Cyclic AMP may not be involved in catabolite repression in Saccharomyces cerevisiae: evidence from mutants unable to synthesize it

Yeast cells with a nonsense adenylate cyclase mutation, cyr1-3, required cyclic AMP for growth. This phenotype was suppressed by the byc1 mutation; however, cyr1-3 bcy1 cells produced no detectable level of adenylate cyclase or cyclic AMP. On induction, the bcy1 and cyr1-3 bcy1 mutant cells produced the same levels of galactokinase and alpha-D-glucosidase as did the wild-type cells and fourfold-higher levels of invertase. Since galactokinase synthesis was severely repressed by glucose in the constitutive GAL81 mutants, irrespective of the cyr1-3 bcy1 genotype, cyclic AMP may not be involved in catabolite repression.     

Abnormal Cell Envelope Ultrastructure of a Saccharomyces Mutant with Invertase Formation Resistant to Hexoses

The most obvious morphological characteristic of Saccharomyces mutant FH4C cells is the tendency to form clumps (production of invertase and alpha-glucosidase by this mutant is highly resistant to repression by hexoses). This peculiar feature arises from the abnormal cell envelope ultrastructure of the mutant. Clumps are formed as a result of the failure of the cell wall of the bud to separate from that of the mother cell. The cell wall also shows irregular thickening. There are many cells with a doughnut shape and with small budlike protrusions. Abnormal septation and wall invagination into vacuoles give rise to cells of differing sizes and irregular profiles. Many vesicles, tubules, and coiled membranous bodies originate from invaginations of the plasmalemma. These structures are frequently observed in the cell wall or the periplasmic space. The cells of mutant FH4C growing in 0.2 M glucose, unlike parent strain 303-67, contain many mitochondria. Large numbers of glycogen deposits are also found in many cells of FH4C.     

A partial defect in carbon catabolite repression in mutants of Saccharomyces cerevisiae with reduced hexose phosphyorylation

Summary Mutants of Saccharomyces cerevisiae with reduced glucose phosphorylation were investigated. They were all recessive and belonged to one gene HEX1, mutant designation hex1. Carbon catabolite repression of alpha-glucosidases, invertase and part of the total malate dehydrogenase was reduced. Repression of the glyoxylate cycle enzymes, isocitrate lyase and malate synthetase, as well as that of gluconeogenetic fructose-1, 6-bisphosphatase was normal. A slight effect on repression of succinate: cytochrome c oxidoreductase and respiration was to be detected. The effect on repression by fructose was much less pronounced but still clear. However, there was a paradoxical effect of hexose concentration with higher concentrations repressing less. Maltose was also less repressing in the mutant. Growth on all sugars degraded via the hexose phosphorylation reaction was reduced and more strongly so at higher concentrations. Intracellular concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate were largely the same in mutant and wild type. The only striking difference between mutant and wild type was a fourfold higher intracellular glucose concentration in maltose grown mutants cells. The data obtained do not support the contention that carbon catabolite repression of the enzymes studied is triggered by intracellular hexoses or their metabolites alone. They rather suggest that it is some component of the hexose phosphorylating system that contributes to carbon catabolite repression.     

Effect of exogenous cycllc amp on catabolite repression of cellulase formation in aspergillus nidulans

The addition of 1 mM cyclic AMP to induced and repressed cultures of Aspergillus nidulans and its mutant strain (CRR 141) resistant to catabolite repression was fully capable of releasing the wild type from catabolite repression while it caused hyperproduction of cellulases in glycerol repressed cultures. The relief of the catabolite repression was also accompanied by a dramatic drop in enhanced protease levels, thereby indicating that the synthesis of proteases (during the catabolite repression) is under the control of cyclic AMP.     

Isolation and characterization of glucose derepressed invertase mutants from Schizosaccharomyces pombe

We have isolated 14 different Schizosaccharomyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression.     

Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling.

Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.     

Regulation of Repressible Acid Phosphatase by Cyclic Amp in SACCHAROMYCES CEREVISIAE

One of the cyr 1 mutants (cyr 1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25 degrees and was unable to derepress acid phosphatase. Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity. The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity. The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase. The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis. Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase. The cyr1-2 mutant produced significantly low levels of invertase and alpha-D-glucosidase. These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.     

Effects of aerobic and anaerobic shock on catabolite repression in cyclic AMP suppressor mutants of Escherichia coli.

Cultures of Escherichia coli K-12 grown on glucose or gluconate under aerobic conditions exhibited catabolite repression of beta-galactosidase synthesis. Depression occurred when these cultures were subjected to anaerobic shock. These states of repression and depression were found to be associated with low and high differential rates of cyclic AMP synthesis, respectively. This observation is consistent with the view that cyclic AMP plays a central role in the catabolite repression phenomenon. We report here, however, that identical stages of repression and derepression occur in mutant strains possessing cya crp(Csm) genotypes and therefore unable to synthesize cyclic AMP. These results suggest that cyclic AMP is not the sole regulator involved in catabolite repression.     

Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.     

The synthesis of beta-galactosidase by constitutive and other regulatory mutants of Escherichia coli in chemostat culture.

