Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.     

Expression of Cryptic β-Fructofuranosidase in Saccharomyces rouxii

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a beta-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a K(m) of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their beta-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii beta-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The beta-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.     

Expression of theSaccharomyces cerevisiae MPR1 gene encodingN-acetyltransferase inZygosaccharomyces rouxii confers resistance tol-azetidine-2-carboxylate

The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.     

Physiological characterization of osmotolerant yeast<Emphasis Type="Italic">Pichia sorbitophila</Emphasis> and comparison with a putative synonym<Emphasis Type="Italic">Pichia farinosa</Emphasis>

The osmotolerant yeast Pichia sorbitophila was found to differ from other yeast species, not only from the conventional ones (Saccharomyces cerevisiae, Schizosaccharomyces pombe), but also from those widely known as osmotolerant (Debaryomyces hansenii, Zygosaccharomyces rouxii). P. sorbitophila was able to survive extremely high extracellular concentrations of salts (e.g., saturated solution of KCl) and other osmolytes (70% glucitol), although it is not classified as halophilic (or osmophilic). P. sorbitophila assimilated a broad range of carbon and nitrogen sources with extreme effectiveness. On solid media, P. sorbitophila created colonies of variable shapes and sizes in relation to media composition, number of colonies on the plate and cultivation conditions. Colonies were able to produce long-distance signals between each other that resulted in growth inhibition of the facing parts of both colonies, but were not inhibited by colonies of other yeast species growing on the same plate. Though sometimes P. sorbitophila has been indicated as a synonym of P. farinosa, comparative physiological studies together with PCR amplification of P. farinosa DNA fragments homologous to known P. sorbitophila genes provided a strong indication that this strain should be classified as a separate species.     

Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts

The microtubular system in growing protoplasts of Saccharomyces uvarum was visualized by immunofluorescence using the monoclonal antitubulin antibody TU 01. We confirmed the coexistence of regular spindle configuration and extensive cytoplasmic networks in growing protoplasts and also observed a distinct distortion of cytoplasmic microtubules in association with wall removal. After a short period for recovery of protoplasts in nutrient medium a restitution of cytoplasmic microtubules and their resumed contact with the protoplast surface was observed. Treatment of growing protoplasts with nocodazole resulted in the disappearance of spindle and cytoplasmic microtubules in the relevant fraction of the protoplast population. In carbendazime (MBC)-arrested protoplasts spindle microtubules were absent but cytoplasmic microtubules associated with spindle pole bodies were clearly visible. Microtubule reassembly on spindle pole bodies occurred within 30 min after washing out nocodazole as well as carbendazime. The approach using protoplasts suggests a simple way in which the differential effect of antimicrotubule agents can be experimentally tested and the microtubule organizing activity of yeast protoplasts visualized at the population level.     

Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid.

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2 microns DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).     

Protoplast fusion in Saccharomyces cerevisiae

The ability of different Saccharomyces cerevisiae yeast strains to form protoplasts and protoplast fusion were studied. The protoplast formation depended mainly on strains used and the time of snail gut enzyme action. The percentages of the regenerating protoplasts varied, depending on strain, from 3 to 33 per cent. From the fusion experiments one can establish that kariogamy is prerequisite for stable for stable diploid formation. The yields of protoplast fusion were higher when both strains were rho+ as compared with rho+ and rho 0 combinations.     

Production of<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Nha2 Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter improves the salt tolerance of<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.     

The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2-22p) playing different roles in cation homeostasis and cell physiology

Antiporters exporting Na(+) and K(+) in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane antiporter transporting only Na(+) (ZrSod2-22p in the CBS 732(T) type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K(+)(Na(+))/H(+)-antiporter capable of both K(+) and Na(+) export. Synteny analyses suggested that ZrSOD2-22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2-22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2-22 antiporter is important for Na(+) detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function.     

