Supersensitivity to levorin and amphotericin B in several Saccharomyces cerevisiae mutants resistant to nystatin]

104 mutants resistant to nystatin were isolated after UV-treatment of two haploid marked strains of Saccharomyces cerevisiae. The analysis of resistance to three polyene antibiotics allowed to determine 8 phenotype classes of mutants including those resistant to nystatin but in various combinations showing hypersensitivity to levorin and (or) amphotericin B. The analysis of UV absorption spectra of sterolic extracts prepared from cells of different mutants showed that similar quality changes in sterol composition could be associated both with polyresistant an supersensitive phenotype. New type of mutants resistant to nystatin and supersensitive to levorin and (or) amphotericin B seems to be promising for studies on the mechanisms of action of polyene antibiotics, the bases of resistance to them and also in consideration of the possibility to increase the efficiency of antimycotic antibiotic therapy.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

Effect of altered sterol composition on the salt tolerance of Zygosaccharomyces rouxii

To obtain mutants containing altered sterol composition and sterol contents, nystatin-resistant mutants were isolated in Zygosaccharomyces rouxii. Two of nine mutants isolated were resistant toward 20 μg of nystatin per ml, while the other seven showed resistance toward 50 μg per ml. However, the seven mutants could not grow at 35°C. TN5, a mutant of the first group, showed the same sterol composition as the wild type strain, with ergosterol and zymosterol as major sterols, whereas it contained free sterols about 70% of those of the wild type. TN1 and TN3, representative mutants of the second group, had altered sterol compositions, containing three major sterols, zymosterol, ergosta-5,7,24-trienol, and an unidentified sterol. TN1 and TN3 could not grow in YPD medium containing more than 8% NaCl, whereas TN5 grew in the same medium containing 15% NaCl after a longer lag phase than the wild type strain. TN1 and TN3, in particular TN3, when incubated in YPD medium containing 15% NaCl, leaked significant amounts of glycerol. Protoplasts of these mutants were more labile than those of the wild-type cells. These facts suggest that the amount and kind of ergosterol in the cell membrane might be concerned with the salt tolerance of Z. rouxii.     

Biochemical basis of varying nystatin resistance of Saccharomyces cerevisiae and Candida maltosa mutants]

Six groups of nystatin resistant mutants of C. maltosa and of haploid and diploid Saccharomyces cerevisiae strains were obtained with the help of genetic and biochemical analysis. It has been shown that every group of the mutants was characterized by a specific level of resistance to nystatin. The dependence of the resistance level upon sterol content has been established. It has been shown that the more the structure of the sterol present differed from ergosterol the higher was the resistance level. The results obtained in vivo permit to make conclusions about the role of different functional groups of sterol molecule in the interaction with nystatin.     

Yeast resistance to polyene antibiotics. IV. A study of the inheritance of changes in the sterol composition of Saccharomyces cerevisiae yeasts caused by mutations of nystatin resistance

Sterol content in haploid and diploid strains of yeast having mutations of resistance to nystatin were studied by UV spectrometry method. Heterozygous diploids carrying one or two nystatin resistance mutations have, as a rule, the sterol content of the wild type strains. Segregants of the same genotype demonstrate differences in sterol content. Double mutants nys1 nys2 and nys1 nys3 have UV spectra typical for single nys2 and nys3 mutants, respectively. Double mutants nys1 nysX are characterized by a "mixed" UV spectra of sterols.     

Genetic, biochemical, and developmental studies of nystatin resistant mutants in Dictyostelium discoideum

Summary Conditions are given for the isolation of nystatin resistant mutants of the cellular slime mold Dictyostelium discoideum. These mutants fall into three phenotypic groups; corresponding to three genes: nysA, nysB, and nysC. Mutants in nysB and nysC affect sterol metabolism since they have altered sterol compositions. Each group contains several unique, but as yet unidentified, sterols in place of the wild type sterol. The nysC strains are most nystatin resistant, display altered sensitivity to some drugs, and grow on nystatin from amoebae or spores. All other mutants are nystatin resistant only as amoebae. Although nysC mutants grow normally, they make small fruiting bodies which appear to result from the formation of smaller aggregates.Supported by N.I.H. grant GM 18476     

Identification of a sterol mutant of Neurospora crassa deficient in delta 14,15-reductase activity.

