Showing papers on "Growth medium published in 1967"

Selective Release of Enzymes from Bacteria

Abstract: A group of hydrolytic enzymes, including phosphatases and nucleases, is selectively released from E. coli and certain other Gram-negative bacteria by a process designated as osmotic shock. This procedure involves exposure of the cells to ethylenediaminetetraacetate (EDTA) in 0.5 molar sucrose followed by a sudden osmotic transition to cold, dilute MgCl(2). Osmotic shock also results in an alteration of the permeability barrier of the bacterial cell and a depletion of the pool of acid-soluble nucleotides, but there is no loss of viability. On being restored to growth medium, the shocked cells recover after a lag period. Formation of spheroplasts by treatment with EDTA and lysozyme leads to selective release of the same group of enzymes. We believe that the selectively released enzymes are confined in a region between the bacterial cell wall and the cytoplasmic membrane. Histochemical studies indicate such a localization. Further, the enzyme activities are measurable with intact cells, even when the substrate is a nucleotide, to which whole cells are impermeable. Another piece of evidence concerns a mutant E. coli with a defective cell wall. In contrast to normal bacteria, this organism loses one of these enzymes into the medium in the course of growth. After osmotic shock, the bacteria show reduced uptake of sulfate,betagalactosides, galactose, and certain amino acids. Furthermore, the shock treatment causes the release of nondialyzable factors able to bind sulfate, galactose, and the same amino acids. A possible interpretation of these observations is the following: the binding proteins occupy sites near the bacterial surface, and they may be components of active transport systems responsible for the concentrative uptake of these nutrients.

Biosynthesis of penicillin N and cephalosporin C. Antibiotic production and other features of the metabolism of a Cephalosporium sp.

Abstract: 1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.

Polyamines and the accumulation of ribonucleic acid in some polyauxotrophic strains of Escherichia coli.

Abstract: The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA. Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.

Path of glucose breakdown and cell yields of a facultative anaerobe, Actinomyces naeslundii.

Abstract: SUMMARY: Actinomyces naeslundii fermented glucose primarily by the Embden-Meyerhof pathway, as based on 14C-glucose fermentation data and enzyme studies. Enzymes of the oxidative pentose phosphate cycle were also present, but functioned only to a minor extent. Growth on glucose was increased 2-to 4-fold in the presence of substrate amounts of CO2 or O2. This increase was attributed to the additional energy (ATP) made available from the breakdown of pyruvate to acetyl coenzyme A. In the absence of CO2 or O2, pyruvate was reduced to lactate. The weight of organism produced/mole ATP (Y ATP) was 15-18 g. units under anaerobic conditions with CO2, dependent on the growth medium, and 20 under aerobic growth conditions.

The synthesis and function of ribosomes in a new mutant of Escherichia coli.

Abstract: the rate of protein synthesis per ribosome is icoonstant over a wide range of growth rates.' That the variation in the number of ribosomes reflects an important regulatory mechanism is emphasized by the immediate effects of transfer to a different growth medium. When the medium is enriched (a shift-up), ribosome synthesis at once increases, and in step with the increase in number of ri-bosomes the rate of protein synthesis rises above the preshift rate.2 Chloramphenicol is known to dissociate the synthesis of RNA from that of protein and it has been proposed that the former may be controlled by the degree of saturation of transfer RNA with amino acids.3 A similar idea was developed from a genetic analysis of strains of E. coli which do not stop synthesizing RNA when deprived of an essential amino acid.4 To analyze this problem further, we decided to search for mutants which for any one of several possible reasons have an RNA con1tent, under defined conditions of growth, significantly different from that of the parent strain. This paper describes the isolatiorn and some of the properties of such a mutant of E. coli. Materials and Methods.-The strain used was Escherichia coli 15 Thy - Pro - Smr (referred to as 15 TP). The selection procedure will be described in detail elsewhere. It is based on the fact that cells with different ratios of RNA to protein differ significantly in density and can be separated by centrifuging to equilibrium in density-gradients of potassium tartrate or Cs2SO4. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, and growth in broth to permit expression of mutations, the bacteria were centrifuged in a density gradient and cells isolated from below the main band of bacteria. Several strains with ratios of RNA to protein greater than that of the parent, when grown under the same conditions, have been isolated, and one of these, strain 15-28, has been studied in detail. It has the same requirements as 15TP and is resistant to streptomycin. Cultures were grown with forced aeration at 37? in a Tris-salts mediuLm2 containing glucose (0.2%), thymirie (10 ,Og/ml), and prolirie (50 jtg/ml), or in the same medium enriched with vitainiri-free casamino acids (1%). Turbidities were measured at 450 mA in 1-cm cuvett,es. The RNA, DNA, and protein conteiits of whole cells were determined as described previously,5 except that lysozyme was used as a stanidard in the measurement of protein anld deoxyadellositic for DNA.

Control of Lysine Biosynthesis in Yeast by a Feedback Mechanism

Abstract: Homocitric acid (β-hydroxy-β-carboxyadipic acid; HC) is accumulated by a lysine-requiring yeast mutant when grown in a chemically defined medium, supplemented with limited amounts of lysine. A study of the formation of HC in relation to the depletion of lysine from the growth medium indicates that HC accumulated only when the concentration of lysine was low. The enzymatic formation of HC from α-ketoglutarate plus acetyl-coenzyme A in cell-free extracts of the same organism was also inhibited by lysine. The inhibitory effect of lysine on the formation of HC in both whole cells and cell-free extracts is indicative of the functional existence of a feedback control mechanism in the pathway for lysine biosynthesis in yeast.