Lytic Action of β(1-3)-Glucanase on Yeast Cells

Candida utilis, Saccharomyces cerevisiae, S. fragilis, Pichia polymorpha, and Hansenula anomala yeast cells, harvested in the early logarithmic phase, were attacked with purified beta(1-3)-glucanase from Micromonospora chalcea, which resulted in the liberation of protoplasts. The treated cells were observed under the electron microscope before the protoplasts were liberated. Differences in the cell walls of the enzyme-treated and untreated cells were observed. The action of the glucanase was also tested against isolated walls of C. utilis. The enzyme attacked the S. cerevisiae cell wall in a uniform manner. The attack on S. fragilis was located in certain zones of the cell wall, where breakage occurred and through which the protoplast emerged. On the other three yeasts, an intermediate attack was observed, not as definitely located as in S. fragilis, yet less uniformly than in S. cerevisiae.     

The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes

The osmotolerant yeast Zygosaccharomyces rouxii CBS732 contains only one copy of the ZrHOG1 and ZrSOD2-22 genes. Both genes were cloned and sequenced (Acc. Nos. AJ132606 and AJ252273, respectively) and their sequences were compared to homologous pairs of genes from Z. rouxii ATCC42981 (genes Z-HOG1, Z-HOG2, Z-SOD2, Z-SOD22). The CBS732 ZrHog1p is shorter than its ATCC42981 counterparts (380 aa residues vs. 407 and 420 aa, respectively) and is more similar to ATCC42981 Z-Hog2p than to Z-Hog1p. Also its promoter region corresponds to that one of Z-HOG2. The CBS732 ZrHOG1 promoter region is recognised by Saccharomyces cerevisiae, and the gene product (MAP kinase ZrHog1p) presence fully complements the osmosensitivity of a S. cerevisiae hog1 mutant strain. The CBS ZrSOD2-22 gene is highly similar to ATCC42981 Z-SOD2 but it contains also a segment of 15 aa residues specific for Z-SOD22. Z. rouxii ZrSod2-22 Na(+)/H(+) antiporter expressed in S. cerevisiae shows better activity toward toxic Na(+) and Li(+) cations than does S. cerevisiae's own Nha1 antiporter, and is efficient in improving the halotolerance of some S. cerevisiae wild types.     

Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina

Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta-mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta-mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Lytic action of strepzymes from micromonospora AS

Summary The effect of lytic enzymes of Micromonospora AS on isolated cell walls and intact or heat killed cells of Candida utilis was investigated. Several substances normally used as stabilizers during protoplast formation were tested for their effect on the lytic action of strepzyme M on intact and dead cells: NaCl and KCl markedly inhibited lysis, sucrose only to 40%. Sorbitol and MgSO₄ have no inhibitory effect. MgSO₄ was selected for further research as it was found to protect the protoplasts. Phosphate buffer pH 6.8 should not be used at concentrations above 0.01 m. When grown submerged in shaking flasks or in pilot fermentation tanks, in liquid medium containing yeast cells and salts, Micromonospora AS gave the highest yield of lytic enzymes. The strepzyme M preparation is thermolabile.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

酿酒酵母和产朊假丝酵母原生质体的形成、再生及属间融合

使用由亚硝基胍诱变所得到的营养缺陷型作为单倍体融合亲株的核基因标记,同时也采用线粒体球红霉素抗性突变株的小菌落形式作为融合亲株的线粒体基因标记。酿酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)两亲株原生质体的制备是用对数生长早期的细胞在蜗牛酶和0.7M KCl及β-巯基乙醇或二巯基苏糖醇的作用下完成的。二者的原生质体的形成率在30—60分钟内达到90—99％。原生质体再生率,酿酒酵母最高为29—35％,产朊假丝酵母为7.5％。两亲株的原生质体在35％PEG(M.W.6,000),10mM CaCl_2条件下被诱导融合。在基础培养基上,长出以营养互补为标记的融合菌株。融合频率为10~(-5)—10~(-6)。试验表明,这些融合菌株具有杂种的性质。其中一株杂合子在同化D-木糖、纤维二糖等的能力上比亲株明显增强。