J. F. T. Spencer

The use of mitochondrial mutants in hybridization of industrial yeasts : III. Restoration of mitochondrial funktion in petites of industrial yeast strains by fusion with respiratory-competent protoplasts of other yeast species.

SummaryProtoplasts of petites of strains 625-CI of Saccharomyces diastaticus and NCYC 1085 of Saccharomyces cerevisiae, originally obtained from the National Collection of Yeast Cultures, England, were fused with protoplasts of Candida pseudotropicalis, Saccharomyces rosei, Yaccharbmycesmontanus, Pichiamembranefaciens, Hansenula anomala, Hansenula capsulata, and Schizosac-charomyces pombe. The respiratory-competent products of the fusions were selected on the basis of using at least one of the carbon sources utilized by the petite parent and not by the other. The products of the fusion of C. pseudotropicalis x 1085(p−) consisted of two cell types; an oval cell which utilized both lactose and maltose and fermented lactose vigorously, and a cylindrical form which fermented maltose slowly. The S. rosei x 1085(p−) hybrids had acquired the ability to metabolize and ferment galactose, and to ferment maltose, from the petite parent. The P. membranaefaciens x 625(p−) hybrids acquired the ability to metabolize galactose, sucrose and maltose, but fermented only glucose, weakly, like the P. membranaefaciens parent strain. The H. capsulate x 625(p−) hybrids, unlike the hybrids with P. membranaefaciens or S. rosei, resembled the petite parent morphologically and also had the fermentative abilities of this strain (galactose, maltose, sucrose and starch), and the ability to ferment starch was considerably enhanced. The S. montanus x 625(p−) hybrids acquired the ability to utilize starch. Schizosaccharomyces pombe x 625(p−) hybrids resembled S. pombe morphologically, but had the ability to metabolize galactose and starch. Some of the asci produced by these hybrids contained abnormal numbers of spores. H. anomala x 624 x(p−) hybrids fermented starch, though weakly.

The use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains

SummaryMethods for the isolation of hybrids in which one or both of the parental strains are industrial yeasts, using mitochondrial mutations as markers for the selection and isolation of the hybrids, are described. The systems used included crosses of industrial strains with auxotrophic laboratory strains which also carried a mitochondrial antibiotic resistance mutation, crosses using an auxotrophic laboratory strain and a petite mutant of an industrial strain carrying a rescuable antibiotic resistance mutation, and crosses using a petite mutant of an industrial strain, carrying a rescuable mitochondrial mutation for antibiotic resistance and a respiratory-competent industrial strain which carried some other marker.

Characterization of hybrids obtained by protoplast fusion, between Pachysolen tannophilus and Saccharomyces cerevisiae

The genes for utilization of xylose were transferred from Pachysolen tannophilus to Saccharomyces cerevisiae. The hybrids resembled the S. cerevisiae parent morphologically and in sugar assimilation. Pulsed field gel electrophoresis showed that the chromosome banding pattern was intermediate between the two parental species.

Yeasts associated with rotting citrus fruits in Tucumán, Argentina

Yeasts were isolated from decaying spots in citrus fruits (oranges, limes, mandarins, grapefruit) produced in the Tucuman region of Argentina. Species isolated from the rotting fruit included Kloeckera apiculata, Candida guilliermondii, Candida stellata, Pichia kluyveri , other Pichia species of the Pichia membranaefaciens group, and Geotrichum candidum. Kloeckera apiculata , appeared to have some pectolytic activity when reinoculated into healthy fruit. The yeasts were probably carried by fruit flies. Larvae of the fruit fly ( Ceratitis capitata ), which is endemic to the region, were found in the decay pockets.

Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion

Summary“Wild” strains of Saccharomyces cerevisiae were isolated from dairy products, bakery goods, fresh fruit and vegetables, and tested for killer activity. Four isolates out of 238 strains possessed killer activity. The best of these was converted to the petite form and hybridized with an industrial strain of Saccharomyces cerevisiae by protoplast fusion. Thirty-eight out of 104 isolates had killer activity, and some of these had good dough-raising activity as well.

Use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains : IV. Characterization of an intergeneric hybrid, Saccharomyces diastaticus x Hansenula capsulata, obtained by protoplast fusion.

