D G Fraenkel

Use of chloramphenicol to study control of RNA synthesis in bacteria

Abstract With a view to deciding whether changes in the rate of synthesis of RNA (relative to that of protein or DNA) in bacteria are dependent on variations in amounts of enzymes concerned with this synthesis, use had been made of the fact that chloramphenicol addition to these cells blocks protein but not RNA synthesis. Measurements have been made of the rate of RNA synthesis before and after chloramphenicol addition (a) to cultures in balanced growth making RNA at different rates relative to their synthesis of protein or DNA, and (b) to cultures in unbalanced growth with changing rates of RNA synthesis. The findings were: 1. 1. Chloramphenicol addition to cultures in balanced growth usually results in an increase in the rate of RNA synthesis. 2. 2. In many cases the rate of RNA synthesis in the presence of chloramphenicol can be markedly increased by enrichment of the medium after chloramphenicol addition. 3. 3. With cultures early in the depressed phase of RNA synthesis during decelerating growth, the addition of chloramphenicol causes a resumption of fast RNA synthesis; later addition of chloramphenicol results in progressively less stimulation. These results are interpreted to mean that the rate of RNA synthesis in Aerobacter aerogenes is not primarily controlled by variations in the amount of enzymes concerned with this synthesis. The amounts of some of these enzymes, however, may be secondarily adjusted to match the actual rate of RNA synthesis.

The specific fructose diphosphatase of Escherichia coli: Properties and partial purification

Abstract Fructose 1,6-diphosphatase (FDPase) has been purified about 10-fold from extracts of Escherichia coli B. The properties of the enzyme are similar to those reported for FDPases from other sources: high specificity for the substrate fructose 1,6-diphosphate, a low value of K m (about 10 −5 M ), requirement for a metal ion, and sensitivity to inhibition by low concentrations of adenosine-5′-monophosphate. The enzyme differs from most other FDPases in having a pH optimum in the physiological pH range.

Fructose bisphosphatase of Saccharomyces cerevisiae: Cloning, disruption and regulation of the FBP1 structural gene

Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis. We have cloned the fructose bisphosphatase (FBP1) structural gene from Saccharomyces cerevisiae by screening a genomic library for complementation of an Escherichia coli fbp deletion mutation. The cloned DNA expresses in E. coli a fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate. Evidence is presented demonstrating that the entire gene, including all cis-acting regulatory sequences, has been cloned. A substitution mutation that disrupts FBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation. The fbp1 mutant strain is a hexose auxotroph, otherwise growing normally. Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated that FBP1 is present in single copy in the haploid genome. Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium. Fructose bisphosphatase activity was not greatly overproduced when the FBP1 gene was present on a multicopy vector in yeast.

Cloning of yeast glycolysis genes by complementation

Abstract The following glycolysis genes have been obtained by complementation in the appropriate yeast mutant from a pool of yeast DNA in the multicopy plasmid YEp13: PGI 1 (phosphoglucose isomerase), TPI 1 (triose-P isomerase), PGK 1 (phosphoglycerate kinase), GPM 1 (phosphoglycerate mutase), PYK 1 (pyruvate kinase), and GCR 1 (glycolysis regulation). The glycolysis enzymes include some of the major proteins of yeast, and in the clones (other than GCR 1) their levels, for the particular one, are 5–10 times normal.

Saccharomyces carlsbergensis fdp mutant and futile cycling of fructose 6-phosphate.

In Saccharomyces, the addition of glucose to cells grown in media lacking sugars causes irreversible inactivation of fructose bisphosphatase. One function of this process might be to prevent a futile cycle of formation and hydrolysis of fructose 1,6-bisphosphate. We tested such cycling by assessing the labeling of the 1-position of glucose in polysaccharides from [6-14C]glucose (J.P. Chambost and D. G. Fraenkel, J. Biol. Chem. 225:2867-2869, 1980) by using mutants impaired in glucose growth and known not to inactivate the phosphatase normally (i.e., the fdp mutant of Saccharomyces carlsbergensis [van de Poll et al., J. Bacteriol. 117:965-970, 1974] and the similar cif mutant of Saccharomyces cerevisiae [Navon et al., Biochemistry 18:4487-4499, 1979] ), as well as in the wild-type strain tested in the 1-h period before inactivation is complete. There was marginal, if any, cycling in any situation, and we conclude that the phosphatase activity is controlled by means other than inactivation or that the extent of cycling is too low to be significant, or both. For the fdp mutant data are also presented on growth, rate of glucose metabolism, metabolite accumulations, enzyme levels, and glucose transport, but the primary lesion is unknown.

