Peter A. Whittaker

Application of protoplast fusion to the nonconventional yeast

Abstract Protoplast fusion has made a significant contribution to our understanding of the genetics and biochemistry of the nonconventional yeasts, and it has facilitated the creation of novel strains of yeast that display enhanced biotechnological potential. This article presents an examination of the means of isolating, reverting, and fusing yeast protoplasts, as well as an analysis of the products resulting from such fusions. Although this review is primarily concerned with the impact protoplast fusion has made on our knowledge of the nonconventional yeasts, where appropriate, reference to data gained from work with the conventional yeasts, i.e., Saccharomyces or Schizosaccharomyces , is made.

Protoplast fusion in a petite-negative yeast, Kluyveromyces lactis

SummaryThe technique of protoplast fusion has been applied to the problem of unstable diploidy in the yeast Kluyveromyces lactis. By protoplast fusion between heterothallic strains of like mating-type, sporulation-deficient hybrids can be obtained. Biochemical, cytological, and genetical characterisation of these hybrids suggests that the majority of fusion products are diploid. Sporulating hybrids can be constructed by protoplast fusion between homothallic strains. Tetrad analysis of these hybrids demonstrates conclusively the diploid nature of fusion products.

Effect of sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of shape Candida albicans

The effect of a sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans was investigated. The total lipid content of this yeast grown in the presence of chlorhexidine was reduced whilst the total sterol content was increased compared with control-grown cells. Lipids and sterol analyses of this yeast grown in the presence and absence of chlorhexidine are presented.Chlorhexidine-grown yeast had a higher level of phosphatidylethanolamine, phosphatidylcholine and monogalactosyldiacylglycerol. Lower proportions of phosphatidylinositol plus phosphatidylserine, phosphatidic acid and cardiolipin were found in C. albicans grown in the presence of the drug when compared with control-grown yeast. The major fatty acids in control-grown cells were C16 and C18. Drug grown-cells had higher proportions of palmitic acid (16 : 0) and stearic acid (18 : 0), but lower proportions of palmitoleic acid (16 : 1) and oleic acid (18 : 1). Chlorhexidine also decreased the unsaturated-to-saturated fatty acid ratio, while the C16/C18 ratios increased compared to control-grown cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug and control-grown yeast were also detected.Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (55.4% wt). A significant increase in ergosterol and obtusifoliol was observed in chlorhexidine-treated cells and a significant decrease in squalene and lanosterol. Our results suggested that chlorhexidine affected the lipid and sterol composition of C. albicans.

Application of the Melle-Boinot process to the fermentation of xylose by Pachysolen tannophilus

The Melle-Boinot process (cell-recycle batch fermentation) was applied to the fermentation of xylose by two strains of the yeast Pachysolen tannophilus for periods of 31 days. This resulted in a significant increase in the ethanol yield and productivity from xylose by P. tannophilus NCYC 614 but not by strain CBS 4045. Analysis of cells of strain NCYC 614 at the end of the process revealed an increased ability to tolerate exogenous ethanol, a characteristic that was lost in the absence of selection pressure. It is postulated that the increased yield of ethanol from xylose by P. tannophilus NCYC 614 is a product of enhanced ethanol tolerance together with increased efficiency of the fermentation process associated with the Melle-Boinot system.

Subinhibitory concentration of octenidine and pirtenidine: influence on the lipid and sterol contents of Candida albicans.

The effect of subinhibitory concentrations of octenidine and pirtenidine on the lipid and sterol composition of Candida albicans was investigated. The total lipid and sterol contents of C. albicans grown in the presence of either octenidine or pirtenidine were reduced compared with control-grown cells. The major differences in the lipid composition of drug-grown and control cells were phosphatidylglycerol, phosphatidylcholine and monogalactosyldiacylglycerol, which increased in the presence of octenidine and pirtenidine. Lower proportions of phosphatidic acid were found in yeasts grown in the presence of the drugs when compared with control C. albicans. Fatty acid analysis of control-grown cells showed that the major fatty acids were C16 and C18. Drug-grown cells had higher proportions of palmitic and linolenic acids but lower proportion of oleic acid. The C16/C18 ratios were higher for octenidine- and pirtenidine-grown cells than control cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug-grown and control yeasts were also observed. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (65.9%). A significant increase in squalene and 4,14-dimethylzymosterol was observed in pirtenidine-treated cells, while octenidine-treated cells showed an increase in zymosterol and obtusifoliol contents. Our results suggest that octenidine and pirtenidine affect the lipids and sterol of C. albicans in different ways. The implications of these findings on the mode of action of these two drugs is discussed.

Isolation and characterization of respiration-deficient mutants from the pathogenic yeastCandida albicans

The isolation of several respiration deficient mutants of the pathogenic yeastCandida albicans is described. These show greatly reduced respiration rates, loss of cytochromesaa3 andb, and reduced growth rates. All of the mutants had lost the ability to assimilate a wide range of carbon sources. Ultrastructural studies showed reduced development of mitochondrial cristae in the mutants. The mutants can be divided into three classes depending on their respiration responses to the addition of cyanide.

The effect of mycophenolic acid on the cell cycle of Candida albicans

Mycophenolic acid inhibited the growth ofCandida albicans. Cultures exposed to a concentration of 8.4 μg ml−1 mycophenolic acid were found to exhibit cell cycle arrest with two or more buds. Nuclear staining revealed that these were nucleate implying a possible defect in cytokinesis.The results are discussed in relation to the possible mode of action of mycophenolic acid.

Autoclaved Polyethylene Glycol decreases yeast protoplast reversion and hybrid production

Autoclaved solutions of the fusogen Polyethylene Glycol (PEG) give reduced yeast protoplast reversion and hybrid formation. Autoclaving has been shown to cause PEG degradation and increase the acetaldehyde content. Consequently, membrane filtration is recommended as the optimum means of sterilising PEG solutions to ensure maximum protoplast reversion and hybrid yield.

A novel technique for studying the adherence of Candida albicans to HEp-2 cells

Abstract Factors which affected the adherence of the pathogenic yeast Candida albicans to HEp-2 cells were investigated. In order to study this phenomenon a novel means of isolating HEp-2 cells with adherent Candida from mixtures of cells was developed. This involved attaching a magnetic bead, with the aid of an antibody, to the surface of the epithelial cell. Initial studies revealed that individual beads could be attached to approximately 70% of HEp-2 cells by varying the original bead to cell ratio. Such bead-HEp-2 complexes could subsequently be collected in a magnetic field. The use of this method for isolating HEp-2 cells with adherent yeast obviated a number of the problems associated with the more conventionally employed technique of differential filtration. A variety of factors were identified as affecting the extent of Candida-HEp-2 adherence and these included the incubation temperature, the phase of yeast cell growth and the carbon source upon which the yeast was grown.