Showing papers on "Growth medium published in 1970"

Steady-State Measurement of the Turnover of Amino Acid in the Cellular Proteins of Growing Escherichia coli: Existence of Two Kinetically Distinct Reactions

Abstract: Turnover of cellular protein has been estimated in Escherichia coli during continuous exponential growth and in the absence of extensive experimental manipulation. Estimation is based upon the cumulative release into carrier pools of free leucine-1-(14)C over a number of time intervals after its pulsed incorporation into protein. Breakdown rates obtained with other labeled amino acids are similar to those obtained with leucine. Two kinetically separate processes have been shown. First, a very rapid turnover of 5% of the amino acid label occurs within 45 sec after its incorporation, most likely indicating maturative cleavages within the proteins after their assembly. A slower heterogeneous rate of true protein turnover follows, falling by 39% in the remaining proteins for each doubling of turnover time. At 36 C, the total breakdown rate of cellular protein is 2.5 and 3.0% per hr over a threefold range of growth rate in glucose and acetate medium, respectively. This relatively constant breakdown rate is maintained during slower growth by more extensive protein replacement, one fifth of the protein synthesized at any time in the acetate medium being replaced after 4.6 doubling times. Intracellular proteolysis thus appears to be a normal and integral reaction of the growing cell. The total rate equals minimal estimates obtained by others for arrested or decelerated growth but is kinetically more heterogeneous. Quantitatively proteolysis is not directly affected by growth arrestment per se as caused by alpha-methylhistidine, chloramphenicol, or uncouplers of oxidative phosphorylation, but qualitatively it can gradually become more homogeneous kinetically as a secondary event of starvation. Under more extreme conditions as with extensive washing, prolonged phosphorylative uncoupling, or acidification of the growth medium, the proteolytic rate can increase severalfold.

Evidence for the control of respiration by DNA in ultraviolet-irradiated Escherichia coli B-r cells.

Abstract: UV irradiation of Escherichia coli B/r cells causes a temporary inhibition of respiration. This inhibition begins about 60 min after irradiation. The duration of the inhibition depends, in part, upon the carbon source on which the cells were grown and its presence or absence at the time of irradiation. In cells grown on a mineral salts and glycerol medium, irradiation with 520 erg/mm 2 at 254 nm almost completely inhibits respiration for 4 h; the addition of exogenous amino acids to the growth medium favors early recovery of respiration. Experiments with caffeine show that excision of pyrimidine dimers is not necessary for the delayed inhibition of respiration, but is necessary for the subsequent recovery of respiration. If chloramphenicol, 5-fluorouracil, or an amber mutant of phage T4 is added to cells immediately after UV, the turnoff of respiration is prevented, but none of these agents reverses the inhibition once it has set in. These results indicate that UV-damaged DNA is transcribed and a protein important for the turnoff of respiration is formed.

A study of the esterases and their function in Candida lipolytica, Aspergillus niger and a yeast-like fungus.

Abstract: SUMMARY: Esterases, determined by polyacrylamide gel electrophoresis, were present when Candida lipolytica was grown in a liquid, shaken, glucose-mineral salts medium. Intracellular esterase activity increased during growth, but extracellular esterase activity was small and increased only marginally. Esterases were not detected in organisms grown on solid glucose-mineral salts medium, but were present when glycerol tributyrin replaced glucose. At the onset of asexual sporulation in a yeast-like fungus, three new esterases occurred. Intracellular esterase activity increased, intracellular lipid utilization occurred, and the respiratory quotient decreased. No extracellular esterase activity was detected. Esterases were only detected in Aspergillus niger at late stages of conidiation when intracellular lipid decreased. Esterase activity was not detected in the mitochondria or in the cell-free growth medium. Esterases of all organisms tested hydrolysed glycerol tributyrin and were arbitrarily classified as lipases; intracellular lipid decreased with increase in esterase activity function. Esterase and profile changes may reflect a role of lipids in sporulation and physiological ageing.

