Metabolite and enzyme profiles of glycogen metabolism in Methanococcoides methylutens.

When a buffered anaerobic cell suspension of Methanococcoides methylutens was maintained under methanol-limited conditions, intracellular glycogen and hexose phosphates were consumed rapidly and a very small amount of methane formed at 4 h of a starvation period. When methanol was supplemented after a total of 20 h of starvation, a reverse pattern was observed: the glycogen level and the hexose phosphate pool increased, and formation of methane took place after a lag period of 90 min. A considerable amount of methane was formed in 120 min after its detection with a rate of 0.18 micromol mg(-1) protein min(-1). When methane formation decreased after 270 min of incubation and finally came to a halt, probably due to complete assimilation of supplemented methanol, the levels of glycogen and hexose monophosphates decreased once again. However fructose 1,6-diphosphate levels showed a continuous increase even after exhaustion of methane formation. In contrast to the hexose phosphate pool, levels of other metabolites showed a small increase after addition of methanol. The enzyme profile of glycogen metabolism showed relatively high levels of triose phosphate isomerase. Glyceraldehyde 3-phosphate dehydrogenase reacted with NADPH with a three-fold higher activity as compared to that with NADH.

Std1, a gene involved in glucose transport in Schizosaccharomyces pombe.

A wild-type strain, Sp972 h-, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [14C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617-2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.

Suppression of pdc2 regulating pyruvate decarboxylase synthesis in yeast.

Mutants lacking pyruvate decarboxylase cannot grow on glucose. We have isolated three different complementation groups of extragenic suppressors that suppress mutations in pdc2, a regulatory locus required for the synthesis of the glycolytic enzyme pyruvate decarboxylase. The most frequent of these is a recessive mutation in the structural gene PFK1 of the soluble phosphofructokinase. The other class XSP18 (extragenic suppressor of pdc2) is a dominant temperature-sensitive suppressor that allows the cells to grow on glucose only at 30 degrees but not at 36 degrees. It also affects the normal induction of the glucose-inducible enolase 2, which can be rescued by providing a copy of wild-type xsp18 in trans-heterozygotes. The pyruvate decarboxylase activity in the triple mutant pdc2 pfk1 XSP18 is nearly equal to the sum of the activities in the two double mutants pdc2 pfk1 and pdc2 XSP18, respectively. This implies that the two suppressors act through independent pathways or that there is no cooperativity between them. In the pdc2 pfk1 XSP18, strain, pfk1 suppresses the loss of induction of glucose-inducible enolase 2 brought about by XSP18 but fails to rescue temperature sensitivity. The third class (xsp37) supports the growth of the pdc2 mutant on glucose but fails to support growth on gluconeogenic carbon sources. All the three suppressors suppress pdc2 delta as well and act on PDC1 at the level of transcription.

Analysis of PFK3--a gene involved in particulate phosphofructokinase synthesis reveals additional functions of TPS2 in Saccharomyces cerevisiae.

The pfk3 mutation of Saccharomyces cerevisiae causes glucose-negativity in a pfk1 genetic background, the mutant is temperature-sensitive for growth and homozygous diploids do not sporulate. It fails to accumulate trehalose, and has an altered glycogen accumulation profile under glucose-starvation conditions. pfk3-6, one of the alleles of pfk3, has an altered morphology, forming long chain-like structures at 36 degrees C. The PFK3 gene was cloned by complementation of the mutant phenotypes. Integrative transformation demonstrated that the complementing fragment encoded the authentic PFK3 gene. The disruption of the gene does not affect viability. Like the EMS-induced pfk3 mutant, the disruptants are temperature-sensitive and in a pfk1 genetic background are also glucose-negative. The PFK3 transcript is induced by heat-shock. Partial DNA sequence shows that PFK3 is identical to TPS2 (De Virgilio et al., 1993). We demonstrate that, apart from being a structural determinant of trehalose 6-phosphate phosphatase, PFK3 (TPS2) is required for PFKII synthesis and normal regulation of S. cerevisiae response to nutrient and thermal stresses.

