Showing papers in "Yeast in 2002"

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Abstract: Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Δ strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2ald6Δ strain was slightly less than that of the other strains from 24 h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains. Copyright © 2002 John Wiley & Sons, Ltd.

A microarray-assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae.

Abstract: Saccharomyces cerevisiae adapts to altered oxygen availability by differentially expressing a number of genes. Under aerobic conditions oxygen control of gene expression is exerted through the activator Hap1 and the repressor Rox1. The Hap1 transcription factor senses cellular heme status and increases expression of aerobic genes in response to oxygen. The repression of hypoxic genes under normoxic conditions results from Hap1-mediated activation of ROX1 transcription. To allow the identification of additional Hap1 and Rox1 target genes, genome-wide expression was analysed in aerobically, chemostat-cultivated hap1 and rox1 null mutants. The microarray results show that deletion of HAP1 causes a lower transcript level of 51 genes. Transcription of 40 genes was increased in rox1 mutant cells compared to wild-type cells. Combining these results with our previously described transcriptome data of aerobically and anaerobically grown cells and with computational analysis of the promoters identified 24 genes that are potentially regulated by Hap1, and 38 genes satisfied the criteria of being direct targets of Rox1. In addition, this work provides further evidence that Rox1 controls transcription of anaerobic genes through repression under normoxic conditions.

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Abstract: Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators

Abstract: We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energy-dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.

Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6.

Abstract: In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ1-3 and SNO1-3. In media lacking vitamin B(6), SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B(6) is essential for growth, copies 2 and 3 seem more related with B(1) biosynthesis during the exponential phase.

Metabolic physiology of aroma-producing Kluyveromyces marxianus

Abstract: Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.

Putative xylose and arabinose reductases in Saccharomyces cerevisiae

Abstract: Saccharomyces cerevisiae mutants, in which open reading frames (ORFs) displaying similarity to the aldo-keto reductase GRE3 gene have been deleted, were investigated regarding their ability to utilize xylose and arabinose. Reduced xylitol formation from D-xylose in gre3 mutants of S. cerevisiae suggests that Gre3p is the major D-xylose-reducing enzyme in S. cerevisiae. Cell extracts from the gre3 deletion mutant showed no detectable xylose reductase activity. Decreased arabitol formation from L-arabinose indicates that Gre3p, Ypr1p and the protein encoded by YJR096w are the major arabinose reducers in S. cerevisiae. The ypr1 deletion mutant showed the lowest specific L-arabinose reductase activity in cell extracts, 3.5 mU/mg protein compared with 7.4 mU/mg protein for the parental strain with no deletions, and the lowest rate of arabitol formation in vivo. In another set of S. cerevisiae strains, the same ORFs were overexpressed. Increased xylose and arabinose reductase activity was observed in cell extracts for S. cerevisiae overexpressing the GRE3, YPR1 and YJR096w genes. These results, in combination with those obtained with the deletion mutants, suggest that Gre3p, Ypr1p and the protein encoded by YJR096w are capable of xylose and arabinose reduction in S. cerevisiae. Both the D-xylose reductase and the L-arabinose reductase activities exclusively used NADPH as co-factor.

Functional analysis of structural genes for NAD+-dependent formate dehydrogenase in Saccharomyces cerevisiae

Abstract: Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD+-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Δ null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Δfdh2Δ double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose–formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate. Copyright © 2002 John Wiley & Sons, Ltd.

PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes.

Abstract: For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.

HXT5 expression is determined by growth rates in Saccharomyces cerevisiae.