A mutation increasing the amount of a constitutive enzyme in Escherichia coli, glucose 6-phosphate dehydrogenase☆

Abstract We describe the selection of a mutation which increases about sixfold the activity of glucose 6-phosphate dehydrogerase in Escherichia coli. In both mutant and wild type the enzyme is constitutive. The new mutation, zwfL1, is closely linked to zwf, the structural gene for the enzyme. According to antibody titration zwfL1 acts to increase the amount of normal enzyme, rather than by specifying an altered enzyme. ZwfL1 is cis-dominant. It might be an “up” promoter mutation.

“Up-promoter” mutations of glucose 6-phosphate dehydrogenase in Escherichia coli

The “up-promoter” mutation, zwfL1, of the constitutive gene (zwf) for glucose 6-phosphate dehydrogenase of Escherichia coli (Fraenkel & Banerjee, 1971), has been mapped with respect to point mutations in the structural gene. Both zwfL1 and two other high-level mutations are at one end of the gene. The direction of transcription of zwf is probably counter-clockwise, as the map is usually drawn. The level of expression of zwf in the wild-type is about 0.1 that of fully induced lac, while one of the promoter mutants, zwfL2, increases its rate of expression by 18-fold, giving perhaps the highest rate for any known bacterial gene.

Allosteric and non-allosteric E. coli phosphofructokinases: effects on growth

Abstract In wild type Escherichia coli K-12 ca. 90% of phosphofructokinase is known to be the allosteric enzyme Pfk-1, and the rest is Pfk-2, a non-allosteric enzyme. An isogenic strain series has now been constructed with varying combinations and amounts of Pfk-1 and Pfk-2 (e.g., no Pfk-1, high level of Pfk-2; normal level Pfk-1, high level Pfk-2, etc.). In minimal medium with glucose, glucose-6-P, and glycerol, aerobically and anaerobically, provided there is adequate total amount of enzyme, what allosteric type it is does not make much difference to growth rate or yield of this organism.

Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli

When E. coli carrying multicopy plasmids for fructose-1,6-P2 aldolase or phosphoglycerate kinase was grown in the presence of 32Pi, there was label at the position of cognate high level polypeptide after SDS-PAGE. As tested for aldolase, the label was resistant to acetone, RNase, and hot TCA treatments, and was also observed by immunoprecipitation, which was competed for by purified aldolase. Incorporation of label also occurred in the presence of chloramphenicol. Immunoprecipitation revealed apparent aldolase labeling in the wild type strain as well.

The Biochemical Genetics of Glycolysis in Microbes

Mutants for most reactions of glycolysis have been described both in Escherichia coli and in Sacchavomyces cevevisiae The pathway between glucose and pyruvate has three irreversible and seven reversible reactions (Figure 1), and most of the intermediates are needed in biosynthesis. Thus, one might expect mutants in an irreversible step to be impaired in growth on glucose but unimpaired gluconeogenically, and mutants in a reversible step to require supplementation even for gluconeogenic growth. To a first approximation this pattern is found but there are deviations (Table I). For example, E, coli mutants blocked between triose-P and phosphoenol-pyruvate do require supplementation (e.g., by glycerol) for growth on lactate (1,2) but phosphoglucose isomerase mutants grow without supplementation by glucose (3) — probably because glucose-6-P and its products are not essential for growth of this organism. Aldolase mutants also do not require supplementation for gluconeogenic growth, and the explanation is unknown; it might relate to other aldolases (see ref. 4). The growth on glucose of a (double) pyruvate kinase mutant occurs because phosphoenolpyruvate is used by the phosphotransferase (PTS) reaction intiating glucose metabolism and such a mutant fails to grown on non-PTS sugars (5).