Genome-wide screen for oxalate-sensitive mutants of Saccharomyces cerevisiae.

Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Delta, ric1Delta, snf7Delta, vps16Delta, vps20Delta, and vps51Delta mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Delta, vps16Delta, vps51Delta, ric1Delta, and rib4Delta mutants, was lethal. With the exception of the rib4Delta mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Delta mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.

SSU1 mediates sulphite efflux in Saccharomyces cerevisiae.

Ssu1p, a plasma membrane protein involved in sulphite metabolism in Saccharomyces cerevisiae, was found to be required for efficient sulphite efflux. An SSU1 null mutant accumulated significantly more sulphite than wild-type, whereas cells expressing multicopy SSU1 accumulated significantly less. Cells expressing FZF1-4, a dominant allele of a transcriptional activator of SSU1 that confers sulphite resistance, also accumulated less sulphite. beta-galactosidase activity in the FZF1-4 strain carrying an SSU1::lacZ fusion was found to be 8.5-fold higher than in a strain carrying wild-type FZF1, confirming that the heightened resistance was correlated with hyperactivation of SSU1. Multicopy SSU1 was also found to increase the sulphite resistance of a number of unrelated sulphite-sensitive strains by a factor of 3- to 8-fold. Rates of efflux of free sulphite from cells expressing multicopy SSU1 or FZF1-4 were significantly greater than that from wild-type or from a SSU1 null mutant. Rates of efflux of bound sulphite from wild-type, a SSU1 null mutant, a FZF1-4 mutant, or cells expressing multicopy SSU1 were not significantly different, suggesting that Ssu1p specifically mediates efflux of the free form of sulphite.

Fzf1p of Saccharomyces cerevisiae is a positive regulator of SSU1 transcription and its first zinc finger region is required for DNA binding.

The FZF1 gene of Saccharomyces cerevisiae encodes a five-zinc-finger transcription factor involved in sulphite tolerance. Previous work based on multicopy suppression analysis placed FZF1 upstream of SSU1, which encodes a plasma membrane protein and putative transporter also implicated in sulphite detoxification. Consistent with this analysis, Fzf1p was found to be a positive regulator of SSU1 transcription. The SSU1 promoter region involved in activation by FZF1 was defined, and the FZF1 protein was shown to bind to it directly in vitro. Deletion of a single, amino-terminal zinc finger of Fzf1p resulted in loss of DNA binding, while the fourth and fifth zinc fingers were found to be dispensable.

Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats.

Lactic acid-producing bacteria prevent carcinogen-induced preneoplastic lesions and tumors in rat colon. Because the mechanisms responsible for these protective effects are unknown, two strains of lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus 191R and Streptococcus salivarius ssp. thermophilus CH3, that are used to produce yogurt, were investigated in vitro and in vivo to elucidate their potential to deactivate carcinogens. Using the "Comet assay" to detect genetic damage, we found that L. bulgaricus 191R applied orally to rats could prevent 1, 2-dimethylhydrazine-induced DNA breaks in the colon in vivo, whereas St. thermophilus CH3 were not effective. However, in vitro, both strains prevented DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in isolated primary rat colon cells. Extracts prepared from milk fermented with St. thermophilus CH3 were as efficient in deactivating MNNG as was L-cysteine. Isolated metabolites arising from bacteria during fermentation in the colon or in milk [L(+) lactate, D(-) lactate, palmitic acid and isopalmitic acid] were not effective. We postulate that thiol-containing breakdown products of proteins, via catalysis by bacterial proteases, could be one mechanism by which MNNG or other carcinogens are deactivated in the gut lumen resulting in reduced damage to colonic mucosal cells.

Use of sulfite resistance in Saccharomyces cerevisiae as a dominant selectable marker.

