Physical and genetic characterization of linear DNA plasmids from the heterothallic yeastSaccharomycopsis crataegensis

Five strains of the heterothallic yeastSaccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1,-2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified inS. crataegensis strains Y-5910 or YB-192. FourS. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules ofS. crataegensis, the 2 M circular DNA ofStaccharomyces cerevisiae, the killer plasmids ofKluyveromyces lactis, or the linear DNA plasmids ofPichia inositovora.In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Sterigmatosporidium gen. n., a new heterothallic basidiomycetous yeast,the perfect state of a new species of Sterigmatomyces Fell

Sterigmatosporidium gen. n. is described as a new basidiomycetous yeast-like fungus with the single species Sterigmatosporidium polymorphum sp. n. for which a Latin diagnosis and a preliminary life cycle are presented. The mean character distinguishing the new genus from the imperfect genus Sterigmatomyces is the development of a dikaryotic mycelium with clamp connections producing sexual spores in ramified whorls and lateral chlamydospores as well as blastospores. The dikaryotic phase could be induced by crossing compatible haploid clones of the heterothallic fungus, which are similar to Sterigmatomyces but not identical with any known species.     

Neurospora discreta,a new heterothallic species defined by its crossing behavior

The species is described and namedNeurospora discreta sp. nov. because of its stringent reproductive isolation. Isolates collected from burned vegetation at a single site near Kirbyville, Texas, include both mating types (Aanda). Experimental criteria based on cross-fertility were used for assigning species status. Crosses between isolates of opposite mating type are highly fertile, producing abundant eightspored asci. In contrast, when the Kirbyville strains are crossed to sexually compatible speciestester strains representingN. crassa, N. intermedia, N. sitophila, andN. tetrasperma, perithecia are rudimentary and no ascospores are produced. The haploid chromosome number is 7. Chromosomes at pachytene resemble those of otherNeurospora species. Biotin is required. Linear growth is slower than for other heterothallic species. When A and a strains from Kirbyville grow toward one another and intersect on crossing medium, there is no barrage. A single homogeneous band of perithecia is formed where they meet, indicating that opposite mating types are vegetatively compatible. The Kirbyville population differs from other heterothallicNeurospora species in ascospore morphology and vegetative traits. Ascospores from Kirbyville parents are larger, and the ribs between confluent parallel grooves are ornamented with dot-like pits. Vegetative cultures from Kirbyville are yellowish rather than orange, and large empty barren protoperithecia or false perithecia are produced abundantly in unfertilized haploid cultures. Isolates from two otherN. discreta populations resemble otherNeurospora species more closely with respect to these morphological traits but are clearly conspecific with the Kirbyville strains on the basis of fertility in crosses.     

Conjugation in the yeast Saccharomycopsis capsularis Schi?nning.

Cells of the yeast Saccharomycopsis capsularis fused in pairs after disolving of part of the cross wall between them near the lateral wall. After nuclear migrations through the opening, the cross wall was closed again and the cells at both sides became asci. The wall of the ascospores developed from a prospore wall. Between the two unit membranes a very thin dark layer broadened to the dark layer of the wall and after that, the light inner layer developed. Immature spores in the strain studied had a ledge which disappeared during maturation.     

Excretion of citric and isocitric acids by the yeast Saccharomycopsis lipolytica

Summary We investigated the excretion of citric and isocitric acids in a strain of Saccharomycopsis lipolytica grown on either n-paraffins, glucose, or glycerol. These acids were excreted in the ratio of 67:33 on n-paraffins and roughly 92:8 on either glucose or glycerol. However, with all the carbon sources used, the relative amount of isocitric acid in the intracellular pool remained below 10%. The assimilation of citric and isocitric acids was prevented when glucose or glycerol were the carbon sources, but not when n-paraffins were used. Citric acid stopped isocitric acid assimilation. These phenomena of selective assimilation and/or uptake might explain the variations observed in the ratio of citric to isocitric acids excreted on different carbon sources.     

Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 X 10(-7) and 5 X 10(-6) mutants per cell. But one class of glucosamine resistant mutants (GAMRA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica

• 1.1.|A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described.
• 2.2.|In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A.
• 3.3.|The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotrophic effect on 2-ketoglutaric binding sites. A second class of effectors is found, including 2-aminoadipic acid, pipecolid acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites.
• 4.4.|Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine.
• 5.5.|No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated.
• 6.6.|Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.

     

Mortierella sugadairana,a new homothallic species related to the firstly described heterothallic species in the genus

A new species, Mortierella sugadairana, is described for a fungus forming homothallic zygospores with a club-shaped macrosuspensor and a microsuspensor originating from the macrosuspensor. The species was isolated from cool regions in Japan and morphologically and phylogenetically close to a heterothallic species M. parvispora, which is the first species reported as a heterothallic species in the genus. Mycelial growth of the species was limited at 30 °C, whereas two isolates of M. parvispora can grow. This may indicate that the species and M. parvispora adapted to different climates from a common ancestor involving differentiation of the manner of reproduction.