Showing papers on "Catabolite repression published in 1986"

A Yeast Gene that is Essential for Release from Glucose Repression Encodes a Protein Kinase

Abstract: The SNF1 gene plays a central role in carbon catabolite repression in the yeast Saccharomyces cerevisiae, namely that SNF1 function is required for expression of glucose-repressible genes. The nucleotide sequence of the cloned SNF1 gene was determined, and the predicted amino acid sequence shows that SNF1 encodes a 72,040-dalton polypeptide that has significant homology to the conserved catalytic domain of mammalian protein kinases. Specific antisera were prepared and used to identify the SNF1 protein. The protein was shown to transfer phosphate from adenosine triphosphate to serine and threonine residues in an in vitro autophosphorylation reaction. These findings indicate that SNF1 encodes a protein kinase and suggest that protein phosphorylation plays a critical role in regulation by carbon catabolite repression in eukaryotic cells.

Cuticle-degrading Enzymes of Entomopathogenic Fungi: Regulation of Production of Chitinolytic Enzymes

Abstract: Summary: Synthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 μg ml−1 h−1) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87% while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase (N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae.

Abstract: Summary: The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since (i) inactivation did not occur during carbon starvation, (ii) when a fermentable sugar was added to starved cells, inactivation began, (iii) when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, (iv) when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.

Proline transport and osmotic stress response in Escherichia coli K-12.

Abstract: Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.

Glucose repression: a complex regulatory system in yeast.

Abstract: Glucose repression is a complex regulatory system controlling numerous biochemical pathways. The triggering signal for glucose repression of many enzymes is given by hexokinase isoenzyme P11. In addition to glucose repression a glucose derepression system exists. Certain upstream activation sites have been identified for glucose derepression.

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Abstract: Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.

Effect of different limitations in chemostat cultures on growth and production of exocellular protease by Bacillus licheniformis

Abstract: Maximal molar growth yields (Y sub max ) and protease production ofBacillus licheniformis S 1684 during NH 4 + -, O2-, and NH 4 + +O2-limitation with either glucose or citrate as carbon and energy source and during glucose-, and citratelimitation in chemostat cultures were determined. Protease production was repressed by excess ammonia when glucose served as C/E-source. Glucose and citrate repressed protease production during NH 4 + -limitation. A low oxygen tension enbanced protease production at low μ-values. It was concluded that, besides ammonia repression, catabolite flux and oxygen tension influence protease production, indicating that the energy status of the cell is important for the level of protease production.Y sub max -values were high during glucose-limitation and indicate a high efficiency of growth caused by a highY ATP max . During NH 4 + -, O2-, and NH 4 + +O2-limitation with glucose as C/E-values were lower than during glucose limitation. The lowerY sub max -values were due to a lower efficiency of energy conservation.Y sub max -values during limitations with citrate as C/E-source were lower than during limitations with glucose as C/E-source.

Catabolite repression mutants of yeast

Abstract: The mechaṅism of catabolite repression in yeast is not well understood, although it has been established that cAMP does not play a role similar to that found in Escherichia coli . To identify the elements implicated in catabolite repression in yeast, a variety of mutants affected in this process have been isolated by different research groups. A systematic review of the results reported in the literature is presented. The conclusion that can be drawn is that the mechanism of catabolite repression is a complex one, with no single gene controlling all the genes subject to repression. The expression of a given gene or set of genes is controlled by several regulatory genes, but it is not yet known whether these genes act cooperatively or sequentially.

Studies of anaerobic and aerobic glycolysis in Saccharomyces cerevisiae.

Abstract: Glucose metabolism was followed in suspensions of Saccharomyces cerevisiae by using 13C NMR and 14C radioactive labeling techniques and by Warburg manometer experiments. These experiments were performed for cells grown with various carbon sources in the growth medium, so as to evaluate the effect of catabolite repression. The rate of glucose utilization was most conveniently determined by the 13C NMR experiments, which measured the concentration of [1-13C]glucose, whereas the distribution of end products was determined from the 13C and the 14C experiments. By combining these measurements the flows into the various pathways that contribute to glucose catabolism were estimated, and the effect of oxygen upon glucose catabolism was evaluated. From these measurements, the Pasteur quotient (PQ) for glucose catabolism was calculated to be 2.95 for acetate-grown cells and 1.89 for cells grown on glucose into saturation. The Warburg experiments provided an independent estimate of glucose catabolism. The PQ estimated from Warburg experiments was 2.9 for acetate-grown cells in excellent agreement with the labeled carbon experiments and 4.6 for cells grown into saturation, which did not agree. Possible explanations of these differences are discussed. From these data an estimate is obtained of the net flow through the Embden-Meyerhof-Parnas pathway. The backward flow through fructose-1,6-bisphosphatase (Fru-1,6-P2-ase) was calculated from the "scrambling" of the 13C label of [1-13C]glucose into the C1 and C6 positions of trehalose. Combining these data allowed us to calculate the net flux through phosphofructokinase (PFK). For acetate-grown cells we found that the relative flow through PFK is a factor of 1.7 faster anaerobically than aerobically.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

Abstract: A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts. Images

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

Abstract: Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.