Induction of Enterococcal L-Forms by the Action of Lysozyme

Abstract: Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.

The role of L-glutamine in the phenotypic change of a rod mutant derived from Bacillus subtilis 168.

Abstract: SUMMARY The morphological mutant rod-4 derived from Bacillus subtilis 168 trp can be changed from a round form to a rod by the addition to the growth medium of sufficient acid-hydrolysate of casein. The hydrolysate can be replaced by a mixture of amino acids, and the only individual amino acids giving similar results are L-glutamic acid, L-proline, L-arginine and L-ornithine. Since the lag in the action of L-glutamate was less than for the other amino acids, this amino acid is likely to be responsible for the effect of the mixture. Experiments with the L-glutamine analogue, γ-L-glutamylhydrazide, strongly suggest that L-glutamine is the active metabolite rather than the amino acid itself. The correcting effect of high ionic strengths of the growth medium on the morphology of this mutant seems to be mostly due to the increased effectiveness of L-glutamate or L-glutamine in the presence of high concentrations of salts.

Control of the production of two protein hormones by rat pituitary cells in culture.

Abstract: A clonal strain of rat pituitary tumor cells (GH3) has continued to produce the pituitary protein hormones growth hormone (GH) and prolactin during 5 years of continued growth in monolayer culture. Studies of the effects of external stimuli have indicated that, in spite of the physical similarity of these protein hormones (each is a single polypeptide of molecular weight ≈23,000), their production is controlled by different mechanisms. Addition of hydrocortisone (HC) (3×10−6 m) to the growth medium leads, after a lag of 12 to 24 hr, to an increased relative rate (rate in experimental cells divided by rate in control cells) of GH production. The relative rate reaches a maximum of 5 to 8 at 30 to 100 hr. Stimulation by HC of GH production is observed in cells growing in either the stationary or the exponential phase of growth. Indirect estimates indicate that, in exponentially growing cells, GH represents about 2% and 14% of the total protein synthesized by control and fully stimulated cells, respectively. Maintenance of the stimulated state requires HC. HC decreases both the growth rate of GH3 cells and their incorporation of amino acids into acid-insoluble material. At the same time that HC stimulates GH production it decreases the relative rate of prolactin production to about 0.2 to 0.3. On the other hand, addition of acid extracts of bovine hypothalamus, cerebral cortex, kidney, or liver (0.3 to 1.0 mg of protein per ml) to the medium leads to an increase of the relative rate of prolactin production to 6 to 9, while decreasing the relative rate of GH production to about 0.5. Chromatographic fractionation of simple extracts of bovine liver has yielded a macromolecular, heatlabile fraction exhibiting these effects at a concentration as low as 20 μg per ml. GH3 cells which have been adapted to growth in suspension culture produce both GH and prolactin. HC is observed to stimulate GH production and suppress prolactin production by cells growing in this state, without affecting the growth rate of the cells.

Mutants of Salmonella typhimurium Lacking Phosphoenolpyruvate Carboxykinase and α-Ketoglutarate Dehydrogenase Activities

Abstract: Two auxotrophic mutants (SM16 and SM51) of Salmonella typhimurium, which for aerobic growth, with hexoses as carbon source, required lysine and methionine (SM51 required also nicotinic acid), were isolated and characterized. The requirement for the amino acids disappeared in anaerobiosis. Neither lipoate nor 4-hydroxybenzoate was effective in supporting aerobic growth of the mutants. The lysine and methionine requirement for aerobic growth was due to the absence in the mutants of the enzymatic activities of the α-ketoglutarate dehydrogenase complex. The mutants could not use succinate as carbon source even after enrichment of the growth medium with acid-hydrolyzed casein and yeast extract. No phosphoenolpyruvate carboxykinase activity was found in the mutants, a phenomenon which explained their inability to use succinate. By interrupted conjugation and by transduction experiments, the positions of the three affected loci, pck, suc, and Nic, were located at approximately 17 to 19 min of the S. typhimurium chromosome; they were found to be closely linked. From different criteria, it appears as if the genetic lesions present in both mutants are due to deletion of a small chromosome fragment.

