Thiamine-induced alteration in sterol composition of Saccharomyces carlsbergensis 4228.

The cells of Saccharomyces carlsbergensis 4228 grown with added thiamine in a vitamin B-6-free medium did not contain ergosterol and zymosterol (the predominant sterols in cells grown without thiamine). Instead, significant amounts of squalene, lanosterol and unidentified sterols accumulated in the thiamine-grown cells. Of the unidentified sterols, the most predominant was delta5,7-ergostadien-3beta-ol at 26.0% of the total sterol. The accumulation of this sterol in the place or ergosterol (delta5,7,22-ergostatrien-3beta-ol) indicates that the desaturation at C-22 of sterol structure is completely blocked in the thiamine-grown cells. On the basis of chromatographic behaviours, the structures of other unidentified sterols were assumed to be 4alpha-methyl-delta8,24(25)-cholestadien-3beta-ol, 4alpha-methyl-delta 8,24(28)-ergostadien-3beta-ol, delta8,24(28)-ergostadien-3beta-ol and delta5,7,24(28)-ergostatrien-3beta-ol. The accumulation of 4alpha-methyl sterols, in addition to that of a large amount of lanosterol (48.4% of total sterol), suggests that the demethylation processes from lanosterol to zymosterol was partially depressed in the thiamine-grown cells.

Effects of thiamine and pyridoxine on the lipid composition of Saccharomyces carlsbergensis 4228.

The lipid composition of Saccharomyces carlsbergensis 4228 cells grown aerobically in the presence of thiamine and absence of pyridoxine was markedly different from that of cells grown without addition of both of the growth factors. In addition to the previous observations showing a reduction in the levels of unsaturated fatty acids (Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem. Biophys. Res. Commun. 59, 777-780) and lack of zymosterol and ergosterol (Nagai, J., Katsuki, H., Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem. Biophys. Res. Commun. 60, 555-560), the thiamine-grown cells were found to contain low levels of total lipids, sterols (especially in the form of esters), triacylglycerols and total phospholipids. However, relative contents of triacylglycerols and phospholipids to total lipids were higher than those of control cells. Hydrocarbons and diacylglycerols accumulated to appreciable degrees. Phospholipid composition was also influenced by thiamine. The ratio of phosphatidylinositol to total phospholipids increased, whereas that of phosphatidylethanolamine decreased. The levels of phosphatidylcholine plus phosphatidylserine decreased in a similar ratio to that of total phospholipids. It was found that unsaturated fatty acid contents were low in all lipid esters tested. The effect of thiamine was particularly noteworthy in the case of sterol esters. Concomitant addition of pyridoxine with thiamine to the medium brought about a normal lipid composition in the yeast cells.

Ultrastructural alterations in Saccharomyces cerevisiae cells in association with elevated temperature-induced autolysis.

By means of the freeze-etching technique ultrastructural alterations in Saccharomyces cerevisiae cells undergoing autolysis at elevated temperature were studied. Wall surfaces of intact cells were smooth. During autolysis wall surfaces became rough with granules of 20-40 nm diameter. This alteration occurred after extensive disintegration of cytoplasmic organelles and after functional and ultrastructural impairments of the plasma membrane, but well before the rupture of the plasma membrane.

[A search for specific genes working on the process of mycelial growth in Candida tropicalis].

Ethanol has been reported to cause mycelial growth in Candida tropicalis Pk233. Cultivation with ethanol in synthetic media containing glucose gave biphasic growth curves. During the first growth phase, there was an accumulation of swollen spherical yeast cells, instead of the oblong ones observed in the control culture, followed by the appearance of spherical daughter cells in chains. During the second growth phase, pseudohyphal cells appeared, projecting from the swollen yeast cells.Subtractive cloning was performed on cDNAs from both cultures to isolate genes expressed during the first phase, correlating to the process of ethanol induced hyphal growth. Subtracted cDNAs identified by homology search included a homologue of URP2 coding ribosomal protein S20, a homologue of nmt1 coding a regulator gene working on thiamine metabolism, and a homologue of MSG5 coding tyrosine phosphatase. Roles of these cloned homologues were discussed on the process of mycelial growth in this organism.

[Subtractive gene cloning using dynabeads oligo(dT)25 for elucidation of pseudohyphal formation in Candida tropicalis].

The dimorphic transition from yeast to pseudohyphae in Candida tropicalis occurs following the addition of ethanol to a synthetic medium containing glucose. We developed a method of subtractive gene cloning to isolate genes, of which the expression was apparently specific for pseudohyphal formation in this organism. Subtraction was performed between sense-strand cDNAs instead of mRNAs from cells of the ethanol culture and anti-sense cDNAs linking to Dynabeads oligo(dT)25 from those of the control culture. Dynabeads oligo(dT)25 are paramagnetic beads with 25 nucleotide-long chains of deoxythymidines covalently linked to their surface and were expected to be easily collected using a magnet. This method using Dynabeads oligo(dT)25 minimizes the degradation of mRNA and makes it easy to construct a cDNA library sufficient to analyze the genetic information on the yeast-to-hyphae transition. Using this strategy, we identified several genes including a homologue of CPP1 coding tyrosine phosphatase and a homologue of nmt1+ encoding protein, which was reported to regulate thiamine biosynthesis.

