Expression of two glutathione S-transferase genes in the yeast Issatchenkia orientalis is induced by o-dinitrobenzene during cell growth arrest

Glutathione S-transferases (GSTs) Y-1 and Y-2 from the yeast Issatchenkia orientalis were purified by passage through a glutathione-agarose column, and the cDNA for GST Y-1 was cloned and sequenced. The deduced amino acid sequence consisted of 188 residues with a total calculated molecular mass of 21,001 Da and showed 36.7% identity to that of GST Y-2, another GST isoenzyme expressed in this strain. Escherichia coli DH5alpha transformed with pUC119 harboring the GST Y-1 gene under the control of the lac promoter exhibited 29-fold-higher GST activity than the same strain with pUC119. Northern blot analysis revealed that both genes were highly expressed in cells cultured in the presence of 200 microM o-dinitrobenzene (DNB), one of the substrates of GST, while only the GST Y-1 gene was expressed, and only slightly, under normal (DNB-free) culture conditions. The DNB in the medium arrested cell growth until it was reduced by conjugation with reduced glutathione. Kinetic analysis of GST gene expression during detoxification of DNB revealed that the levels of expression of both genes were elevated within 3 h after the addition of DNB and that they further increased until 12 h postaddition. The levels of expression of both genes were decreased markedly when the DNB concentration in the culture medium was lowered. These results suggest that I. orientalis cells sense xenobiotics and arrest cell growth as a mechanism for preventing the induction of mutations by these compounds, while the levels of expression of the GST genes are up-regulated for detoxification.     

A new method of isolation and a new form of transketolase from baker's yeast]

A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed. The enzyme yield is 90% of its total content in the original yeast extract. The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

Degradation of malic acid by Issatchenkia orientalis KMBL 5774, an acidophilic yeast strain isolated from Korean grape wine pomace

Several yeast strains degrading malic acid as a sole carbon and energy source were isolated from Korean wine pomace after enrichment culture in the presence of malic acid. Among them, the strain designated as KMBL 5774 showed the highest malic acid degrading ability. It was identified as Issatchenkia orientalis based on its morphological and physiological characteristics as well as the nucleotide sequences of the internal transcribed spacer (ITS) I-5.8S rDNA-ITS II region. Phylogenetic analysis of the ITS I-5.8S rDNAITS II sequences showed that the KMBL 5774 is the closest to I. orientalis zhuan 192. Identity of the sequences of the KMBL 5774 was 99.5% with those of I. orientalis zhuan 192. The optimal pH of the media for the growth and malic acid degradation by the yeast was between 2.0 and 3.0, suggesting that the strain is an acidophile. Under the optimized conditions, the yeast could degrade 95.5% of the malic acid after 24 h of incubation at 30 degrees in YNB media containing 2% malic acid as a sole carbon and energy source.     

Continuous flow method for extraction and bioluminescence assay of ATP in baker's yeast

Summary The intracellular ATP of baker's yeast (Saccharomyces cerevisiae) was measured using the bioluminescent firefly luciferase assay. Benzalkonium chloride and trichloro-acetic acid served in the experiments as extracting agents and optimal conditions for the extraction and assay of the intracellular ATP are reported. Using the results obtained from manually performed experiments two continuous flow systems were designed for the measurement of ATP in yeast cells during cell growth. Good correlation between the amount of cellular ATP and cell growth was found during the exponential growth phase.     

Mutarotase in galactose-induced baker's yeast

Cell-free extracts of baker's yeast possess mutarotase activity only after induction of cells in the presence of galactose. The mutarotase activity appears 1 h after transfer to a galactose-containing medium and rises in synchrony with the utilization of galactose. Cycloheximide blocks the induction completely at a concentration of 100 μg/ml. InSaccharomyces fragilis the mutarotase is constitutive but its activity is strikingly increased after growth on galactose. The yeast mutarotase resembles in some respects analogous enzymes from other cells (pH dependence, substrate specificity, heat lability). Its affinity ford-galactose is substantially greater than ford-glucose. There may exist a coupling between mutarotase activity and the anomer-specific galactokinase.     