Abstract: In the yeast Saccharomyces cerevisiae, hexose transporter (Hxt) proteins transport glucose across the plasma membrane. The Hxt proteins are encoded by a multigene family with 20 members, of which Hxt1-4p and Hxt6-7p are the major hexose transporters. The remaining Hxt proteins have other or unknown functions. In this study, expression of HXT5 under different experimental set-ups is determined. In glucose-grown batch cultures, HXT5 is expressed prior to glucose depletion. Independent of the carbon source used in batch cultures, HXT5 is expressed after 24 h of growth and during growth on ethanol or glycerol, which indicates that growth on glucose is not necessary for expression of HXT5. Increasing the temperature or osmolarity of the growth medium also induces expression of HXT5. In fed-batch cultures, expression of HXT5 is only observed at low glucose consumption rates, independent of the extracellular glucose concentration. The only common parameter in these experiments is that an increase of HXT5 expression is accompanied by a decrease of the growth rate of cells. To determine whether HXT5 expression is determined by the growth rate, cells were grown in a nitrogen-limited continuous culture, which enables modulation of only the growth rate of cells. Indeed, HXT5 is expressed only at low dilution rates. Therefore, our results indicate that expression of HXT5 is regulated by growth rates of cells, rather than by extracellular glucose concentrations, as is the case for the major HXTs. A possible function for Hxt5p and factors responsible for increased expression of HXT5 upon low growth rates is discussed.

Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1.

Abstract: Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide. Three genes, MET3, MET14 and MET16, are essential for this reduction. We investigated the level of transcription of these genes in strains of S. cerevisiae with high, medium and low sulphite formation. The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains. We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation. Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16. The overexpression of these two genes leads to a two- to three-fold sulphite formation. In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph. The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold. Furthermore, sulphite formation strongly depends on growth conditions, e.g. yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media. The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions.

Characterization of the putative maltose transporters encoded by YDL247w and YJR160c

Abstract: The maltose permease family of Saccharomyces cerevisiae comprises five proteins, three of which are characterized, MAL31, MAL61 and AGT1 and two putative permeases, YDL247w (MPH2) and YJR160c (MPH3). The two uncharacterized permeases share 100% identity and have 75% identity with MAL31 and MAL61 and 55% identity with AGT1. Characterization of the genes YDL247w and YJR160c confirmed that they encode α-glucoside permeases capable of transporting maltose, maltotriose, α-methylglucoside and turanose. Analysis of the promoter regions identified regulatory elements, binding sites for the transcriptional activator, Malx3p and the inhibitory protein, Mig1p. Further analysis of the flanking sequences located blocks of identity covering five open reading frames, indicating that this region was involved in chromosomal block duplication. The members of the maltose permease family are proteins that have strongly overlapping but nevertheless distinct functions, which is a selective advantage for yeast, as it reflects successful adaptation to the variety of environmental conditions to which the yeast cells are exposed; such adaptability is very important in an industrial context. Copyright © 2002 John Wiley & Sons, Ltd.

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae.

Abstract: Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.

Phenotypic analysis of gene deletant strains for sensitivity to oxidative stress.

Abstract: Ascertaining the impact of inhibitors on the growth phenotype of yeast mutants can beuseful in elucidating the function of genes within the cell. Microtitre plates and robotics have been used to screen over 600 deletions from EUROSCARF, constructed in an FY1679 strain background, for sensitivity to various oxidants. These included the inorganic hydroperoxide, H2O2, an organic peroxide (cumene hydroperoxide) and a lipid hydroperoxide (linoleic acid hydroperoxide). These produce within the cell several different reactive oxygen species that can cause damage to DNA, proteins and lipids. Approximately 14% of deletants displayed sensitivity to at least one of the oxidants and there was also a distribution of deletants that showed sensitivity to all or different combinations of the oxidants. Deletants included genes encoding proteins involved in stress responses, heavy metal homeostasis and putative cell wall proteins. Although global mechanisms have been identified that provide general stress responses, these results imply that there are also distinct mechanisms involved in the protection of the cell against specific damage caused bydifferent oxidants. Further analysis of these genes may reveal unknown mechanisms protecting the cell against reactive oxygen species. Copyright © 2002 John Wiley & Sons, Ltd.

Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast.