Induction of specific enzymes of the oxidative pentose phosphate pathway by glucono-delta-lactone in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on D-glucono-delta-lactone (delta gl) was found to be associated with a specific coordinate induction of the synthesis of two enzymes of the oxidative pentose phosphate pathway--6-phosphogluconate dehydrogenase and 6-phosphogluconolactonase--together with that of a third enzyme, gluconokinase. The gnd1 mutation, responsible for an approximately 80% loss of 6-phosphogluconate dehydrogenase activity and the inability of the cells to grow on delta gl, completely abolished the induction of all three enzymes, while the gnd2 mutation affected this only partially. One class of gnd1 revertants, selected for growth on delta gl, was found to have recovered normal dehydrogenase activity and the ability to synthesize the three enzymes when induced by delta gl. Another class of delta gl-positive revertants possessed constitutively elevated levels of gluconokinase. In contrast, glucose-positive revertants of gnd1, with restored constitutive dehydrogenase activity, continued to remain deficient in induction of the three enzymes and also failed to grow on delta gl. Induction of 6-phosphogluconate dehydrogenase activity was associated with increased transcription of the gene coding for the major isoenzyme; the transcript remained undetectable in the gnd1 mutant. Induction of these specific enzymes thus appears to be essential for growth of S. cerevisiae on delta gl.

Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae.

Ethanol and CO2 production from glucose by non-proliferating suspensions of aerobically-grown, glucose-derepressed wild-type Saccharomyces cerevisiae is inhibited by O2; monitoring by mass spectrometry provides a direct method for measurement of the Pasteur effect. Under aerobic conditions, that part of the CO2 evolved equivalent to the O2 consumed, is produced by respiration: subtraction of this respiratory CO2 from the total gives CO2 produced by aerobic glycolysis. Pasteur quotients (anaerobic CO2/aerobic glycolytic CO2) were within the range 1.2 to 3.0. The Pasteur effect was not observed in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of mitochondrial energy metabolism, or in a rho degree cytoplasmic petite mutant. A 'non-allosteric' mutant with an altered regulatory subunit of phosphofructokinase showed no Pasteur effect. Strains bearing a nonsense mutation pfk1 in the catalytic subunit of soluble phosphofructokinase (PFKI) also showed no Pasteur effect; the residual fermentative activity of this strain was dependent on PFKII, the particulate phosphofructokinase. A double mutant lacking both PFKI and glucose-6-phosphate dehydrogenase showed similar characteristics to those of the single pfk1 mutant; this indicates that the hexose monophosphate shunt is not acting to bypass the phosphofructokinase block. A 'hyper-allosteric' mutant altered in the regulatory subunit encoded by the gene PFK2 showed characteristics of glucose fermentation and ethanol oxidation very similar to those of wild-type organisms. These results indicate that either of the two phosphofructokinases can carry out glycolysis.

Complementation of soluble phosphofructokinase activity in yeast mutants.

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit. The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24 degrees C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.

Nature of primary product(s) of D-glucose 6-phosphate dehydrogenase reaction. 13C and 31P NMR study.

Glucose 6-phosphate dehydrogenase catalyzes the oxidation of glucose 6-phosphate, resulting in the formation of 6-phosphogluconolactone. As this compound is unstable, it has not been characterized directly. NMR provides a way to directly monitor all components of a reaction and study their structure. Here we report some results on the glucose 6-phosphate dehydrogenase reaction using 31P and 13C-NMR. Our results indicate that two different lactones, namely gamma (1-4) and delta (1-5) 6-phosphogluconolactones, are formed as products in this reaction. This is in contrast to an earlier suggestion that glucose 6-phosphate dehydrogenase produces only the delta-lactone. On the basis of these results, a new mechanisms for dehydrogenation of the sugar phosphate is proposed.

Gluconeogenic mutations in Pseudomonas aeruginosa: genetic linkage between fructose-bisphosphate aldolase and phosphoglycerate kinase.