Influence of Culture Media on the Radiation Resistance of Micrococcus radiodurans

Abstract: The addition of NZ-case (a tryptic digest of casein) to a growth medium (PC) consisting of tryptone, glucose, and yeast extract caused a significant decrease in γ radiation resistance of Micrococcus radiodurans. The level of radiation resistance was inversely related to the concentration of NZ-case. The ld50 for this organism was approximately 700 krad when grown in tryptone, glucose, yeast extract, and dl-methionine (TGYM) broth, but it was approximately one-half as resistant when grown in a PC medium containing 0.5% NZ-case (PCNZ). The resistance to ultraviolet light was also reduced. Cultures transferred from PCNZ to TGYM media regained the high level of resistance.

Comparison of Effects of Simple Sugars on 3T3 Mouse Fibroblasts and 3T3 Cells Transformed by Infection with Oncogenic Viruses

Abstract: Summary 3T3 mouse fibroblasts, a cell line which ordinarily exhibits a high degree of contact inhibition in tissue culture, were markedly altered by addition of l-fucose to the growth medium. Such effects were not observed when equal amounts of different closely related sugars were substituted. When 3T3 cells were transformed by oncogenic viruses [simian virus 40 (SV40) or polyoma virus or sequential infection with both viruses], their susceptibility to changes caused by l-fucose was greatly reduced. Thus, virus-transformed cells grown in the presence of l-fucose showed much less of a change in the morphology of individual cells, their pattern of association, their rate of growth, and incorporation of protein and RNA precursors as compared to ordinary 3T3 cells. In the different cell lines tested, there was a general parallelism between degree of susceptibility to alteration by l-fucose and degree of contact inhibition. The mechanism by which l-fucose alters ordinary 3T3 cells, and the reasons for the difference in virus-transformed lines are unknown. However, the results raise the possibilities that l-fucose alters ordinary 3T3 cells by combining with complementary sites of these cells and that interactions between natural cellular sugar constituents and complementary sites play a role in mediating cell contact inhibition.

Macromolecular Synthesis and Radiosensitivity in Escherichia coli

Abstract: A number of strains of E. coli develop radioresistance in the stationary phase when glucose is present in a natural growth medium. Cells grown in such a medium have a large excess of macromolecules per cell, compared with cells grown to the same phase of the growth cycle in the same medium without this carbon source. The amounts of the various classes of ribosomes found in extracts of resistant and sensitive cells are different; the excess RNA in resistant cells is accounted for largely by the amount of 30S and 50S ribosomal particles. A correlation has been made between radioresistance and ability to initiate rapid new protein synthesis. In several strains this capacity to synthesize protein correlates with the types of ribosomes present in the cell. We have not been able to quantitate in any precise manner many of the apparent correlations demonstrated here. Such data can result only from better understanding of the multicomponent systems involved in these complex syntheses.

Stimulation of the growth of microorganisms

Abstract: PROCESS FOR STIMULATING THE GROWTH OF MICROORGANISMS BY EMPLOYING AN EFFECTIVE AMOUNT OF DIMETHYL SULFOXIDE IN THE GROWTH MEDIUM.

Lipid metabolism in cultured cells VII. Linoleic acid content of cells grown on lipid-free synthetic medium.

Abstract: SummaryThe fatty acid composition of L-strain mouse fibroblasts growing both in serum and in lipid-free synthetic medium was measured. Linoleic acid comprised about 17% of the total fatty acids in cells grown on serum-supplemented medium and about 6% in cells grown for prolonged periods in lipid-free chemically defined medium. In contrast to other fatty acids however linoleic acid was not synthesized from C14-acetate added to the growth medium. The most probable explanation of these anomalous findings is that cells under deficiency conditions can conserve traces of linoleic acid which are present below detectable levels in the culture environment. No synthesis of positional iso-mers of linoleic acid similar to that which may occur in linoleic acid deficiency in vivo was observed.

Changes in Sensitivity to Acriflavine of Escherichia coli Grown in Media of Different Glucose Contents

Abstract: SUMMARY: Organisms of Escherichia coli K-12 strains sensitive and resistant to acriflavine were plated on media with and without acriflavine after growth in media containing different concentrations of glucose. Proportionally more organisms produced colonies, in the presence of acriflavine, after growth in media containing a high concentration of glucose than in media with lower glucose contents. The final pH value of the growth medium was low with the high glucose media. With the resistant strain, the number of bacteria which survived acriflavine increased as the final pH value of the medium from which bacteria were harvested was decreased, but the initial glucose concentration rather than the final pH value of the culture was more influential in increasing survival with the sensitive strain. The acriflavine-binding capacity of the bacteria was affected by the initial glucose concentration of culture medium probably indirectly through a change of pH. Acriflavine sensitivity of the bacteria varied with the amount of acriflavine bound. The acriflavine-binding capacity of bacteria modified by the pH value of culture medium was stabilized in the course of several doublings of bacteria in that medium. The glucose concentration of the medium affected the acriflavine sensitivity of the sensitive strain through some mechanism other than the change of pH.

Chemically Characterized Media for Study of Foot-and-Mouth Disease Virus in Baby Hamster Kidney Cells

Abstract: Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.

Homoaconitic Acid Accumulation by a Lysine-Requiring Yeast Mutant

Abstract: Homoaconitic acid, the second intermediate of the proposed pathway for lysine biosynthesis in yeast, is accumulated in the growth medium of a lysine-requiring mutant. This acid has been identified on paper and column chromatography by comparing it with authentic cis-homoaconitic acid. The infrared spectrum of the isolated material was identical with that of synthetic cis-homoaconitic acid. In addition, the chemical structure of the enzymatic product has been verified by degradation to glyoxylic and α-ketoglutaric acids after treatment with KMnO4 and HIO4 and by catalytic reduction to the saturated acid 1,2,4-butanetricarboxylic acid. The isolated homoaconitic acid was also identified as a substrate for a purified enzyme preparation of homoaconitase.