Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Regulation of the β-ketoadipate pathway in Rhizobium japonicum and bacteroids by succinate

Rhizobium japonicum 61-A-101 and its bacteroids catabolize phenol and p-hydroxybenzoate. With phenol as a carbon source, utilization started only after a prolonged lag phase while p-hydroxybenzoate was almost instantancously metabolized. Succinate, which supports rapid growth of Rhizobium japonicum, completely repressed respication of phenol; the oxidation of p-hydroxybenzoate was partially inhibited. Pyruvate, supporting slower growth than succinate, retarded the onset of phenol consumption but did not affect its maximum rate.Catabolite repression of phenol utilization by succinate appears to be a characteristic feature of rhizobia. In Pseudomonas putida which also actively metabolizes phenol, succinate had no effect on phenol utilization.     

Contributions of XyIR, CcpA andcre to diauxic growth ofBacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulatexyl operon expression on diauxic growth and expression of axylA-lacZ fusion.xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation ofxylR yields a two-fold increase in expression ofxylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. WhenxylR andcre are inactivated together a residual two-fold repression ofxylA is found. Inactivation ofxylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion ofccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivatedcre site inxylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.     

Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5 end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

Sucrose transport and hydrolysis inRhizobium tropici

TheRhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN₃) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, thatR. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.     

Selection of a mutant strain of Lipomyces kononenkoae with dextranase synthesis resistant to catabolite repression

A mutant strain of Lipomyces kononenkoae 2896-3 synthesizing dextranase but resistant to catabolite repression was obtained using N-nitroso-N-methylurea treatment. Enzyme biosynthesis in media with dextran and other carbon sources was then characterized. The capacity of the mutant to produce dextranase when grown on hydrolysed corn starch is demonstrated.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.