Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases

Summary We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.     

Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants.

Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Summary A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the K _m values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990)     

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.     

Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle.

Fifteen-fold overexpression of phosphoenolpyruvate synthase (Pps) (EC 2.7.9.2) in Escherichia coli stimulated oxygen consumption in glucose minimal medium. A further increase in Pps overexpression to 30-fold stimulated glucose consumption by approximately 2-fold and resulted in an increased excretion of pyruvate and acetate. Insertion of two codons at the PvuII site in the pps gene abolished the enzymatic activity and eliminated the above-described effects. Both the active and the inactive proteins were detected at the predicted molecular weight by polyacrylamide gel electrophoresis. Therefore, the observed physiological changes were due to the activity of Pps. The higher specific rates of consumption of oxygen and glucose indicate a potential futile cycle between phosphoenolpyruvate (PEP) and pyruvate. A model for the stimulation of glucose uptake is presented; it involves an increased PEP/pyruvate ratio caused by the overexpressed Pps activity, leading to a stimulation of the PEP:sugar phosphotransferase system.     

Effects of phosphoenol pyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum

The phosphoenol pyruvate carboxylase gene (ppc) of lysine-producing Corynebacterium glutamicum and C. lactofermentum strains was inactivated by marker exchange mutagenesis. The mutants lacked completely phosphoenol pyruvate carboxylase (PEP carboxylase) activity, but grew in minimal medium containing glucose as the sole carbon source. In addition, the ppc ^– strains produced equivalent titers of lysine in shake flasks and in 10-l fermentation experiments as their parent strains. To address the question of how ppc ^– Corynebacterium strains generate oxaloacetate (OAA) for their own metabolism as well as for high-level lysine production, we measured the activities of enzymes leading to OAA synthesis. Whereas pyruvate carboxylase activity was not detected in any of the strains, phosphoenol pyruvate carboxykinase (PEP carboxykinase) activity was found to be significantly higher in C. glutamicum ppc mutants compared to the parent strains. On the other hand, PEP carboxykinase activity in C. lactofermentum was essentially absent. As glyxylate cycle enzymes are strongly repressed by glucose, they are not likely to compensate for the lack of PEP carboxylase activity. PEP carboxykinase, among several candidates, could play this role. Correspondence to: M. Gubler     

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.     

Induction of pyruvate decarboxylase in glycolysis mutants of Saccharomyces cerevisiae correlates with the concentrations of three-carbon glycolytic metabolites

Pyruvate decarboxylase, PDCase, activity in wild-type yeast cells growing on ethanol is quite low but increases up to tenfold upon addition of glucose, less with galactose and only slightly with glycerol. PDCase levels in glycolysis mutant strains growing on ethanol or acetate were higher than in the wild-type strain. These levels correlated with the sum of the concentrations of three-carbon glycolytic metabolites. The highest accumulation was observed in a fructose bisphosphate aldolase deletion mutant concomintant with the highest PDCase activity wild-type level. On the other hand, the PDCase levels in the different mutants again correlated with the sum of the concentrations of the three-carbon glycolytic metabolites. This was interpreted to mean that full induction of PDCase activity requires the accumulation of hexose-and triosephosphates.Abbreviations PDCase pyruvate decarboxylase - dw dry weight - PEP phosphoenolpyruvate - WT wild-type     

Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.