A fluorescent-cytochemical method for selective detection of yeast mitochondrial DNA

Yeast mitochondrial DNA (mDNA) can selectively be detected using a specific dye (DAPI). Nuclear DNA (nDNA) was stained along with mDNA only in three out of the fifteen studied yeast strains. Visualisation with a luminescent microscope showed that mDNA content varied among different yeast species as well as the size and shape of fluorescent mitochondrial nuclei. Intensive nDNA fluorescence was detected when a fixed specimen was treated with DAPI. Under these conditions, mDNA was revealed only in yeast cells with its high content. The process of fixation was shown to interfere with the integrity of mitochondria. It is also possible that the structure of DNA was modified to affect its interaction with the dye and thus the level of fluorescence. The developed technique of selective mDNA staining and the experimental results make it possible to gain a more accurate quantitative information about DNA content at the cellular level using cyto- and spectrofluorimetric techniques. This study involves important aspects pertinent to the dye interaction with yeast nuclear and mitochondrial DNA, both native and subjected to different fixation procedures.     

On a disturbance of the normal Pasteur reaction in baker's yeast

Resting cells of baker's yeast, suspended in phosphate buffer pH 5.0 with glucose give initially a normal Pasteur reaction, which means that fermentation is repressed under aerobic conditions by the respiratory process.However, after 1 to 2 hours fermentation a disturbance of the Pasteur reaction sets in, the aerobic fermentation rising to the anaerobic level or sometimes above this level without a corresponding decrease in respiration. It is demonstrated that this disturbance is closely related to an aerobic growth pattern in which the yeast in its final growth stage before harvesting obtains its energy exclusively from the respiratory process.The interrelation of fermentation and respiration is discussed. In this discussion the aerobic fermentation is defined as the metabolism of the excess of intermediates formed along the Embden-Meyerhof pathway and unable to enter the Krebs cycle due to the limited capacity of the electron transfer system.The author is greatly indebted to Prof. Dr. T. O. Wikén for his interest in this study and for offering him the opportunity to conduct the investigations in his laboratory.     

Properties of the sugar carrier in baker's yeast

A total of 16 hexoses and pentoses were investigated with respect to transport intoSaccharomyces cerevisiae cells. All monosaccharides were transported across the cytoplasmic membrane but only those with an equatorial hydroxyl group in positions 1 and 4 of theC1 chair conformation and those with an equatorial hydroxyl group in position 2 and an equatorial −CH₂OH group in position 5 of the1C chair conformation reached an equilibrium distribution in the entire cell water volume. Other monosaccharides reached a distribution in only 20–66% of the intracellular water. The two groups of sugars are apparently transported by different carriers (either in parallel or in series), each of them showing countertransport and an apparent activation energy of 6,700–7,800 cal/mol. The carrier transporting the perfectly distributing sugars (Group 1) is affected by uranyl nitrate but not by 2,4-dinitrophenol, the other carrier (Group 2) is apparently not susceptible to uranyl ions but is influenced by 2,4-dinitrophenol. The space of distribution of the Group 1 sugars is reduced in hypertonic media in accordance with changes of intracellular water, that of the Group 2 sugars is altered only very slightly. The carriers differ in their kinetic parametres (mobility of the loaded carriers, maximum rate of transport). There is only a very indistinct competition for transport between representatives of the two groups. Preincubation with d-galactose induces the formation or unmasking of a transport system whereafter even the Group 2 sugars reach equilibrium in the entire cell water. Part I. Fol. microbiol. 10: 30, 1965.     

Properties of the sugar carrier in baker's yeast

A kinetic test evolved for distinguishing between mobile carrier transport in which one or two substrate molecules are transported at a time was applied to sugar transport inSaccharomyces cerevisiae and it was found that the mechanism here involves attachment of one sugar molecule to one molecule of carrier. Incidental to the test, the dissociation constants of some sugar-carrier complexes were determined. The diversity of sugar transport mechanisms in different cells is discussed.     

Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine

Grape must was fermented by a mixed culture of Saccharomyces cerevisiae W-3 (a wine yeast) and Issatchenkia orientalis KMBL 5774 (a malic acid-degrading yeast). Co-fermentation with 1:1 (v/v) inoculum ratio of W-3 and KMBL 5774 decreased malic acid to 0.33 mg/ml from 1.1 mg ml with W-3 alone. Ethanol production was the same in both cases (7.8%, v/v). Acetaldehyde, 1-propanol, 2-butanol and isoamyl alcohol all decreased, with an increase in methanol, in the co-fermented wine. Sensory evaluation showed a higher score in the wine fermented with 1:1 (v/v) inoculum ratio than those obtained by 4:1 (v/v) inoculum ratio or W-3 alone.