Spheroplast Fusion Research Articles

Spheroplast fusion as a mode of genetic recombination in mycobacteria.

Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR9 and A11, which were obtained by the chemical mutagenesis of the wild type strain A+ with N-methyl-N'-nitro-N-nitrosoguanidine. The carotenoid pigments and the alpha- and beta-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on Löwenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 degrees C. The frequency of NgR9 X A11 recombination in optimal conditions was about 2.5 X 10(-3). The recombinants selected on the basis of their carotenoid pigment profile were also tested for their alpha- and beta-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions for spheroplast fusion as a mode of genetic recombination in M. aurum are described.

Construction of Dextrin Fermentative Yeast Strains That Do Not Produce Phenolic Off-Flavors in Beer

The successful construction by hybridization or spheroplast fusion of novel brewing-yeast strains that possess the ability to utilize wort dextrins was previously hampered by the presence of a comp...

Increased osmotolerance of genetically modified ethanol producing strains of Saccharomyces Sp.

A stable spheroplast fusion product of the polyploid brewing strain Saccharomyces uvarum (cares bergensis) , strain 21 and a genetically constructed diploid Saccharomyces diastaticus, strain 1384 has been shown to have improved ethanol producing capability in defined media (Panchal et al., 1982). This fusion product, strain 1400 was further subjected to fermentations in defined media containing glucose substrate and varying concentrations of the non-metabolized sugars sorbitol or mannitol.

Genetic analysis of Candida albicans: identification of different isoleucine-valine, methionine, and arginine alleles by complementation.

By using the spheroplast fusion technique as a tool for genetic analysis, we have demonstrated complementation among three of four isoleucine-valine mutants, two of three methionine mutants, and two arginine mutants of independent origin from two different Candida albicans isolates. The two adenine mutants derived from the same parent strain did not complement. Complementation resulted predominantly from heterokaryon formation and, in some cases, from heterozygote formation. In either case, most fusion products were unstable and showed nuclear as well as chromosomal segregation, in a few cases resulting in recombination of parental auxotrophic markers. However, some fusion products were fairly stable.

The genetic manipulation of industrial yeast strains

ABSTRACT Hybridization, spheroplast (protoplast) fusion and transformation have been studied as means to genetically manipulate brewing and related yeast strains. Hybridization is a secondary technique for verifying the gene composition of recombinants produced by fusion and transformation. Further, it can be used to study gene dosage and gene suppression effects. Fusion is more empirical than manipulative since little control can be exerted over the genotypic makeup of the fusion product. Transformation with native DNA is a more subtle technique than fusion since the introduction of single traits into strains can be controlled. 2μm DNA plasmids are present in all brewing strains examined but the growth temperature of some strains is critical before significant numbers of plasmid can be detected. KEYWORDS Saccharomyces cerevisiae;, Sacch. uvarum (carls bergensis);, Kluyveromyces lactis;, hybridization, fusion, transformation, plasmid.

Parasexual genetic analysis of Candida albicans by spheroplast fusion.

Doubly auxotrophic strains of Candida albicans were selected from mutagenized cultures. Spheroplasts prepared from the auxotrophic strains were fused with polyethylene glycol. Prototrophic derivatives formed by this fusion protocol from auxotrophic strains were selected by complementation on minimal medium. These prototrophs had a cell volume twice that of the original strain and were shown to be heterozygous at four loci. Prototrophs obtained by this procedure infrequently gave rise to auxotrophic recombinants whose cell volume remained twice that of the original strain. It is suggested that these auxotrophic recombinants arise from mitotic crossing-over. This paper is the first report of a parasexual cycle in C. albicans.