Two S. cerevisiae genes were found to exhibit dominant phenotypes useful for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZF1-4, which confers sulfite resistance, was originally isolated and identified as RSU1-4, but the two genes are shown here to be allelic. Cysteine 57 in wild-type Fzf1p was found to be replaced by tyrosine in Fzf1-4p. Multicopy SSU1, which also confers sulfite resistance, was found to be somewhat less efficient. In both cases, a period of outgrowth in non-selective medium following transformation was found to be necessary. The number of transformants obtained was found to be strain-dependent, and also to depend on the sulfite concentration used during selection. Undesirable background growth of non-transformants was not observed at cell densities as high as 2.5 x 10(7)/plate. In two ura3 laboratory strains where selection for URA3 was applied independently of that for sulfite, the transformation efficiency for sulfite resistance was about 50% that for uracil prototrophy.

Effects of hydrolysis of milk glycerides on the antimutagenicity of a hexane extract of milk.

Reconstituted nonfat dry milk was treated with different amounts of lipase from Pseudomonas fluorescens. Hexane extracts of treated milks were dissolved in dimethylsulfoxide and assayed for antimutagenicity using the Ames test (Salmonella typhimurium TA 100) against N-methyl, N'-nitro, N-nitrosoguanidine. Anti-N-methyl, N'-nitro, N-nitrosoguanidine activity increased significantly as the amount of added lipase increased. At the highest lipase concentration tested, activity increased 5-fold, suggesting that liberated fatty acids contributed to the increased antimutagenicity. The activities of mixtures of pure fatty acids on antimutagenesis were examined using the Ames test. At the lowest concentrations tested, mixtures of palmitic and stearic acids and mixtures of palmitic and isopalmitic acids exhibited greater activity than did the individual acids. At all doses tested, mixtures of the monoacylglycerides of palmitic and stearic acids exhibited the same activity as the individual components. Quantification of fatty acids in milk and yogurt by gas chromatography indicated a 2 to 20-fold greater content of free fatty acids in yogurt. The increase in free fatty acids may contribute to the increase in antimutagenicity of yogurt relative to that of milk.

Antimutagenic effects of milk fermented by Lactobacillus helveticus L89 and a protease-deficient derivative.

The antimutagenic effects of whey, acetone extracts, and protein fractions isolated from milk that had been fermented by Lactobacillus helveticus L89 were investigated using the mutagen 4-nitroquinoline-N'-oxide in the Ames test (Salmonella typhimurium TA 100). Fermented milk significantly inhibited mutagenesis induced by 4-nitroquinoline-N'-oxide. However, milk fermented by a nonproteolytic variant of the same strain showed no inhibitory effects. Results were similar for the whey fractions and acetone extracts of the fermented milks. After fermentation, milk proteins were fractionated by size-exclusion HPLC and were tested for antimutagenicity. The fraction showing the greatest activity was further analyzed by reverse-phase HPLC. Our results indicate that antimutagenic compounds are produced in milk during fermentation by L. helveticus, and the release of peptides is one possible contributing mechanism.

SSU1 encodes a plasma membrane protein with a central role in a network of proteins conferring sulfite tolerance in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SSU1 gene was isolated based on its ability to complement a mutation causing sensitivity to sulfite, a methionine intermediate. SSU1 encodes a deduced protein of 458 amino acids containing 9 or 10 membrane-spanning domains but has no significant similarity to other proteins in public databases. An Ssu1p-GEP fusion protein was localized to the plasma membrane. Multicopy suppression analysis, undertaken to explore relationships among genes previously implicated in sulfite metabolism, suggests a regulatory pathway in which SSU1 acts downstream of FZF1 and SSU3, which in turn act downstream of GRR1.

Ethanol production from spent cherry brine.

Spent cherry brine is an acidic byproduct of maraschino cherry processing and typically consists of variable amounts of glucose and fructose of up to 11% fermentable solids, 0.5-1.5% CaCl2, up to 0.4% sulfur dioxide, sorbitol, and lesser amounts of other cherry constituents. Disposal of brine represents a significant cost to processors because of its high biological oxygen demand. As an alternative, brine was tested as a substrate for ethanol production. Initially, the toxic level of sulfur dioxide was reduced by raising brine pH to 8.0 to precipitate calcium sulfite. Because alkalinization was subsequently found to result in a 10-fold reduction in phosphorous, brines were titrated with phosphoric acid to pH 6.0 prior to inoculation with Saccharomyces cerevisiae. All strains of Saccharomyces cerevisiae tested were able to ferment all lots of Ca(OH)2-treated and phosphorous-enriched brines efficiently. One lot of brine containing 10% (w/v) fermentable sugar yielded 4.7% (w/v) ethanol in 4 days.

