Development of genetic techniques and the genetic map of the yeast Saccharomycopis lipolytica

Summary Genetically useful strains of the hydrocarbon-utilizing yeast Saccharomycopsis lipolytica were developed through extensive inbreeding. Spore viability and the percentage of 4-spored asci were increased to the point where tetrad analysis was possible. Procedures for mutant isolation and scoring, maintenance of stocks, mating, sporulation, complementation, tetrad and random spore analysis have been developed for these inbred strains. Sixty seven mutations in fiftyeight genes have been isolated and utilized in mapping studies. Twenty-two cases of linkage have been detected among the 278 gene pairs investigated. Six linkage fragments have been established and a few genes ordered in these fragments. No centromere, linked markers have yet been detected. Evidence for gene conversion, mitotic recombination and diploidization in S. lipolytica is presented.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Genetic strains of Hansenula polymorpha

Six centromeric linkage groups and four non-centromeric fragments are revealed in the genetic stocks of Hansenula polymorpha which were obtained by intratetrad breeding in several generations of two genetically different parental strains progeny. Fourteen nuclear markers are mapped, including auxotrophic mutations, mating regulation loci, determinants of sporulation and heat tolerance. Complex origin of the haploid genome of these stocks leads to affinity interactions and to 14 per cent increase in DNA content in haploid stocks, as compared with the parental strains.     

Mannitol 1-phosphate metabolism is required for sporulation in planta of the wheat pathogen Stagonospora nodorum

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wild-type strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.     

Subject index vol. 70 (1980)

Antibiotic-resistant (either to erythromycin or chloramphenicol) temperature-sensitive mutants were isolated with about the same frequency in 2 strains of the petite negative yeast K. lactis.The ery^R and cap^R mutants isolated in the strain K. lactis CBS 2359 showed with high frequency both a lethal-conditioned (lc) or a petite temperature-sensitive (pts) phenotype, whereas amongst the many ery^R and cap^R mutants isolated in the strain K. lactis CBS 2360 only lc phenotypes appeared. In the mutants isolated from K. lactis CBS 2360, one growth cycle in the presence of ethidium bromide irreversibly blocked the transmission of antibiotic resistance and temperature sensitivity (lc and pts), whereas at least 2 growth cycles were required to give the same results for the mutants isolated in K. lactis CBS 2359.The spontaneous reversion frequencies for the temperature sensitivity were about the same for the lc mutants isolated in the 2 strains, but the frequencies of co-reversion of the antibiotic resistance were higher in ery^Rlc and cap^Rlc mutants isolated from K. lactis CBS 2360.The analysis of the effect of the exposure to erythromycin or to the temperature of 36°C on protein synthesis carried out by isolated mitochondria of 2 ery^Rlc mutants of K. lactis CBS 2360 and CBS 2359 showed that, in these mutants, mitochondrial protein synthesis became resistant to the drug and sensitive to temperature. The exposure at 36°C, before protein synthesis was inactived, determined in these mutants a condition of sensitivity to the antibiotic, suggesting that even though the 2 K. lactis strains differ in some aspects concerning the behaviour of their mitochondrial information they might depend, as to their petite-negative character, on the role that mitochondrial protein synthesis has in cell division.     

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

The ascomycete Magnaporthe grisea is a model species for the study of plant fungal interactions. As in many filamentous fungi, targeted gene replacement occurs at low frequency in M. grisea (average 7%). mus52/KU80 is a gene essential for non-homologous end joining (NHEJ) of DNA double-strand breaks. Its deletion increases the frequency of targeted gene replacement in fungi [Ninomiya, Y., Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining. Proc. Natl. Acad. Sci. USA 101(33), 12248-53]. M. grisea KU80 deletion mutants were constructed and displayed wild-type phenotypes regarding pathogenicity, growth, sporulation and mating. MgADE4 targeted gene replacement frequency was increased in Deltaku80 mutants (80% vs 5%) and high frequencies (>80%) were observed at seven other loci. However, the deletion of MgKU80 did not increase the frequency of ACE1 replacement indicating that this locus has an intrinsic reduced ability for gene replacement. These results open the way to large-scale reverse genetics experiments in M. grisea facilitating the study of the infection process.     

Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.     

From yeast genetics to biotechnology

Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology [6]. Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field. Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.     

Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus.

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.     

Analysis of homothallic Saccharomyces cerevisiae strain mating during must fermentation

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.     

Analysis of Homothallic Saccharomyces cerevisiae Strain Mating during Must Fermentation

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.     