Fusion ofSaccharomyces UvarumwithSaccharomyces Cerevisiae:Genetic Manipulation and Reconstruction of a Brewer's Yeast

Genetic manipulation of brewer's yeast is hampered by the yeast's inability to mate, low frequency of sporulation, and poor spore viability—all of which are partly because of its polyploid or aneuploid consititution. In the present study, we introduced genetic markers into a brewing strain of Saccharomyces uvarum by conventional mutagenic procedures and then crossed the marked strain with auxotrophic S. cerevisiae haploids, using spheroplast fusion. Fusion of the auxotrophic brewing strain with an a mating type S. cerevisiae produced hybrids that were nonsporulating and nonmating when tested with a and α tester strains. Fusion of the same brewing strain with an α mating type S. cerevisiae produced hybrids that were nonmaters but sporulated well, producing principally four-spored asci of high viability. Tetrad analyses of three hybrids recovered from the latter fusion proved that karyogamy had occurred. Asci from two of these hybrids showed regular segregation of all markers, including mating type. The third hybrid, however, exhibited regular segregation in only four of 18 asci dissected and had from one to three nonviable spores in the remaining 14; of these, seven single ascospores did not mate but sporulated. Genetic data, as well as DNA analyses, were used to estimate ploidy. Haploid ascospores were used to construct triploid brewing strains carrying 0, 1, 2, or 3 maltose genes using spheroplast fusion and conventional mating techniques. These triploid strains exhibited a gene dosage effect with respect to fermentation rate corresponding to the number of maltose genes present in each. These results suggest a model for the construction of genetically improved brewing strains.

Erratum

"Erratum." Journal of the American Society of Brewing Chemists, 38(2), p. 76This article refers to:Use of Spheroplast Fusion and Genetic Transformation to Introduce Dextrin Utilization into Saccharomyces Uvarum

Use of Spheroplast Fusion and Genetic Transformation to Introduce Dextrin Utilization into Saccharomyces uvarum

By two methods of genetic manipulation, the ability to use dextrin was introduced into Saccharomyces uvarum and S. cerevisiae. Both methods obtained the genetic information for dextrin utilization from S. diastaticus. With the fusion technique, spheroplasts were formed by the enzymatic digestion of the cell walls of the species to be fused. Fusion took place in polyethylene glycol; complete cells were then regenerated in a hypertonic media containing 3% agar. Successful fusion products were capable of using dextrin and melibiose as carbohydrate sources. The transformation procedure involved the extraction and purification of DNA from S. diastaticus. The S. uvarum and S. cerevisiae recipients were spheroplasted and then incubated with the extracted DNA. Complete cells were subsequently regenerated in hypertonic media containing 3% agar. The transformants were selected by their ability to utilize and ferment dextrin.

Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.

SPHEROPLAST FUSION OF BREWER'S YEAST STRAINS

Spheroplasts of brewing polyploid yeast strains have been successfully fused with spheroplasts of haploid yeast strains. After regeneration of the cell wall, stable fusion recombinants were isolated. Genetic analysis of these recombinants revealed that they contained the genotype of both parents, sporulated well with each ascus containing four spores and were indeed diploid. Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.

Application of Yeast Genetics within the Brewing Industry. A Review

A brewing yeast strain must remove specific nutrients from wort and impart desired flavor to beer, and after fulfilling their metabolic role, the microorganisms must be effectively removed from the fermented wort. The strain's heredity and environment influence these functions. Environmental influences on yeast performance have been studied extensively, but the influence of genetic makeup has been neglected until recently. Although strains of Saccharomyces generally are diploid in the vegetative phase and haploid spores can be induced, genetic work involving brewer's yeast strains is complicated by their frequent triploid, polyploid or aneuploid nature. The strains sporulate poorly and rarely form spores in fours in the ascus, and many of the spores that form are nonviable. However, hybridization, transformation, spheroplast fusion, mutation and DNA recombination could potentially be used to genetically manipulate brewer's yeast strains; review of the methods indicates that spheroplast fusion apparently has the greatest potential. Guidelines for handling DNA recombinant material are reviewed, and an attempt is made to place the potential hazards of DNA recombination in proper perspective.

Spheroplast fusion and heterokaryon formation in Mucor racemosus.

Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to spheroplasts by using commercial chitinase and Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed in a buffered Ca(NO3)2 solution and fusion occurred. After cell wall regeneration, prototrophs were isolated. The frequency of heterokaryon formation was 1.45 X 10(-4). Prototrophic isolates segregated parental nuclei at a high frequency, indicating that heterokaryons had formed.

Fusion of yeast spheroplasts

Spheroplasts of two different auxotrophic strains of Saccharomyces cerevisiae, both of mating type a, were fused with the aid of polyethylene glycol and calcium ions. After reversion to vegetative cells in solid media, the resulting zygotes were shown to be diploid cells of mating type aa.