Abstract: Counter-selection is a useful gene manipulation technique for repeated gene disruptions, gene shufflings and gene replacements in yeasts. We developed a novel counter-selection system using a galactose-inducible growth inhibitory sequence (Kawahata et al.1999. Yeast 15: 1-10). This counter-selection marker, named GAL10p-GIN11, has several advantages over previous counter-selection markers, i.e. use of an inexpensive galactose medium for counter-selection, combined use with any transformation markers for gene introduction, and no requirement of specific mutations in the host strains. The GIN11 sequence, which is a part of an X-element of the subtelomeric regions, contained a conserved autonomously replicating sequence, causing the possibility of inefficient chromosomal integration. We isolated GIN11 mutants that lost the replication activity but retained the growth-inhibitory effect when overexpressed. A mutant GIN11M86 sequence was selected and fused to the CUP1 promoter for the counter-selection on a copper-containing medium. The GALp-GIN11M86 and the CUPp-GIN11M86 were used for constructing sets of integrating plasmids containing auxotrophic markers involving HIS3, TRP1, LEU2, URA3 or ADE2, or a drug-resistant marker PGKp-YAP1. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the GALp-GIN11M86, which were flanked by direct repeats of a hisG sequence, were constructed. The counter-selectable integrating plasmids and the gene disruption cassettes can allow the markers to be used repeatedly for yeast gene manipulations.

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers.

Abstract: Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces.

The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation.

Abstract: We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1–7 under enological conditions. A strain in which the sugar carrier genes HXT1–HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions. No growth or fermentation was observed in winemaking conditions for the hxt1-7Δ strain. The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation. However, these two carriers have different functions. The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone. The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation. The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation. These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation. This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle. Copyright © 2002 John Wiley & Sons, Ltd.

A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis.

Abstract: Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1ppz2 double disruptant showed temperature-sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl2. The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl2. The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases. Copyright © 2002 John Wiley & Sons, Ltd.

ARL1 and membrane traffic in Saccharomyces cerevisiae.

Abstract: To examine the functions of the Arf-like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1Δ::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature-sensitive (ts) growth (suppressed on high-osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1Δ strains secreted less protein as measured by TCA-precipitable radioactivity found in the media of [35S]-labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluid-phase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1Δssd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic. Copyright © 2002 John Wiley & Sons, Ltd.

Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens.

Abstract: Insertional mutagenesis is a powerful tool for the isolation of novel mutations. The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants. To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S. cerevisiae genome. The position of 54 of these T-DNAs was determined. The T-DNA showed no clear preference for certain DNA sequences or genomic regions. We have isolated insertions in the coding regions of the genes YGR125w, YDR250c, YGR141w, YGR045c, YPL017c, YGR040w, YDL052c, YJL148w, YCL033c, YFL061w, YJR033c, YDR175c and YLR309c confirming that these genes are non-essential for S. cerevisiae haploid growth on minimal medium. Given the advantages of T-DNA, we propose its use as an ideal mobile DNA element for insertional mutagenesis in yeasts.

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Abstract: Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.

Siderophore uptake by Candida albicans: effect of serum treatment and comparison with Saccharomyces cerevisiae