A study of copulation,sporulation and meiotic segregation in Candida lipolytica

Summary We have attempted to optimize conjugation and sporulation in Candida lipolytica, by studying the conditions of culture and growth.Copulation between compatible strains is a rare event, particularly in the case of auxotrophic mutants. However, diploids can be selected for on minimal medium provided parents are suitable auxotrophs. These diploids can multiply vegetatively for many generations. They can also be induced to sporulate at a very high frequency.Free ascospores were isolated by means of paraffin oil and segregations of markers could be studied. At first quite irregular, these segregations improved following a number of brother-sister matings. At the same time, the mean number of spores per ascus as well as spore germinability were considerably increased.     

Two temperature-sensitive mutants of Saccharomyces cerevisiae with altered expression of mating-type functions

Two mutants of Saccharomyces cerevisiae have been isolated from normal haploid MAT alpha strains and characterized as having temperature- sensitive, pleiotropic phenotypes for functions associated with mating. At the permissive temperature, 23 degrees C, they were found to behave as normal MAT alpha haploids with respect to mating efficiency, sporulation in diploids formed with MAT a strains, secretion of alpha- factor, and failure to secrete the MATa-specific products, a-factor and Barrier. At higher temperatures they were found to decline in mating and sporulation efficiency and to express the a-specific functions. Genetic analysis established that one of these mutants, PE34, carries a temperature-sensitive allele of the MAT alpha 2 gene and that the other, PD7, carries a temperature-sensitive allele of the TUP1 gene.     

A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1.     

粉被虫草无性型单孢子株间和单孢子株内的营养亲和性

在含5％氨酸钾的KMM和KPS培养基上,粉被虫草无性型以3种途径产生不利用硝酸盐的突变株（nit突变株）。（1）由菌落基质菌丝形成的快速生长气生菌丝角变;（2）菌落表面快速生长的气生菌丝;（3）菌落基质菌丝缓慢生长形成的基质菌丝角变。来自18个单孢子株的94个nit突变株中,64.8％的突变株是稳定的。配对试验结果表明:在全部19个配对中,单孢子株内配对率为57.9％,单孢子株间配对率为42.1％。在全部nit突变株中,Cp-14c3突变株与其它突变株间的配对率最高（18.2％）。单孢子株间配对率高的孢子株是Cp-14Cp-7,Cp-5和Cp-6,将来自Cp-14同一单孢子株的Cpe-14C3分别与Cp-14cl和Cp-14c4nit突变株配对后发现,它们形成的浓密生长配接线的颜色是不相同的,前者橙色,后者白色。统计结果发现,所试全部的单泡子株可分成11个营养亲和群（VCGs）,那些含有易与其它菌株配对的nit突变株的单孢子株,如Cp-1,Cg-4,Cp-5,Cp-6,Cp-7,Cp-13,和Cp-14等皆在同一营养亲和群内。用Hochest33258荧光染色观察发现,野生型菌株的菌丝和分生孢子单核,nit突变株的少量分生孢子中可见双核,互补配对形成的浓密菌丝丛中的分生孢子则常见双核。     

Vacuolar accumulation and extracellular extrusion of electrophilic compounds by wild-type and glutathione-deficient mutants of the methylotrophic yeast Hansenula polymorpha

The methylotrophic yeast Hansenula polymorpha CBS4732 leu2 detoxifies electrophilic xenobiotics by glutathione (GSH)-dependent accumulation in vacuoles, as shown by fluorescence microscopy. GSH-dependent and GSH-independent export of xenobiotic derivatives were also demonstrated by high-performance liquid chromatography (HPLC). Conjugates of GSH and N-acetylcysteine with monobromobimane and N-[1-pyrene]maleimide were observed among the HPLC fractions, along with unidentified derivatives.     

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.     

Gene Transfer in CAULOBACTER CRESCENTUS: Polarized Inheritance of Genetic Markers

Recombination frequencies were determined for 15 independently isolated auxotrophs of C. crescentus crossed pairwise in all possible combinations. The results indicate that the mutants may be grouped into at least two types: "fertile" strains, which recombine with all other mutants at frequencies ranging from less than 10-6 to 3 times 10-2, and "nonfertile" strains which recombine with fertile strains at high frequencies and with other nonfertile strains at low or negligible frequencies. Several lines of evidence indicate a polarized inheritance of markers. Two of these are (1) the preferential inheritance of unselected markers from the nonfertile parent in fertile times nonfertile crosses, and (2) the consistent ordering of markers based on the frequency at which the mutants recombine with each of the three fertile strains. Although the evidence is not conclusive at this point, the results are most consistent with conjugation at the mechanism of gene transfer in these bacteria.