Role of pyruvate metabolism in the growth of Streptococcus faecalis in the presence of propionate.

The growth of Streptococcus faecalis is inhibited by propionate, and the inhibition is reversed by lipoic acid or acetate. A study of the role of pyruvate oxidation in S. faecalis showed that propionate inhibited the lipoic acid-dependent aerobic oxidation of pyruvate in resting cells. Pyruvate dehydrogenation with neotetrazolium as a hydrogen acceptor in cell-free extracts also required lipoic acid and was markedly inhibited by propionyl phosphate as well as sodium propionate. Some lipid substances, such as palmitate, oleate, behenate, and lecithin, had a lipoic acid-replacing effect on growth of the organism. Biotin or bicarbonate promoted the lipoic acid-dependent growth. Acetate-2-(14)C added to the medium was mainly incorporated into the lipid fraction of the cells. Evolution of (14)CO(2) from pyruvate-2-(14)C was not observed in resting cells of the organism, even under aerobic conditions. From the above findings, it is concluded that lipid synthesis through pyruvate oxidation plays a very important role in bacterial growth in medium containing propionate.

Increase in cyclic AMP content with enhanced phosphatidylinositol turnover in the cells of Candida tropicalis during mycelial growth caused by ethanol.

Ethanol causes mycelial growth of Candida tropicalis Pk 233, which is associated with enhanced metabolism of phosphatidylinositol at the mid-log phase of growth, and the effects of ethanol are prevented by concomitant addition of myo-inositol (FEBS Lett. 214, 127-129, 1987). Ethanol induced also a marked increase in cellular content of cAMP at the mid-log phase, and myo-inositol abolished this effect of ethanol. The elevated level of cAMP content caused by ethanol was gradually lowered through the late-log and stationary phases and reached to control level. Very similar effects of ethanol and myo-inositol were observed in adenylate cyclase activity, while the activity of cAMP phosphodiesterase was not affected by ethanol. The ethanol-induced change in cAMP content was therefore ascribed to that in adenylate cyclase activity. These results suggested that cAMP plays an important role in combination with phosphatidylinositol turnover in the development of mycelial form in this dimorphic yeast.

Accumulation of cAMP in the cells of Candida tropicalis at an early stage of ethanol-induced filamentous growth and its prevention by myo-inositol.

Phosphatidylinositol metabolism is enhanced in the cells of Candida tropicalis Pk 233 at an early stage of filamentous growth caused by ethanol, and myo-inositol prevents the ethanol-induced changes in the metabolism and morphology [Uejima et al. (1987) FEBS Lett. 214, 127-129]. The accumulation of cAMP and an increase in adenylate cyclase activity were observed in the cells grown with ethanol to the mid-log phase. Myo-inositol abolished these effects of ethanol also. The activity of cAMP phosphodiesterase was affected by neither ethanol nor myo-inositol. These results suggest that the inositol phospholipid-linked and cAMP-linked signaling pathways may be involved in the mechanism of ethanol-induced filamentous growth of this yeast and also that myo-inositol would affect morphogenesis by controlling these pathways.

Activation by monovalent cations of dissimilatory nitrate reductase in the cells of Pseudomonas denitrificans.

Cellular activity of nitrate reductase in Pseudomonas denitrificans which had been grown under denitrifying conditions was increased several times upon incubation of cell suspension with monovalent cations. The enhancement of nitrate reductase activity caused by monovalent cations was ascribed to the activation of the enzyme, since the membrane fraction isolated from the cells after the cation treatment retained the elevated levels of enzyme activity. However, monovalent cations had no effect when added directly to cell-free homogenate, suggesting an important role of some definite structure of membrane in the expression of the effect of monovalent cations.

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast.

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.

Mechanism of thiamine-induced respiratory deficiency in Saccharomyces carlsbergensis.

Cells of Saccharomyces carlsbergensis 4228 grown aerobically with added thiamine (1 microgram . ml-1) in a vitamin B6-free medium contained no detectable heme precursors, such as delta-aminolevulinate, coproporphyrin III, or protoporphyrin IX. The deficiency in heme precursors in the thiamine-grown cells was accompanied by previously reported phenomena, i.e., growth depression, vitamin B6 deficiency, and respiratory deficiency due to a marked decrease in the activities of heme-containing enzymes and cytochrome level (I. Nakamura et al., FEBS Lett. 62: 354-358, 1976). It has been reported that all of the effects of thiamine are abolished by adding pyridoxine to the medium. delta-Aminolevulinate was found to have quite similar effects to those of pyridoxine, except that growth was partially improved by delta-aminolevulinate, whereas it was fully restored by pyridoxine. Incubation of the thiamine-grown cells with delta-aminolevulinate resulted in the appearance of the heme precursors and the heme-containing enzymes. Consistent with the lowered amount of vitamin B6, the thiamine-grown cells had a lowered activity of delta-aminolevulinate synthase, a pyridoxal phosphate-dependent enzyme. Not only the holoenzyme activity but also the apoenzyme activity was very low in these cells. These results indicate that the thiamine-induced vitamin B6 deficiency brings about the decrease in delta-aminolevulinate synthase activity, which leads to heme deficiency and therefore to respiratory deficiency.