Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae

Galactose does not allow growth of pyruvate carboxylase mutants in media with ammonium as a nitrogen source, and inhibits growth of strains defective in phosphoglyceromutase in ethanol–glycerol mixtures. Starting with pyc1, pyc2, and gpm1 strains, we isolated mutants that eliminated those galactose effects. The mutations were recessive and were named dgr1-1 and dgr2-1. Strains bearing those mutations in an otherwise wild-type background grew slower than the wild type in rich galactose media, and their growth was dependent on respiration. Galactose repression of several enzymes was relieved in the mutants. Biochemical and genetic evidence showed that dgr1-1 was allelic with GAL2 and dgr2-1 with GAL4. The results indicate that the rate of galactose consumption is critical to cause catabolite repression.     

Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae

The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU·mg^-1 at this induced state. In glucoselimited, derepressed cells, 20 mU·mg^-1 were detected and under repressed conditions isocitrate lyase activity was not detected.The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1

We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide     

Repression of asexual embryogenesis in vitro by some plant growth regulators

Summary A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Anne's Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above. This paper is part of B. Tisserat's Ph.D. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast

Summary The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose. Glucose repression in the transformants was like that in the wild type. The HEX2 gene was localized within a 2.15 kb fragment. The restriction map was confirmed by Southern hybridization of genomic DNA. Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM. Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1. Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.     

Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii

The production of xylitol by the yeast Candida guilliermondii was investigated in batch fermentations with aspenwood hemicellulose hydrolysate and compared with results obtained in semi-defined media with a mixture of glucose and xylose. The hemicellulose hydrolysate had to be supplemented by yeast extract and the maximum xylitol yield (0.8 g g^–1) and productivity (0.6 g l^–1 h^–1) were reached by controlling oxygen input.     

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EII^man), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (K _m=11 M) and a low-affinity proton motive force driven glucose permease (GP) (K _m=170 M). (ii) Both 9R-4 and X-160 had only the low-affinity system (K _m=181 M for 9R-4, 278 M for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EII^man) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.Abbreviations GCR glucose-mediated catabolite repression - FCR fructose-mediated catabolite repression - PEP phosphoenolpyruvate - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - glc:PTS phosphoenolpyruvate:glucose phosphotransferase system - GP glucose permease - CCCP carbonylcyanide mchlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - P proton motive force - G-6-P glucose-6-phosphate - 2DG 2-deoxyglucose - IAA iodoacetic acid - EII^man enzyme II component of man:PTS - EIII^man enzyme III component of man:PTS - EII^glc enzyme II component of glc:PTS - EIII^glc enzyme III component of glc:PTS