The synthesis of beta-galactosidase by an E. coli constitutive mutant was examined in a chemostat using glucose-, glycerol-, succinate- or N-limited growth media. Except for glucose-grown bacteria, the steady-state intracellular level of beta-galactosidase was maximal at dilution rates between 0-2 and 0-3 h-1. At higher dilution rates enzyme synthesis was reduced by catabolite repression, which could be relieved by the addition of cyclic AMP. With a catabolite-resistant mutant (UV5c), no decrease in enzyme level at high dilution rates were observed. All mutants examined were constitutive and gave decreased enzyme levels at low dilution rates, with the exception of lac-/F'lac UV5c mutants where the enzyme levels rose at low dilution rates. Hyper-producing mutants were isolated but were unstable. A constitutive mutant growing on glycerol-limited media was considered the most suitable for large-scale production of beta-galactosidase in a chemostat.     

Genes Affecting the Regulation of SUC2 Gene Expression by Glucose Repression in SACCHAROMYCES CEREVISIAE

Mutants of Saccharomyces cerevisiae with defects in sucrose or raffinose fermentation were isolated. In addition to mutations in the SUC2 structural gene for invertase, we recovered 18 recessive mutations that affected the regulation of invertase synthesis by glucose repression. These mutations included five new snf1 (sucrose nonfermenting) alleles and also defined five new complementation groups, designated snf2, snf3, snf4, snf5, and snf6. The snf2, snf4, and snf5 mutants produced little or no secreted invertase under derepressing conditions and were pleiotropically defective in galactose and glycerol utilization, which are both regulated by glucose repression. The snf6 mutant produced low levels of secreted invertase under derepressing conditions, and no pleiotropy was detected. The snf3 mutants derepressed secreted invertase to 10-35% the wild-type level but grew less well on sucrose than expected from their invertase activity; in addition, snf3 mutants synthesized some invertase under glucose-repressing conditions.--We examined the interactions between the different snf mutations and ssn6, a mutation causing constitutive (glucose-insensitive) high-level invertase synthesis that was previously isolated as a suppressor of snf1. The ssn6 mutation completely suppressed the defects in derepression of invertase conferred by snf1, snf3, snf4 and snf6, and each double mutant showed the constitutivity for invertase typical of ssn6 single mutants. In contrast, snf2 ssn6 and snf5 ssn6 strains produced only moderate levels of invertase under derepressing conditions and very low levels under repressing conditions. These findings suggest roles for the SNF1 through SNF6 and SSN6 genes in the regulation of SUC2 gene expression by glucose repression.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Hexose-transport-deficient mutants of Chlorella vulgaris

Autotrophically grown cells of Chlorella vulgaris show a strong increase in the uptake rates for hexoses and for seven amino acids when incubated in the presence of hexoses. This increase is due to de-novo synthesis of three transport proteins: one forhexoses and two for amino acids. Mutants deficient in hexose transport were obtained after treatment of wild-type cells with acridine orange, followed by a selection procedure using the toxic hexose analogue, 2-deoxy-D-glucose. Moreover, the two amino-acid-transport systems could not be induced in these mutants by hexoses. The capacity to phosphorylate hexoses was identical in mutants and in the wild-type strain. The loss of transport activities can be correlated with the loss of certain radiolabeled protein bands on fluorograms of sodium dodecylsulfate-polyacrylamide gels. These proteins are assumed to be responsible for the different transport systems in the wild-type strain. With the help of additional mutants defective in one or two of the different aminoacid-transport systems, it has been attempted to assign the different transport activities to individual protein bands on the gel.Abbreviations AUP arginine-uptake-defective mutant - 2-DG 2-deoxy-D-glucose - 6-DG 6-deoxy-D-glucose - HUP hexose-uptake-defective mutant - PUP^- proline-uptake-defective mutant - SDS sodium dodecyl sulfate - WT wild type     

Effects of crp mutations on adenosine 3'',5''-monophosphate metabolism in Salmonella typhimurium.

Wild-type Salmonella typhimurium could not grow with exogenous cyclic adenosine 3',5'-monophosphate (AMP) as the sole source of phosphate, but mutants capable of cyclic AMP utilization could be isolated provided the parental strain contained a functional cyclic AMP phosphodiesterase.All cyclic AMP-utilizing mutants had the growth and fermentation properties of cyclic AMP receptor protein (crp) mutants, and some lacked cyclic AMP binding activity in vitro. The genetic defect in each such mutant was due to a single point mutation, which was co-transducible with cysG. crp mutants isolated by alternative procedures also exhibited the capacity to utilize cyclic AMP. crp mutants synthesized cyclic AMP at increased rates and contained enhanced cellular cyclic AMP levels relative to the parental strains, regardless of whether or not cyclic AMP phosphodiesterase was active. Moreover, adenylate cyclase activity in vivo was less sensitive to regulation by glucose, possibly because the enzyme II complexes of the phosphotransferase system, responsible for glucose transport and phosphorylation, could not be induced to maximal levels. This possibility was strengthened by the observation that enzyme II activity (measured both in vitro by sugar phosphorylation and in vivo by sugar transport and chemotaxis) was inducible in the parental strain but not in crp mutants. The results suggest that the cyclic AMP receptor protein regulates cyclic AMP metabolism as well as catabolic enzyme synthesis.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.