Ribosomal DNA spacer probes for yeast identification: studies in the genus Metschnikowia

To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7.7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0.65 kb BamHI-HpaI fragment containing non-transcribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclusively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G + C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.     

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.     

Identification and characterization of amidase- homologous AMI1 genes of bottom-fermenting yeast

It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. bayanus AMI1 gene, while AMI1-B on chromosome X is most homologous to the Saccharomyces paradoxus AMI1 gene. Overproduction of AMI1 in S. cerevisiae resulted in a slow-growth phenotype. Although a hydropathy plot shows that Ami1p has a putative signal sequence, it was located in the cell, not secreted into the medium. By metabolome analysis of intracellular compounds, the amount of histidine and arginine is increased, and the amount of threonine, lysine and nicotinic acid is decreased in the Ami1p-overproducing strain as compared with the control, suggesting that Ami1p may hydrolyse some amides related to amino acid and niacin metabolism in the cell.     

Aneuploidy,copy number variation and unique chromosomal structures in bottom‐fermenting yeast revealed by array‐CGH

DNA microarray for comparative genome hybridization (CGH) of bottom‐fermenting yeast was performed based on our in‐house DNA sequence data. Aneuploidy, copy number variation and unique chromosomal structures were observed among bottom‐fermenting yeast strains. Our array experiments revealed a correlation between copy number variation and mRNA expression levels. Chromosomal structures in a Saccharomyces carlsbergensis‐type strain and in a S. monacensis‐type strain that both belong to S. pastorianus phylogenetically differed greatly from those in contemporary industrial bottom‐fermenting yeast strains. The knowledge gained in this study contributes to a more precise genomic characterization of bottom‐fermenting yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA

The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism.     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

甜酒酿中酵母菌多样性的研究

从福建酒曲、苏州酒曲和安琪酒曲中分离出6株酵母菌.通过对所分离的酵母菌的18S rDNA进行分析鉴定,同时利用形态、生理生化的分析,最终确定这6株菌属于二个类群,其分别属于酿酒酵母属,复膜酵母属.利用18S rDNA的序列,进行聚类分析,这6株酵母菌其中福建酒曲中有4株酵母菌,分别为F J-2、F J-5、F J-6、F J-7,其中FJ-2与Saccharomyces cerevisiae strain OC 11的相似性达到99％、F J-5与S.cerevisiae S288c相似性达99％、F J-6与S.cerevisiae strain OC11相似性达99％,F J-7与Saccharomycopsis fibuligera strain YW12相似度达到99％.苏州酒曲中有1株,为SZ-2与S.cerevisiae的相似性达99％.安琪酒曲中有1株,该菌株与酿酒酵母的相似度为100％.由此可见,在酒酿中发挥作用的酵母菌是酿酒酵母属和复膜酵母属中的菌株.     

Identification and characterization of Clostridium perfringens using single target DNA microarray chip

A DNA microarray method was developed to identify the presence of toxin genes: encoding beta toxin (cpb), epsilon toxin (etx), enterotoxin (cpe), alpha toxin (cpa), and iota toxin (iA) in Clostridium perfringens. To build the DNA chip, each gene sequence was represented by one approximately 22-bp amino-modified oligonucleotide printed twice on aldehyde-coated slides. Multiplex PCR with Cy3 and Cy5-dCTP derivatized fluorescent nucleotides was used to label five genes and fluorescent probes were prepared. The PCR probes were denatured and single-strand-labeled DNAs were separated and purified using magnetic beads. The presence of toxin genes in C. perfringens was detected by hybridization of amplified ssDNA probes to oligonucleotides on the chip representing one target sequence of each toxin gene. The DNA chip was able to identify eight strains of C. perfringens.     

Molecular cloning and DNA analysis of a gene encoding alpha mating pheromone from the yeast Saccharomyces naganishii

A DNA fragment encoding the precursor peptide for alpha mating pheromone was isolated from the S. naganishii genome based on the amino acid sequence of the mature pheromone. The precursor peptide contains three copies of the pheromone. Hydrophobicity analysis of the precursor peptide revealed an N-terminal signal sequence for translocation into the lumen of the endoplasmic reticulum and several signals for a series of secretion-related processes. However, upstream regulatory sequences necessary for expression of the S. cerevisiae alpha mating pheromone gene were not found, suggesting the divergence of systems that regulate alpha mating pheromone gene expression in S. naganishii and S. cerevisiae. Hybridization of a probe corresponding to the S. naganishii alpha mating pheromone nucleotide sequence to S. naganishii chromosomal DNA revealed a single gene located on either chromosome VI or VII. The S. naganishii alpha mating pheromone sequence has been deposited in the DDBJ/EMBL/GenBank data library under Accession No. AB086431.     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species