Multicopy FZF1 (SUL1) suppresses the sulfite sensitivity but not the glucose derepression or aberrant cell morphology of a grr1 mutant of Saccharomyces cerevisiae.

An ssu2 mutation in Saccharomyces cerevisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants, the effect of CLN1 overexpression on sulfite sensitivity was investigated. Overexpression in GRR1 cells resulted in sulfite sensitivity, suggesting a connection between CLN1 and sulfite metabolism. Multicopy FZF1, a putative transcription factor, was found to suppress the sulfite sensitive phenotype of grr1 strains, but not the glucose derepression or aberrant cell morphology. Multicopy FZF1 was also found to suppress the sensitivity of a number of other unrelated sulfite-sensitive mutants, but not that of ssu1 or met20, implying that FZF1 may act through Ssu1p and Met20p. Disruption of FZF1 resulted in sulfite sensitivity when the construct was introduced in single copy at the FZF1 locus in a GRR1 strain, providing evidence that FZF1 is involved in sulfite metabolism.

Palmitic acid is the major fatty acid responsible for significant anti-N-methyl-N'-nitro-N-nitroguanidine (MNNG) activity in yogurt.

We describe here the isolation and identification of palmitic acid as being responsible for significant anti-N-methyl-N'-nitro-N-nitroguanidine (MNNG) activity in yogurt. The Ames test (Salmonella typhimurium TA100) was used to direct fractionation of activity. Yogurt was freeze-dried and extracted with acetone to yield a crude extract. The crude extract was purified by normal phase silica gel, Sephadex LH-20, and reversed phase medium pressure liquid chromatographies. The major compound in the active medium pressure liquid chromatographic fractions was determined to be palmitic acid on GC and high pressure liquid chromatography (HPLC) systems, and by nuclear magnetic resonance (NMR) analysis. Other saturated straight chain and methyl branched fatty acids were detected by GC/MS and were later shown to possess anti-MNNG activity. Of the straight chain fatty acids, palmitic acid had the highest anti-MNNG activity. All omega - 1 methyl branched fatty acids tested were more active than their straight chain counterparts. A trace amount of isopalmitic acid (14-methyl pentadecanoic acid), a minor milk lipid, was detected in one of the active fractions, and was later shown to be five times more active than palmitic acid. Isopalmitic acid also inhibited mutagenesis induced 4-nitroquinoline-N-oxide (4NQO), and 7, 12-dimethyl benz[a]anthracene (DMBA), and was found to inhibit the metabolic activation of DMBA.

Antimutagenicity of yogurt.

Yogurt is milk fermented by a mixture of two bacteria: Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus. Epidemiological studies have correlated a reduced risk of colon cancer with yogurt consumption. Independent studies have established that yogurt and extracts thereof are antimutagenic. Although multiple explanations can account for yogurt's putative anticarcinogenicity, we are interested in testing the hypothesis that antimutagenic compounds produced during fermentation are responsible. We recently reported on the antimutagenicity of an acetone extract of yogurt against the experimental carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 3.2'dimethyl-4-aminobiphenyl (DMAB) (Mutation Res. (1995) 334, 213-224). We are now aware that palmitic acid is an active ingredient against MNNG.

Antimutagenicity of an acetone extract of yogurt.