Mutants of mucoid Pseudomonas aeruginosa defective in fructose-bisphosphate aldolase (FBA), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAP) or 3-phosphoglycerate kinase (PGK) were unable to grow on gluconeogenic precursors like glutamate, succinate or lactate. The gap and pgk mutants could grow on glucose, gluconate or glycerol, but fba mutants could not. This suggests that the metabolism of glucose or gluconate does not require either PGK or NADP-linked GAP but does require the operation of the aldolase-catalysed step. For gluconeogenesis, however, all three steps are essential. Recombinant plasmids carrying genes for FBA, PGK, GAP or phospho-2-keto-3-deoxygluconate aldolase (EDA) activities were constructed from a genomic library of mucoid P. aeruginosa selecting for complementation of deficiency mutations. Analysis of their complementation profile indicated that one group of plasmids carried fba and pgk genes, while another group carried eda, 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate dehydrogenase (zwf) genes. The gap gene was not linked to any of these markers. Partial restoration of FBA activity in spontaneous revertants of Fba- mutants was accompanied by a concomitant loss of PGK activity. These experiments indicate a linkage between the fba and pgk genes on the P. aeruginosa chromosome.

Fructose 1,6-bisphosphate aldolase activity is essential for synthesis of alginate from glucose by Pseudomonas aeruginosa.

We have isolated a mutant of Pseudomonas aeruginosa deficient in fructose 1,6-bisphosphate aldolase activity. This mutant, similar to the mutants deficient in any of the Entner-Doudoroff pathway enzymes, does not allow appreciable alginate formation from glucose and gluconate, but allows alginate synthesis from mannitol and fructose. This suggests that glucose and gluconate must be converted to fructose 1,6-bisphosphate via the Entner-Doudoroff pathway enzymes and fructose 1,6-bisphosphate aldolase.

Mutations in the regulatory subunit of soluble phosphofructokinase from yeast.

Mutant alleles of the gene PFK2 have been obtained that alter the sensitivity to ATP inhibition of the soluble yeast phosphofructokinase. One of the alleles makes the enzyme sensitive to micromolar concentrations of ATP. Intragenic revertants of PFK2 mutants confirm that the PFK2 gene determines not only the regulatory properties of the soluble enzyme but also the catalytic activity of particulate phosphofructokinase.

Multiple genes control particulate phosphofructokinase of yeast.

Mutants of Saccharomyces cerevisiae lacking the particulate phosphofructokinase define at least four unlinked genes, PFK2, PFK3, PFK4 and PFK5. A structural role of PFK2 is indicated. Mutations in the other three have pleiotropic effects.

Genetics of yeast glucokinase.

Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose. Reversion studies with glucokinase mutants indicate that the yeast S. cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase. Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes. The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme. In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent. These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase. Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate. Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation. The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost. Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1. --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene. In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants. These results raise the possibility that the protein products of these genes have some other essential function. --An earlier mapping result for hxk2 has been corrected. The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2.

Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.

Phosphofructokinase mutants of yeast. Biochemistry and genetics.

Mutants of Saccharomyces cerevisiae completely lacking the soluble glycolytic enzyme fructose-6-P kinase are described. The mutations are semidominant, do not complement one another, and define a gene PFK1 located 28-cm distal to rna1 on the extended right arm of chromosome XIII. Of 10 independent mutants, 3 can be suppressed by ochre suppressors. All mutants examined synthesize proteins that cross-react to the antibody against the purified yeast P-fructokinase. The enzyme in spontaneous revertants is distinguishable from the wild type enzyme with respect to thermolability and ATP inhibition. The locus PFK1 thus defines the structural gene of the enzyme. The pfk1 mutants are not leaky in vivo. All the glucose consumed by a double mutant lacking both P-fructokinase and 6-P-gluconate dehydrogenase ends up as 6-P-gluconate, yet the pfk1 mutants can glycolyze and grow on glucose in air. The cell mass produced per unit of glucose also remains unchanged. Anaerobically, however, growth does not take place, nor does glycolysis. P-fructokinase is thus a dispensable enzyme for aerobic growth, but indispensable for anaerobic growth. The properties of pfk1 mutants suggest that yeast has an alternative mechanism for the aerobic metabolism of fructose-6-P, presumably through the recently reported particulate P-fructokinase (Lobo, Z., and Maitra, P. K. (1982) FEBS Lett. 137, 279-282).

Pentose phosphate pathway mutants of yeast.

A glucose-negative mutant of Saccharomyces cerevisiae lacking 6-phosphogluconate dehydrogenase, the second enzyme of the pentose phosphate pathway, has been obtained by inositol starvation. Suppression of this mutant for growth on glucose takes place by the loss of glucose 6-phosphate dehydrogenase. A lesion in the latter enzyme alone leaves growth practically unaffected. The mutations define the respective structural genes.