Uninduced Cultures Research Articles

ISOLATION OF BACTERIOPHAGE‐LIKE PARTICLES FROM UNINDUCED CLOSTRIDIUM TETANI CULTURES

The isolation of hexagonal-headed, tailless, bacteriophage-like particles from uninduced cultures of Clostridium tetani is described. Clear, round, 1--3 mm diameter plaques were noted on Clostrisel agar plates, which were overlaid with soft agar inoculated with 7--14 day broth cultures. Particles were detected by transmission electron microscopy from broth cultures seeded with scrapings from the plaques. Both electron dense and electron lucent heads were noted. An electron dense head was observed attached to the surface of a dividing bacterium.

Regulation of Synthesis of Benzyl Alcohol Dehydrogenase in Acinetobacter calcoaceticus NCIB8250

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second type of repression was observed during rapid growth or after the addition of compound such as succinate which are rapidly and completely metabolized.

Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida

Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems.

Testosterone uptake by membrane vesicles of Pseudomonas testosteroni

Uptake of testosterone was demonstrated in membrane vesicles prepared from Pseudomonas testosteroni grown on testosterone. In contrast, membrane vesicles from uninduced cultures revealed no significant transport activity for steroids. The K m of the reaction was 2 · 10 −6 M and the V 28.5 nmoles/min per mg protein. Steroid uptake was maximal within the pH range of 8 to 9 and at incubation temperatures between 30 and 37 °C. Transport of steroid was dependent upon NAD + and was reduced by NADH, dinitrophenol, and inhibitors of electron transport, such as N 3 − · CN − and amytal. The intravesicular steroid concentration was approx. 800 times the steroid concentration present in the medium at the start of the incubation.

A Kinetic Study of Glycolytic Enzyme Synthesis in Yeast

Abstract The kinetics of induced synthesis of glycolytic enzymes of the hybrid yeast Saccharomyces fragilis x Saccharomyces dobzhanskii in response to the addition of glucose or galactose has been studied in a basal medium containing peptone, acetate, and yeast extract. Glucose and galactose bring about a 3- to 100-fold increase in specific activity of various glycolytic enzymes during a 7- hour period. The smallest increase is observed in the case of P-glucoisomerase (EC 5.3.1.9) and the largest with glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12). In the stationary state of cells grown in the presence or absence of glucose, the glycolytic enzymes display a coordinate relationship to one another. The time course of enzyme synthesis by glucose and galactose, as well as of enzyme disappearance on removal of the sugars, however, suggests a kinetic heterogeneity. With galactose as the inducing carbohydrate, the enzymes increase in specific activity in the following sequence: pyruvate kinase (EC 2.7.1.40), within 20 min after addition of galactose; P-glucomutase (EC 2.7.5.1), P-fructokinase (EC 2.7.1.11), and glyceraldehyde-3-P dehydrogenase, within 1 hour; P-glucoisomerase, P-glycerate kinase (EC 2.7.2.3), P-glycerate mutase (EC 2.7.5.3), and enolase (EC 4.2.1.11), between 1 and 3 hours; hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.7), triose-P isomerase (EC 5.3.1.1), and pyruvate decarboxylase (EC 4.1.1.1), between 2 and 5 hours. Alcohol dehydrogenase (EC 1.1.1.1) falls outside any of these groups; after an initial period of repression, galactose causes increased synthesis of this enzyme during later periods. The increase of glycolytic enzyme activity elicited by sugars seems to be due to increased production of the same enzyme species already existing in uninduced cultures. The glucose-induced increase in glycolytic enzyme activity in Saccharomyces cerevisiae is completely prevented by cycloheximide, an inhibitor of protein synthesis in this yeast. P-Fructokinase and pyruvate decarboxylase decay rapidly inside the cells in the presence of this antibiotic.

Properties of the deoxyribonucleic acid contained in the defective particle coliphage 15.

Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm(2)) or to mitomycin C (5 mug/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm(3) followed by components with densities of 1.5 and 1.49 g/cm(3). The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 +/- 1% and 46.6 +/- 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 x 10(6) daltons.

Nature of lactose-fermenting Salmonella strains obtained from clinical sources.

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.

Changes in the specific activities of the galactose enzymes in E. coli under different growth conditions.

An increase in the specific activity of kinase and transferase is observed when late growth phase is compared with early exponential phase. The increase is more pronounced in the presence of saturating inducer concentrations than in uninduced cultures. Epimerase does not increase in late growth phase in the presence of D-fucose, provided the other enzymes of the galactose pathway are present. It is believed that increases observed are due to an increased rate of synthesis of the galactose enzymes in late growth phase, and that the lack of coordination found for epimerase is due to an inactivation of the latter enzyme, which occurs in the presence of D-fucose and needs an intact galactose pathway.

Relation between iron uptake, pH of growth medium, and penicillinase formation in Staphylococcus aureus.

The uptake of iron and the formation of penicillinase was examined in cultures of wild-type Staphylococcus aureus. Uptake of iron was about twice as great at pH 4.7 as at pH 7.4 At pH 4.7, increase in iron uptake in the range of 1.0 to 4.0 mug per mg of bacterial protein was associated with a progressive increase in the rate of penicillinase formation, but a direct correlation between cellular iron content and rate of enzyme formation was not demonstrated. Addition of iron to deferrated medium enhanced penicillinase formation at pH 6.5 to 7.4 two- to fourfold in cultures induced with benzylpenicillin and in uninduced cultures. To demonstrate an effect on the uninduced cells, it was necessary to increase iron uptake by preliminary incubation of cells with iron in buffer. Calcium and certain other ions depressed iron uptake at acidic and at neutral pH, and, presumably as a result of this action, depressed the formation of penicillinase. Iron did not enhance penicillinase formation at pH 4.7 by two penicillinase constitutive mutants nor by wild-type cells undergoing induction at pH 6.5 by cephalosporin C or methicillin. After removal of cephalosporin C or methicillin during an early phase of induction, residual synthesis of enzyme was increased by prior uptake of iron. The results are considered compatible with the concept that uptake of iron, especially at acidic pH, interferes with the formation or function of penicillinase repressor.

THE DISTRIBUTION AND FORMATION OF PENICILLINASE IN A BACTERIAL POPULATION OF BACILLUS LICHENIFORMIS.

Summary: The distributions of penicillinase in populations of uninduced, induced and constitutive cultures of Bacillus licheniformis strains 749 and 749/c each showed a large positive skew. Analysis of the enzyme distribution in the uninduced population as a Poisson distribution showed that penicillinase molecules were produced not as independent random events, but in clusters containing 1100 molecules. There were 0.4 (average 0.85) clusters/organism. The fundamental random event resulting in the formation of a cluster of penicillinase molecules may be the formation of one enzyme-forming site capable of producing 1100 such molecules. Analysis of the enzyme distribution in the constitutive population showed that in this case penicillinase was produced in groups of 55,000 molecules, or fifty of the clusters of penicillinase found in the organisms of the uninduced population. This may be the number of enzyme-forming sites made by a constitutive penicillinase gene during the period between its formation and replication. Analysis of the enzyme distribution in the induced population showed that the organisms did not respond equally after induction, but all organisms probably increased their rates of penicillinase synthesis. Enzyme partition can be highly asymmetric when an organism divides; the enzyme molecules do not appear to be able to diffuse freely within the cell cytoplasm, but may be associated with some structurally rigid material in the organisms.