepared from E.coli following the method of Kaback (14), except that a 20-cc syringe equipped with an 18-gauge needle, rather than a Teflon and glass homogenizer, was used for resuspension of the membrane pellet (H. R. Kaback, personal communication). The membrane-vesicle preparations were shown to be free of bacterial contamination by incubation on nutrient agar. Protein concentrations were determined by the procedure of Bradford (7)
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