Reconstituted non-fat dry milk powder, fermented by a mixture of Streptococcus thermophilus CH3 and Lactobacillus bulgaricus 191R to produce yogurt, was freeze-dried and extracted in acetone. After evaporation of the acetone, the extract was dissolved in dimethyl sulfoxide (DMSO) and tested for antimutagenicity. In the Ames test, significant dose-dependent activity was observed against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-quinoline-N-oxide (4NQO), 3,2'-dimethyl-4-aminobiphenyl (DMAB), 9,10-dimethyl-1,2-benz[a]anthracene (DMBA), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2). Weak activity was observed against 1,2,7,8-diepoxyoctane (DEO), and no activity was observed against methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), or aflatoxin B1 (AFB1). In a related assay (Saccharomyces cerevisiae D7), significant antimutagenic activity was detected against MNNG and 4NQO. Activity against the experimental colon carcinogens MNNG and DMAB was examined further, as assayed in the Ames test (Salmonella typhimurium TA100). Compounds responsible for both activities were less soluble in aqueous solutions than in DMSO. Adjustment of yogurt pH to 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the amount of anti-MNNG activity recovered. In contrast, extractability of anti-DMAB activity was significantly greater at acidic pH. Conjugated linoleic acid, a known dairy anticarcinogen, failed to inhibit mutagenesis caused by either mutagen, suggesting that other yogurt-derived compound(s) are responsible. Unfermented milk was treated with lactic acid, yogurt bacteria without subsequent growth, or both, to determine if formation of antimutagenic activity required bacterial growth. Extracts of the milk treatments exhibited the same weak antimutagenicity observed in unfermented milk, approximately 2.5-fold less than in the yogurt extracts, suggesting that antimutagenic activity is associated with bacterial growth.

Antimutagenicity of fermented milk.

Reconstituted nonfat dry milk was fermented by Lactobacillus helveticus CH65, Lactobacillus acidophilus BG2FO4, Streptococcus salivarius ssp. thermophilus CH3, Lactobacillus delbrueckii ssp. bulgaricus 191R, and by a mixture of the latter two organisms. The fermented milks were then freeze-dried, extracted in acetone, dissolved in dimethylsulfoxide, and assayed for antimutagenicity in the Ames test (Salmonella typhimurium TA 100) against N-methyl, N'-nitro, N-nitroso-guanidine, and 3,2'-dimethyl-4-amino-biphenyl. Dose-dependent activity was significant against both mutagens in all extracts. Maximal inhibitory activity against 3,2'-dimethyl-4-aminobiphenyl and N-methyl, N'-nitro, N-nitroso-guanidine was 2- and 2.7-fold greater, respectively, than that exhibited by extracts of unfermented milk. Extracts of milk fermented by L. delbrueckii ssp. bulgaricus 191R were examined further. Compounds that were responsible for activity against both mutagens were less soluble in aqueous solutions than in dimethylsulfoxide. Adjustment of milk fermented by L. delbrueckii ssp. bulgaricus 191R to pH 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the activity specific for 3,2'-dimethyl-4-aminobiphenyl. In contrast, compounds with activity specific for N-methyl, N'-nitro, N-nitrosoguanidine were less extractable at pH 7.6. The weak antimutagenicity of unfermented milk was not increased by addition of 2% L-lactic acid.

Isolation and characterization of sulfite mutants of Saccharomyces cerevisiae.

Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.

The chromosomal constitution of wine strains of Saccharomyces cerevisiae.

A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII.

Conversion of Wine Strains of Saccharomyces cerevisiae to Heterothallism.

A general method to convert homothallic strains of the yeast Saccharomyces cerevisiae to heterothallism is described which is applicable to genetically well-behaved diploids, as well as to strains that sporulate poorly or produce few viable and mating-competent spores. The heterothallic (ho) allele was introduced into three widely used wine strains through spore x cell hybridization. The resultant hybrids were sporulated, and heterothallic segregants were isolated for use in successive backcrosses. Heterothallic progeny of opposite mating type and monosomic for chromosome III produced by sixth-backcross hybrids or their progeny were mated together to reconstruct heterothallic derivatives of the wine strain parents. A helpful prerequisite to the introduction of ho was genetic purification of the parental strains based on repeated cycles of sporulation, ascus dissection, and clonal selection. A positive selection to isolate laboratory-wine strain hybrids requiring no prior genetic alteration of the industrial strains, coupled with a partial selection to reduce the number of spore progeny needed to be screened to isolate heterothallic segregants of the proper genotype made the procedure valuable for genetically intractable strains. Trial grape juice fermentations indicated that introduction of ho had no deleterious effect on fermentation behavior.