Yeast production of hepatitis b surface antigen

Abstract: Novel DNA constructs comprising regulatory regions plus the structural coding region for hepatitis B surface antigen (HBsAg) are disclosed. The regulatory regions employed are responsive to methanol, non-catabolite repressing carbon sources and catabolite repressing carbon sources followed by carbon source starvation. The novel constructs are incorporated into a variety of linear and circular plasmids. Such plasmids are used to transform suitable hosts and ultimately used for the production and isolation of hepatitis B surface antigen in high yields.

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Abstract: Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

Transcript analysis of the citrate synthase and succinate dehydrogenase genes of Escherichia coli K12.

Abstract: Summary: A transcript analysis of the citrate synthase and succinate dehydrogenase genes (gltA-sdhCDAB) of Escherichia coli was done by nuclease S1 mapping. Evidence was obtained for two monocistronic gltA transcripts extending anti-clockwise, to a common terminus, from independent promoters with start points 196 bp (major) and 299 bp (minor) upstream of the gltA coding region. Evidence was also obtained for two polycistronic sdh transcripts, sdhCDAB (minor) and sdhDAB (major), extending clockwise, from sites 219 bp upstream of sdhC and 1455 bp upstream of sdhD (i.e. within sdhC), to a common terminus. The synthesis of all of the transcripts was repressed by growth in the presence of glucose, and this is consistent with the well-established fact that both enzymes are subject to catabolite repression. Sequences resembling known binding sites for the cAMP-CRP (cyclicAMP-cyclicAMP receptor protein) complex occur in the vicinity of each promoter suggesting that they are activated by the cAMP-CRP complex.

Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoenzyme composition in Salmonella typhimurium.

Abstract: Hydrogen metabolism in Salmonella typhimurium is differentially regulated by mutations in the two anaerobic regulatory pathways, defined by the fnr (oxrA) and oxrC genes, and is controlled by catabolite repression The synthesis of the individual hydrogenase isoenzymes is also specifically influenced by fnr and oxrC mutations and by catabolite repression in a manner entirely consistent with the proposed role for each isoenzyme in hydrogen metabolism Synthesis of hydrogenase isoenzyme 2 was found to be fnr dependent and oxrC independent, consistent with a role in respiration-linked hydrogen uptake which was shown to be similarly regulated Also in keeping with such a respiratory role was the finding that both hydrogen uptake and the expression of isoenzyme 2 are under catabolite repression In contrast, formate hydrogenlyase-dependent hydrogen evolution, characteristic of fermentative growth, was reduced in oxrC strains but not in fnr strains Hydrogenase 3 activity was similarly regulated, consistent with a role in hydrogen evolution Unlike the expression of hydrogenases 2 and 3, hydrogenase 1 expression was both fnr and oxrC dependent Hydrogen uptake during fermentative growth was also both fnr and oxrC dependent This provided good evidence for a distinction between hydrogen uptake during fermentation- and respiration-dependent growth and for a hydrogen-recycling process The pattern of anaerobic control of hydrogenase activities illustrated the functional diversity of the isoenzymes and, in addition, the physiological distinction between the two anaerobic regulatory pathways, anaerobic respiratory genes being fnr dependent and enzymes required during fermentative growth being oxrC dependent

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth.

Abstract: We report the development of an efficient Bacillus subtilis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain. Images

Regulatory Mutations Affecting the Synthesis of Pectate Lyase in Erwinia chrysanthemi

Abstract: Pectate-lyases (PL) are extracellular enzymes secreted by Erwinia chysanthemi. By their attack of pectin, a constituent of plant cell walls, they are the major factor involved in the phytopathogenicity of this bacterial species. We described the isolation and characterization of mutants in the regulatory systems controlling the synthesis of PL. The regulation of these proteins appeared complex and submitted to at least two important controls. Three classes of mutants constitutively synthesizing PL were found after a chemical mutagenesis of the wild-type strain B374. The cri mutations (class IV) conferred a resistance to the catabolite repression exerted by glucose to various proteins of the cell such as PL, cellulases, proteases, B-galactosidase and the hexuronate transport system. In the cri mutants the synthesis of PL remained inducible by pectin degradative products but were no longer repressible in presence of glucose. In contrast, the gpiR mutants (class I) were always sensitive to glucose repression but no longer inducible in presence of pectin derivatives. In these mutants, the regulatory gene responsible for the induction by pectin derivatives was inactivated. This gene is probably gpiR itself. Moreover the gpiR mutation led to a synthesis of PL independent of the growth phase. In liquid cultures of E. chrysanthemi, PL synthesis increases only at the end of the exponential growth, while in the gpiR mutants the PL synthesis is proportional to the cell density.

