Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.     

Characterization of plasma membrane H+-ATPase from salt-tolerant yeast Candida versatilis

Plasma membrane was isolated from the salt-tolerant yeast Candida versatilis and the ATPase in plasma membrane was characterized. The ATPase was a typical H⁺-ATPase with similar properties to the Saccharomyces cerevisiae and Zygosaccharomyces rouxii enzymes. It was reacted with antibody (IgG) raised against S. cerevisiae plasma membrane H⁺-ATPase. The ATPase activity was not changed by adding NaCl and KCl to the assay solutions, but was increased by NH, especially by ammonium sulfate. In vivo stimulation of ATPase activity was observed by the addition of NaCl into the culture medium, as observed in Z. rouxii. No in vivo activation of H⁺-ATPase by glucose metabolism was observed in C. versatilis cells and the activity was independent of the growth phase, like Z. rouxii and unlike S. cerevisiae cells.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响

为了提高鲁氏接合酵母抗高盐胁迫能力,作者探索了添加吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响。通过测量甘油、海藻糖含量以及Na⁺/K⁺-ATP酶活性等一系列生理表征来验证鲁氏接合酵母高盐适应性的变化。实验结果表明,鲁氏接合酵母在高盐胁迫条件下外源添加吡哆醇,在延滞期时,菌株提前24 h积累细胞内甘油,并且加快了细胞内海藻糖分解代谢速率;在对数期时,细胞内钠钾比率（Na⁺/K⁺）降低82.3%,细胞膜上Na⁺/K⁺-ATP酶活性增加16.9%,2-苯乙醇产量（质量浓度）提高6.42倍;在稳定期时,生物量提高10.6%,乙醇产量（质量浓度）提高5%,2-苯乙醇产量（质量浓度）提高1.26倍。综上所述,吡哆醇的添加能有效提高鲁氏接合酵母的高盐胁迫耐受能力,进一步增强了鲁氏接合酵母在酱油等高盐发酵食品中的应用前景。     

Characterization of plasma membrane proton-ATPase from salt-tolerant yeast Zygosaccharomyces rouxii cells

Plasma membrane was isolated from the salt-tolerant yeast Zygosaccharomyces rouxii and from Saccharomyces cerevisiae. The ATPase in the plasma membrane of Z. rouxii cells was a typical proton-ATPase as judged by testing with various ATPase inhibitors. There were slight differences in the pH optima of activities and in the sensitivity to sodium chloride (NaCl) and potassium chloride (KCl) of the ATPase from Z. rouxii and S. cerevisiae. The specific ATPase activity from Z. rouxii was higher in cells grown in a medium containing 2 _M-NaCl than in those not containing NaCl. No in vivo activation by incubation with glucose was observed in Z. rouxii cells and the specific ATPase activity was independent of the growth phase, unlike S. cerevisiae cells.     

Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐intensity pulsed UV light irradiation

The relationship between prior growth of food‐spoilage yeast in high‐sugar environments and their subsequent survival postpulsed UV (PUV) irradiation was investigated. Test yeast were separately grown to early stationary phase in YPD broth containing increasing concentrations of glucose (1–50% w/v) and were flashed with ≤40 pulses of broad‐spectrum light at lamp discharge energy settings of 3.2, 7.2 and 12.8 J (equivalent to UV doses of 0.53, 1.09 and 3.36 μJ cm^?2, respectively) and their inactivation measured. Findings showed that prior growth in high‐sugar conditions (≥30% glucose w/v) enhanced the sensitivity of all nine representative strains of Zygosaccharomyces bailii, Z. rouxii and Saccharomyces cerevisiae yeast to PUV irradiation. Significant differences in inactivation amongst different yeast types also occurred depending on amount of UV dose applied, where the order of increasing sensitivity of osmotically stressed yeast to PUV irradiation was shown to be Z. rouxii, Z. bailii and >S. cerevisiae. For example, a 1.2‐log order difference in CFU mL^?1 reduction occurred between Z. bailii 11 486 and S. cerevisiae 834 when grown in 50% w/v sugar samples and treated with the uppermost test UV dosage of 3.36 μJ cm^?2, where these two yeast strains were reduced by 3.8 and 5.0 log orders, respectively, after this PUV treatment regime compared to untreated controls. The higher the UV dose applied the greater the reduction in yeast numbers. For example, a 1.0‐, 1.4‐ and 4.0‐log order differences in CFU mL^?1 numbers occurred for S. cerevisiae 834 grown in 15% w/v sugar samples and then treated with PUV dose of 0.53, 1.09 and 3.36 μJ cm^?2, respectively. These findings support the development of PUV for the treatment of high‐sugar foods that are prone to spoilage by osmotolerant yeast.     

Characterization of MEL genes in the genus Zygosaccharomyces

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78·6% and 78·2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus α-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0·4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel⁺ Z. fermentati strains but failed to hybridize to any chromosome of Mel⁺ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces. The sequence has been deposited in the EMBL Data Library under Accession Number L24957.     

An overview of membrane transport proteins in Saccharomyces cerevisiae

All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to ～60 transport proteins whose function was at least partially known, ～100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K⁺ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na⁺-solute symporters, a protein very similar to electroneural cation-chloride co-transporters, and a putative Na⁺-H⁺ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.     

Comparative study of physiological adaptation to salt stress in the genome shuffled Candida versatilis and a wild-type salt-tolerant yeast strain

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na⁺, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na⁺/H⁺-antiporter played a primary role in Na⁺ extrusion and H⁺-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na⁺, enhance H⁺-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.     

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732^T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMX^R and ClonNAT^R as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATα^P expression locus with the loxP–kanMX–loxP cassette.     

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe. Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca²⁺-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca²⁺-binding motifs. The KlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. © 1998 John Wiley & Sons, Ltd.     

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1α-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82·4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part of a region homologous to EF-1α. The comparison of amino acid sequences of the Sup2 proteins reveals high conservations (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae where found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.     

Monovalent cation transporters at the plasma membrane in yeasts

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H⁺-ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na⁺, K⁺, and H⁺, both under normal growth conditions and in response to stress.     

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K⁺/H⁺ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K⁺ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae

A 6·8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22⁺ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5′ end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl-carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNA^Leu (UAA) isoacceptor located near a delta sequence.