Abstract: Iron uptake systems often function as virulence factors in pathogenic organisms. Candida albicans is a fungal pathogen that infects immunocompromised hosts, such as AIDS patients or granulocytopenic bone marrow transplant recipients. Here we show that iron uptake from siderophores occurs in C. albicans and is mediated by one or more high-affinity transport systems. Iron carried on ferrioxamine B, triacethyl-fusarinine, ferrichrome, or ferricrocin was actively taken up via a high-affinity mechanism. The kinetic parameters of uptake were similar to those found in S. cerevisiae. Furthermore, for ferrichrome and ferrioxamine B, cellular uptake of fluorescent analogues was observed. In C. albicans, iron uptake from siderophores was regulated by iron availability, with iron deprivation inducing uptake. Serum exposure, which induces a morphogenic shift from yeast to filamentous forms known to be required for virulence, also resulted in induction of iron transport from ferrichrome-type siderophores. In a tup1/tup1 strain which grows constitutively in the filamentous form, iron transport was derepressed for all siderophores tested. The genes mediating uptake and utilization of iron from siderophores in C. albicans have not been identified; however, the transcript abundance for CaSIT1 was regulated in a manner consistent with the pattern of iron uptake from ferrichrome-type siderophores. Furthermore, CaSIT1 overexpression in S. cerevisiae resulted in inhibited siderophore iron uptake, suggesting that the expressed protein may interact with proteins of S. cerevisiae involved in iron uptake from siderophores. In summary, iron uptake from ferrichrome-type siderophores was induced in filamentous C. albicans, and a potential role of this iron acquisition system in pathogenicity should be considered. Copyright © 2002 John Wiley & Sons, Ltd.

Characterization of Ypr1p from Saccharomyces cerevisiae as a 2-methylbutyraldehyde reductase.

Abstract: The metabolism of aldehydes and ketones in yeast is important for biosynthetic, catabolic and detoxication processes. Aldo-keto reductases are a family of enzymes that are able to reduce aldehydes and ketones. The roles of individual aldo-keto reductases in yeast has been difficult to determine because of overlapping substrate specificities of these enzymes. In this study, we have cloned, expressed and characterized the aldo-keto reductase Ypr1p from the yeast Saccharomyces cerevisiae and we describe its substrate specificity. The enzyme displays high specific activity towards 2-methylbutyraldehyde, as well as other aldehydes such as hexanal. It exhibits extremely low activity as a glycerol dehydrogenase. The enzyme functions over a wide pH range and uses NADPH as co-factor. In comparison to other mammalian and yeast aldo-keto reductases, Ypr1p has relatively high affinity for D,L-glyceraldehyde (1.08 mM) and hexanal (0.39 mM), but relatively low affinity for 4-nitrobenzaldehyde (1.07 mM). It displays higher specific activity for 2-methylbutyraldehyde than does yeast alcohol dehydrogenase and has a K(m) for 2-methyl butyraldehyde of 1.09 mM. The enzyme is expressed during growth on glucose, but its levels are rapidly induced by osmotic and oxidative stress. Yeast in which the YPR1 gene has been deleted possess 50% lower 2-methylbutyraldehyde reductase activity than the wild-type strain. This suggests that the enzyme may contribute to 2-methyl butyraldehyde reduction in vivo. It may therefore play a role in isoleucine catabolism and fusel alcohol formation and may influence flavour formation by strains of brewing yeast.

Systematic analysis of sporulation phenotypes in 624 non-lethal homozygous deletion strains of Saccharomyces cerevisiae

Abstract: A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non-lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL- and DL-dityrosine, sporulation-specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non-essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype.

HSP12 is essential for biofilm formation by a Sardinian wine strain of S. cerevisiae

Abstract: Sardinian sherry strains of S. cerevisiae form a biofilm on the surface of wine at the end of the ethanolic fermentation, when grape sugar is depleted and when further growth becomes dependent on access to oxygen. A point mutation in HSP12 or deletion of the entire gene results in inability to form this film. HSP12 encodes a heat-shock protein previously foundby others to be active during stationary phase, in cells depleted for glucose, and in cells metabolizing ethanol and fatty acids, all conditions associated with sherry biofilms. The DNA sequence of HSP12 allele of strain Ar5-H12 has GenBank Accession No. AY046957. Copyright © 2002 John Wiley & Sons, Ltd.