We recently discovered, on the chromosome of Saccharomyces cerevisiae sigma 1278b, novel MPR1 and MPR2 genes required for resistance to a toxic analogue of L-proline, L-azetidine-2-carboxylic acid. The MPR genes, which were absent in the S. cerevisiae genome project strain S288C, encoded a novel acetyltransferase of 229 amino acids that detoxifies the analogue by acetylating it. The MPR1 gene homologue found in Schizosaccharomyces pombe was also shown to encode a similar acetyltransferase. To further analyse the origin and the physiological role of the yeast novel gene, we report here the comparative analysis of the MPR1 gene in the S. cerevisiae complex spp. which belong to the Saccharomyces sensu stricto group. Only the type strain of S. paradoxus exhibited resistance and acetyltransferase activity to L-azetidine-2-carboxylic acid. PCR was then used to isolate the new MPR1 homologue (Spa MPR1) from S. paradoxus with the primers based on the sequence of the MPR1 gene. Gene expression and enzymatic analysis showed that the cloned Spa MPR1 gene encodes an L-azetidine-2-carboxylic acid acetyltransferase of 231 amino acids, which has 87% identity to the MPR1 protein. We also found in the protein databases that S. bayanus contains a DNA fragment that is partly homologous to the MPR1 gene. However, the gene product was considered to lose the enzymatic activity, possibly due to the gene truncation or the base substitution(s) at the important region for catalysis. Further, genomic PCR analysis showed that most of the S. cerevisiae complex spp. have the sequence highly homologous to the MPR1 gene.     

A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.     

CYS3基因的敲除及其对Saccharomyces cerevisiae3-甲硫基丙醇合成代谢的影响

研究胱硫醚γ-裂解酶基因CYS3敲除对S. cerevisiae 3-甲硫基丙醇合成代谢的影响。将编码胱硫醚-γ-裂解酶的CYS3基因和抗性标记基因Zeocin克隆,构建敲除组件CYS3Δ:Zeocin,醋酸锂法将其转化导入S. cerevisiae S288C表达,构建CYS3基因敲除的工程菌。结果表明：摇瓶发酵120 h时,工程菌S. cerevisiae C3和S. cerevisiae S288C的3-甲硫基丙醇生成量分别为0.60 g/L和0.94 g/L,S. cerevisiae C3较野生型S288C的3-甲硫基丙醇生成量降低36.2%。说明CYS3基因敲除对S. cerevisiae的3-甲硫基丙醇有较大影响,并呈现负调节作用。     

Identification of bottom-fermenting yeast genes expressed during lager beer fermentation

It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.     

DNA sequence of the beta-isopropylmalate dehydrogenase gene and phylogenetic analysis of the yeast Saccharomyces exiguus Yp74L-3

The beta-isopropylmalate dehydrogenase (LEU2) gene from a homothallic wild-type yeast, Saccharomyces exiguus Yp74L-3, was analyzed to estimate the phylogenetic position of this strain in yeasts. The beta-isopropylmalate dehydrogenase gene of Yp74L-3 was first isolated as a clone complementing the leu2 mutation of Saccharomyces cerevisiae, and then confirmed to complement the haploid leu2 mutant derived from strain Yp74L-3 through genetic transformation. The nucleotide sequence of the cloned DNA revealed an open reading frame (ORF) encoding the beta-isopropylmalate dehydrogenase composed of 365 amino acids. The beta-isopropylmalate dehydrogenase coding sequence from the Yp74L-3 strain displayed 76.7% similarity to that of S. cerevisiae. Candidates for a UAS and a TATA-box in the 5'-upstream region and for a poly-A attachment site in the 3'-downstream region were found. A phylogenetic tree constructed from the nucleotide sequences of the beta-isopropylmalate dehydrogenase coding regions revealed that Yp74L-3 is located between S. cerevisiae and the Kluyveromyces yeasts. The LEU2 gene cloned from Yp74L-3 will serve as an effective genetic marker for constructing the transformation system in S. exiguus Yp74L-3.     

Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells

In the presence of polyethylene glycol (PEG), budding cells of Saccharomyces cerevisiae in the early log phase were transformed by exogenous plasmid DNA without additional specific chemical or physical treatments. This capacity of the yeast cells to become competent was strictly dependent on the growth phase, being induced in the early log phase, becoming maximum between the early and mid log phases and then disappearing rapidly in the mid log phase. The transformation was most efficient at pH 6 and the frequency increased with increasing DNA and cell concentrations. PEGs with average molecular sizes between 1000 and 3500 showed almost the same effects and were used most efficiently at 35%. The transformation frequency of S. cerevisiae was markedly enhanced when the oxidized form of glutathione (GSSG), but not the reduced form, was included in the mixture comprising early log phase cells, plasmid DNA, and PEG, and the transformation system with GSSG could be used as a convenient transformation method for the yeast S. cerevisiae.     

Characterization of the cytochrome c gene from the starch-fermenting yeast Schwanniomyces occidentalis and its expression in Baker's yeast

A cytochrome c protein gene, CYC10, of the dextran- and starch-fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein-coding region shares close homologies to the cytochrome c genes. A S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein-encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiae. The CYC10 gene was oxygen-induced but not subject to catabolite repression. The expression of the CYC10 gene was studied in the heterologous yeast S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indicating that these two genes share similar or closely related cis- and trans-acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. occidentalis cells. Search in the 5' untranslated region of the CYC10 gene revealed some homologies at -425 to -405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae. Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.     

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae,S. uvarum and a new species,S. douglasii

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10⁴ to 10⁵ asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by chance, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80–90% spore viability, thus establishing a second member of the S. douglasii species.