Protein utilization by basidiomycete fungi

Abstract: Three species of basidiomycete fungi, Agaricus bisporus, Coprinus cinereus and Volvariella volvacea , were grown on defined liquid media under conditions of proteinase induction (in the presence of protein as sole source of carbon, nitrogen or sulphur) and derepression (in the absence of any source of one of these major elements). All three species utilized protein as sole source of carbon, nitrogen and sulphur. Protein was utilized as efficiently as was glucose when provided as a sole source of carbon. When supplied together as carbon sources both protein and glucose were utilized more rapidly, and growth was greater, than when either protein or glucose were supplied separately as sole sources of carbon. Thus no catabolite repression was observed in the presence of glucose and, similarly, no catabolite repression was observed when media were supplemented with ammonium chloride as well as protein.

Identification and nucleotide sequence of the promoter region of the Bacillus subtilis gluconate operon.

Abstract: The nucleotide sequence (742 bp) of the promoter region of the Bacillus subtilis gluconate (gnt) operon is presented. Nuclease Sl mapping revealed the start point of the transcription and suggested that the expression of this operon is probably regulated at the transcriptional level. The sequences of the -35 and -10 regions suggested that RNA polymerase possessing sigma-43 may recognize this structure. The 223 bp fragment containing 100 bp upstream from the transcription start site actually exhibited a promoter activity when cloned in a promoter probe vector of pPL603B. This promoter activity was highly derepressed and although still under catabolite repression. The fragment on a high copy plasmid could titrate a regulator of the gnt operon so that the expression of the operon on the host chromosome also became derepressed.

N2-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa

Abstract: Arginine-nonutilizing (aru) mutants of Pseudomonas aeruginosa strain PAO converted L-arginine to N2-succinylarginine or N-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of N2-succinylornithine. Enzyme assays with crude P. aeruginosa extracts established the following pathway: L-arginine + succinyl-CoA → N2-succinylarginine → N2-succinylornithine → N_succinylglutamate 5-semialdehyde → N-succinylglutamate → succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified N2-acetylornithine 5-aminotransferase (N2-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated N2-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables P. aeruginosa to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in P. aeruginosa fulfill different functions.

The use of catabolite derepression as a means of improving the fermentation rate of brewing yeast strains

Abstract: The selective isolation of spontaneously arising derepressed Saccharomyces diastaticus and S. cerevisiae strains yielded mutants capable of improved maltose utilization, ethanol production, and glu...

Synthesis and regulation of extracellular β (1–3) glucanase and protease by cytophaga sp. In batch and continuous culture

Abstract: Lytic enzyme systems with the ability to break whole cells of yeast are a mixture of several enzymes and virtually all contain beta(1-3)glucanases and some protease. It appears that the presence of these two enzyme activities is necessary to break the two layers of the rigid cell wall. The enzyme system of Cytophaga NCIB 9497 has a high activity towards the walls of yeast and also of bacteria. This article describes the production of this extracellular lytic enzyme system in batch and continuous culture-it was found to be inducible. The synthesis and regulation of the two main constituent enzymes, beta(1-3)glucanase and protease, have been investigated. The synthesis of beta(1-3)glucanase is regulated by bothinduction (by an unknown inducer) and catabolite repression. Highbeta(1-3)glucanase activities were obtained in continuous culture at low dilution rates over a narrow range (0.05-0.10 h(-1)), and there is evidence of the presence of more than one glucanase enzyme. Proteolytic activity appears subject to catabolite repression and made up of the activities of more than one protease enzyme. Productivity and enzyme concentration were increased several fold in continuous culture when compared to batch culture.

Regulation of Bacterial Metabolism

Abstract: The discussion of the metabolism of aerobic heterotrophs has shown that a number of catabolic and anaplerotic enzymes are needed by microorganisms only under certain growth conditions. β-Galactosidase is required by E. coli during growth on lactose but not if glucose serves as substrate. A Pseudomonas strain confronted with phenol in its environment requires several specific enzymes in order to take advantage of this compound. These enzymes are not needed if organic acids such as malate or succinate were the substrates utilized by this microorganism. Thus, it is reasonable and economical that organisms do not synthesize all the time all the enzymes they are able to make but only those that are necessary for their metabolism under current physiological conditions. This regulation of enzyme synthesis is accomplished by induction and repression.

Positive and negative regulation of CAR1 Expression in Saccharomyces cerevisiae

Abstract: We localized the chromosomal targets of several of the regulatory controls of expression of theCAR1 gene. Fusion tolacZ of several fragments of the 5′ non-coding region showed that induction ofCAR1 by arginine is positively regulated by the products of theARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affectsCYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory allelesCARGRII andCARGRIII is not situated in the 5′ non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5′—nitrogen catabolite repression—activation by arginine—CARGRI-mediated repression—CAR1.