Inhibitory effect of nikkomycin Z on chitin synthases in Candida albicans

Abstract: Nikkomycin Z is a competitive inhibitor of chitin synthases in fungi. It has been reported that it inhibits chitin synthases (Chs) 1 and 3, but not 2, of Saccharomyces cerevisiae. In our study, we found that: (a) nikkomycin Z inhibited all three Chs isozymes of Candida albicans (CaChs). The IC50 value for CaChs1 is 15 µM, for CaChs2 0.8 µM, and for CaChs3 13 µM; (b) nikkomycin Z inhibits vegetative growth of C. albicans differently in different growth media; growth inhibition was observed on Spider and corn meal agar plate, but not on Lee's plate; (c) growth inhibition by nikkomycin Z accompanied by the absence of septum and cell wall chitin, which in turn brought about cell lysis. Nikkomycin Z did not lyse cells in Lee's media and lysis was partially prevented in the presence of sorbitol as an osmostabilizer in Spider medium. Therefore, nikkomycin Z prevented the formation of septum and cell wall chitin by inhibiting chitin synthase activities in a growth medium-dependent manner. Copyright © 2002 John Wiley & Sons, Ltd.

SRC1: an intron-containing yeast gene involved in sister chromatid segregation

Abstract: Analysis of a three-member gene family in the yeast Saccharomyces cerevisiae has allowed the discovery of a new gene that comprises two contiguous open reading frames previously annotated as YML034w and YML033w. The gene contains a small intron with two alternative 5′ splicing sites. It is specifically transcribed during G2/M in the cell cycle and after several hours of meiosis induction. Splicing of the mRNA is partially dependent on NAM8 but does not vary during meiosis or the cell cycle. Deletion of the gene induces a shortening of the anaphase and aggravates the phenotype of scc1 and esp1 conditional mutants, which suggests a direct role of the protein in sister chromatid separation. Copyright © 2002 John Wiley & Sons, Ltd.

Mutant analysis reveals complex regulation of sphingolipid long chain base phosphates and long chain bases during heat stress in yeast.

Abstract: Sphingolipid long chain bases (LCBs) in Saccharomyces cerevisiae, dihydrosphingosine (DHS) and phytosphingosine (PHS) and their phosphates (DHS-P and PHS-P) are thought to play roles in heat stress. However, quantitative studies of LCBs and LCBPs have been limited by analytical methods. A new analytical procedure allowed us to measure changes in all known LCBPs and LCBs in wild-type and mutant cells during heat shock and to correlate the changes with heat stress resistance. All five molecular species of LCBPs increased rapidly but transiently when log and stationary phase cells were heat-stressed and when log-phase cells were induced for thermotolerance, suggesting that LCBPs play a role in heat stress. In support of this hypothesis, cells lacking the minor LCB kinase, Lcb5p, but not the major kinase, Lcb4p, were two-fold less resistant to killing when log-phase cells were induced for thermotolerance. Thus, LCBPs seem to play a minor role in heat-stress resistance. However, their role may be masked by LCBs, which are elevated in mutant strains, such as one lacking Lcb4p. This elevation demonstrates that one function of Lcb4p is to regulate LCB levels. Two new compounds, C16 DHS and C16 DHS-P, were identified, with the latter being degraded by the Dpl1p lyase. Our data provide a basis for determining how the basal and heat-induced levels of individual species of LCBs and LCBPs are governed by the Lcb4p and Lcb5p kinases, the Dpl1p lyase and the Lcb3p phosphatase. Copyright © 2002 John Wiley & Sons, Ltd.

SHAM‐sensitive alternative respiration in the xylose‐metabolizing yeast Pichia stipitis

Abstract: SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-Δ deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c1 and a·a3 did not change in the sto1-Δ mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-Δ deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis. Published in 2002 by John Wiley & Sons, Ltd.

Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria

Abstract: Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactisrag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria tooxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Δ strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Δ mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Δ mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Δ mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent ‘acetaldehyde reductase’ was also increased. These observations indicate that two mechanisms may participate inmitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP+- and NAD+-dependent alcohol dehydrogenases. Copyright © 2002 John Wiley & Sons, Ltd.