SummaryA hybrid of Saccharomyces diastaticus x Hansenula capsulate was obtained by conversion of the S. diastaticus parent to a petite, protoplasting and fusion of the strains and isolation of the hybrids on selective media, containing glycerol or galactose. All of the single-cell clones obtained resembled S. diastaticus in morphology, and fermented glucose, galactose, maltose, sucrose and starch. The hybrid sporulated, producing spores resembling those of S. diastaticus, but in asci which differed somewhat morphologically. The stability of the hybrid depended on the composition of the maintenance media, as hybrid clones maintained on glycerol medium lost their ability to sporulate after a few transfers and the cellular morphology altered to a form more closely resembling H. capsulate, and those maintained on glucose-containing medium lost the ability to utilize glycerol. Cultures maintained on yeast extract-starch medium retained the ability to sporulate and utilize glycerol. However, dissection of asci from the hybrid yielded clones, some of which had lost the ability to metabolize glycerol but which still sporulated. These clones had an unaltered cytochxome spectrum, and in other ways appeared to be respiratory competent. Washed cells of hybrids grown on galactose or starch as sole carbon source metabolized starch, as shown by respirometric measurements, but cells maintained on glycerol as sole carbon source had lost this ability. There was some evidence that the addition of vitamins stimulated oxygen uptake on starch by the hybrids. The formation of sporulating hybrids in the cross may permit the genetic analysis of other Saccharomyces x Hansenula crosses.

Yeasts associated with algarrobo trees (Prosopis spp.) in northwest Argentina : a preliminary report

SummaryYeasts were isolated from exudates from trees at three sites in northwest Argentina, two between the towns of Amaicha del Valle and Cafayate and one in the Quebrada de Cafayate, a deep river valley north of Cafayate. The majority of the yeasts were identified asCandida famata andRhodotorula graminis, though isolates of other species ofRhodotorula, Candida boidinii, Pichia membranaefaciens, and occasional isolates of other species were obtained. None of the species was the same as those isolated in Crete, from pods of the carob (European algarrobo). Of 96 cultures investigated, 26 utilized methanol as sole carbon source. The frequency of isolation of methylotrophic yeasts from this habitat may prove to be of considerable scientific and technological interest.

Inability of Petite Mutants of Industrial Yeasts to Utilize Various Sugars, and a Comparison with the Ability of the Parent Strains to Ferment the Same Sugars Microaerophilically

A number of industrial strains of Saccharomyces cerevisiae were converted to the petite form and tested for the ability to utilize galactose, maltose, sucrose, α-methyl glucoside and raffinose. The parent strains all metabolized these sugars aerobically. Twelve of the petite forms did not utilize galactose, six failed to utilize maltose, 17 did not utilize x-methyl glucoside, and 18 did not utilize raffinose. The petites of two distiller’s yeast strains did not utilize sucrose. The respiratory-competent parent strains nearly all fermented galactose, maltose, sucrose and raffinose, though 19 strains did not ferment α-methyl glucoside microaerophilically. Three strains did not ferment galactose, two fermented it only after several days adaptation, one did not ferment raffinose, and two did not ferment sucrose under microaerophilic conditions. Six respiratory-competent strains which did not utilize galactose when in the petite form fermented higher (10%) concentrations of glucose and maltose under microaerophilic conditions, but only three of these fermented galactose. The implications of these findings for the use of such strains in industry are discussed briefly.

Apparent bisexual behavior of yeast strains obtained from hybridization of industrial yeasts of the genusSaccharomyces with auxotrophic diploids

During a genetic study of some hybrids of brewers and distillers yeast strains with impaired sporulation characteristics and genetically marked auxotrophic aa and alpha alpha diploids, strains which showed positive mating reactions with both a and alpha haploid tester strains were observed. These strains proved to be homothallic and sporulated freely. The original hybrids, which appeared to be tetraploid, usually yielded sporulating single-spore clones on dissection of asci formed from them, with few or no mating strains among them. Dissection of asci from these clones yielded some single-spore clones which showed mating reactions with one or the other or both haploid tester strains, and further selection produced strains which on sporulation and dissection yielded single-spore clones which were apparently bisexual and sporulated freely. These strains proved to be homothallic, yielding single-spore clones which were all of the a mating type, and in which the mating reaction and, possibly, the action of the genes for homothallism were impaired, so that sporulating, non-mating diploids and haploids of both mating types were present in cultures originally obtained as single-spore clones.

Two new methods for recovery and genetic analysis of hybrids after fusion of yeast protoplasts

A method for regeneration of yeast protoplasts and fusants in a gelatin-agar mixture, followed by total recovery of the regenerated cells from the gelatin-agar mixture and isolation of the fusants, is described. A one-step method for obtaining intergeneric fusants in which the greater part of the genome is derived fromSaccharomyces cerevisiae, and in which the fusant can be sporulated directly and tetrad analysis carried out without construction of further hybrids, is also described.