Biochemistry of ungerminated and germinated spores of the vesicular-arbuscular mycorrhizal fungus, Glomus caledonius: changes in neutral and polar lipids.

Neutral and polar spore lipids of the vesicular-arbuscular (VA) endophyte Glomus caledonius, were identified and quantitatively determined during spore germination, germ tube growth, and germ tube senescence. There are no previous reports detailing the spore lipid components of any member of the Endogenaceae, which is in the Zygomycotina. The fungus contained 45 to 72% total lipid depending upon its stage of growth. The concentration of neutral lipids decreased during germination while the polar lipids increased. Triacylglycerides were the most abundant neutral lipid, and lesser amounts of diacylglycerides, monoacylglycerides, free fatty acids, bound fatty acids, hydrocarbons, and sterols. The major fatty acids identified by gas--liquid chromatography and mass spectrometry were 16:1, 16:0, and 18:1. The minor fatty acids identified were n-3 and n-6 polyunsaturates. The n-3 polyunsaturated fatty acids have not been reported before in Zygomycetes. The fatty acid composition of the individual lipid classes was examined. The major phospholipids were phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, with smaller amounts of diphosphatidylglycerol and phosphatidic acid. The free sterol fraction was in greater quantity than sterol esters during germination and germ tube elongation. The capacity to synthesize sterols was demonstrated. Approximate net rates of change in the different lipid components were calculated. During spore germination and early germ tube growth, there was a net synthesis of lipids, with a large production of free fatty acids, in the germinating spore. Later in the growth period there was a net degradation of lipid, characterized by a large conversion of free fatty acids to unidentified compounds. During this period net free sterol synthesis ceased and sterol ester synthesis continued using the existing free sterol.

Production of aseptic spores of vesicular-arbuscular endophytes and their viability after chemical and physical stress.

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30 degrees C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30 degrees C for Glomus caledonius or at 35 degrees C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 mug of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 mug/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40 degrees C for 80 min, 55 degrees C for 10 min, and 60 degrees C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 x 10 ergs cm from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.

Preservation of spores of vesicular-arbuscular endophytes by L-drying.

The spores of four species of vesicular-arbuscular endophytes were L-dried at 22 degrees C, and their viability was tested after heating at 80 degrees C for up to 40 min. L-drying of spores in the soil in which they developed was a very effective method of preservation of all spore types examined. Slow L-drying of spores separated from soil and supported on glass fiber filters also gave high viability for spores of some species. A scheme for the long-term preservation of vesicular-arbuscular endophyte spores is proposed.

Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion.

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 microgram mL-1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

Death of microbial cells: rate constant calculations.

The previously published procedure for calculation of rate constants associated with the death of microbial cells is shown to be so sensitive to variation in experimental data as to render it impractical for this application. The only obvious modification to the published procedure met with only limited success. It is concluded that a fresh search should be undertaken for the solution to this fundamental problem. The success of such further attempts may depend upon an alternative description of the normal probability integral rather than upon refined experimentation.

Effects of proteolytic enzymes on invertase secretion in sphaeroplasts of Saccharomyces: inhibition by trypsin.

Exogenously added trypsin arrested invertase secretion by sphacroplasts of Saccharomyces strain 1016. The mechanism of inhibition is presumed due to attack on plasma membrane protein(s). Gross membrane damage by trypsin was not apparent, as evidence by the absence of leakage of intracellular alkaline phosphatase, after trypsin treatment. Trypsin treatment did induce an increased sensitivity to lysis, observed only when changes in osmotic pressure were made and fresh glucose added. While synthesis of invertase was eventually inhibited by trypsin, a greater than twofold increase in internal invertase was observed, due to complete inhibition of secretion. This is the first report of the uncoupling of synthesis and secretion in yeast.

A modified procedure for studying enzyme secretion in yeast sphaeroplasts: subcellular distribution of invertase.

A significantly modified procedure for investigating enzyme secretion from yeast sphaeroplasts, and results from its application are described. Sphaeroplasts were derepressed for invertase biosynthesis in the presence of helicase and fractionated to reveal the distribution of high and low molecular weight forms of invertase. Secreted enzyme was found to be of high molecular weight, exclusively. Less than 10% of the total invertase activity was present in washed sphaeroplasts and of this, 43% was soluble, consisting of both high and low molecular weight forms of invertase. Washed membranes retained 32% of the internal invertase activity, and on solubilization with Triton X-100 the enzyme was found to be of an intermediate molecular weight. These results are consistent with the hypothesis that invertase is glycosylated at the plasma membrane.

Sub-cellular location of mercury in yeast grown in the presence of mercuric chloride.

The distribution of 203Hg in Saccharomyces cerevisiae grown in the presence of mercuric chloride has been examined by physical and chemical fractionation procedures and autoradiography. The major fraction of the bound mercury is tightly bound to the wall. A significant quantity of mercury penetrates to the cytoplasm but only a minor fraction is present as low molecular weight components. The wall associated mercury is not readily released by extraction with sodium hydroxide or ethylenediamine but a major fraction is solubilized by pronase and Helicase treatment. Isolated walls are capable of binding their own weight of mercury to high-affinity adsorption sites. The major role of the cell envelope in the in vivo binding of mercury and the penetration to the cytoplasm of mercury was confirmed by autoradiography.