A mutant (erg-3) of Neurospora crassa resistant to the polyene antibiotic nystatin was compared with its sensitive, wild-type parent to detect differences in sterol composition using gas chromatography-mass spectrometry. The major sterol in wild-type mycelia, comprising 80% of the total, was ergosterol. The major sterols in mutant mycelia, comprising 86% of the total, were delta 8,14-sterols. It is proposed that the nystatin-resistant strain is unable to synthesize ergosterol because it lacks delta 14,15-reductase activity as a result of a mutation in the erg-3 gene.     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Effects of pisatin on Dictyostelium discoideum: its relationship to inducible resistance to nystatin and extension to other isoflavonoid phytoalexins

Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by pisatin is probably not via an alteration of membrane sterols. The inducible pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation. Received: 23 February 1998 / Accepted: 26 June 1998     

Yeast mutants requiring ergosterol as only lipid supplement

Mutants of $\text{Saccharomyces cerevisiae}$ were isolated which required ergosterol or cholesterol as the only lipid supplement. They also required methionine, were petite, and showed complete absence of respiratory cytochromes. Revertants of these strains grew without ergosterol and methionine, were grande, and had respiratory cytochromes. Most revertants did not make ergosterol and were nystatin resistant. Sterol analysis and enzyme assays suggested a block in sterol formation after lanosterol.     

Study of the resistance of Candida guilliermondii to polyene antibiotics

250 Candida guilliermondii strains resistant to the polyene antibiotics nystatin, levorin and amphotericin B were obtained using UV irradiation. When the mutant strains became resistant to one of the polyene antibiotics, their resistance to the other ones changed. Phenotypic analysis showed that the resistance of the strains to polyene antibiotics did not make them susceptible to a rise in osmotic pressure and to a change of the temperature of incubation. Some of the polyene-resistant strains were stained in a medium with methylene blue. Analysis of the sterol composition in the mutants by UV spectroscopy showed that the resistance to polyene antibiotics sometimes involved changes in the sterol composition. Two new UV spectrum types were recorded for the sterols of the mutant strains; they differed from the UV spectrum for the sterols of the parent sensitive strain.     

Yeast resistance to polyene antibiotics. III. Phenotypic analysis of polyene-resistant sterol mutants of Saccharomyces cerevisiae yeasts

The pleiotropic effects of nystatin resistant mutations have been studied. It has been shown that resistance to nystatin is often accompanied by temperature-sensitivity and, in some cases, by osmotic remedial sensitivity. The dependence of the expression of the mutations on temperature is demonstrated. A new type of conditional nystatin-dependent mutants is found. All nys2 mutants have a defected membrane and are stained on a methylene blue containing media. At the same time, the mutations under investigation do not affect the respiratory competence and the exoenzyme activity of acid phosphatases.     

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.     

Overlapping functions of the yeast oxysterol-binding protein homologues

The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.     

Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation

Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae. Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S. cerevisiae. Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants. While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions. Also, the wild type grown anaerobically was not affected by iron-limited conditions. Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants. During iron limitation, the wild type showed a reduced oxygen uptake rate. Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol. Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

Yeast genes involved in growth inhibition by Pseudomonas syringae pv. syringae syringomycin family lipodepsipeptides

Abstract Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv. syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses. Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups. Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol. All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin. The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action.     

Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae

A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism. The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell. There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability. When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin. When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased. Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.     

The effect of altered sterol composition on cytochrome oxidase and S-adenosylmethionine: delta 24 sterol methyltransferase enzymes of yeast mitochondria

Arrhenius kinetics of two mitochondrial enzymes, cytochrome oxidase and S-adenosylmethionine: Δ 24 sterol methyltransferase were analyzed in wild-type and sterol mutant strains of yeast. Temperature effects on the enzymes isolated from the ergosterol producing wild-type and nystatin resistant mutants (major sterol Δ⁸(9), 22 ergostadiene-3-β-ol) were compared. Transition temperatures were lower in both mutant strains compared to wild-type. Lipid analysis shows a relationship between sterol content and the temperature dependent transition phases.