Resistance of Clostridium perfringens to Varying Degrees of Acidity during Growth and Sporulation

Abstract: SUMMARY: Viable cells of each of 6 strains of Clostridium perfringens were exposed to 4 levels of acidity during phases of the growth cycle. The selected strains included 4 which had been recovered in association with food poisoning outbreaks and 2 strains not so associated. The growth media tested included Fluid Thioglycollate Medium, DS-sporulation medium and the CP-2V medium proposed by Hauschild. The level of acidity, length of exposure of the cells, the growth medium employed and the phase in the growth curve influenced the survival of C. perfringens. Exposure of cells grown in DS-sporulation medium to buffers pH 6.0 had little effect on the survival over the 8-hr test period, with somewhat greater sensitivity of cells being demonstrated at pH 4.5. Exposure of cells, similarly produced, to buffer pH 1.0 or 2.0 was much more effective in reducing the percentage of survival, particularly during early log phase and at the onset of sporulation. Based on the 3 growth media utilized, calculated survival curves resulting from exposure of cells to pH 1.0 or 2.0 were erratic in shape, and percentage survival was almost universally less than 10%. Source of the strain, whether food poisoning or non-food poisoning associated, appeared to have no significant effect on the acid resistance of the cells. The comparatively regular increase in the percentage of survivors after the initiation of sporulation suggests that spores exhibit a greater resistance to acid stress than vegetative cells. Incubation at temperatures of 37° or O°C, during the time of treatment with the test buffers pH 1.0 or 2.0, produced no consistent change in the percentage of survivors when the cells were grown in FTG.

Nitrogen requirements of the genus Linderina

Abstract: The influence of different nitrogen sources on the growth of Linderina was examined in liquid culture. Both species of Linderina were unable to assimilate nitrate nitrogen and nitrite seemed to be toxic. Ammonium nitrogen was used but the growth response was considerably lower than that with some organic nitrogen materials. Inclusion in the growth medium of succinic acid as a carbon source failed to improve the assimilation of ammonium.Amino nitrogen as aspartic acid and asparagine gave good growth though not as good as with L- or DL-glutamic acid. The response to DL-glutamic acid was markedly better than to the L-isomer whereas the D-isomer gave relatively poor growth.

Enhanced CO2 production by yeast exposed to elevated temperatures.

Abstract: SUMMARY: After starvation, yeast exposed to elevated temperatures produced CO2 twice as fast as unexposed organisms. The lag which preceded linear CO2 production by starved yeast was essentially eliminated by heat treatment. Uptake and retention of sorbose was greater in heated yeast. Heating was accomplished by brief immersion of the organisms in heated solutions and by growth for 2 h. at 35°. Short heat treatments increased the production of CO2 when glucose was included in the suspending medium, whereas heating in water or in growth medium without glucose resulted in a decreased production of CO2.

Regulation of Ribonucleic Acid Synthesis by Histidine and Methionine During Recovery of Escherichia coli from Magnesium Starvation

Abstract: During magnesium starvation of Escherichia coli B, most of the ribosomes break down to low-molecular-weight components. When magnesium is restored to the medium, the cells recover. The rate of recovery can be increased greatly by supplementing the growth medium with a mixture of 21 amino acids. This increased rate of recovery is shown to be due to the effect of only two amino acids, histidine and methionine, which initially stimulate accumulation of cellular ribonucleic acid without increasing the rate of protein synthesis. In contrast, histidine and methionine supplementation to logarithmically growing E. coli B is not as effective in stimulating growth as is the complete amino acid mixture. Since cells recovering from magnesium starvation preferentially synthesize ribosomes, it is possible that histidine and methionine play a special role(s) in ribosomal ribonucleic acid synthesis or stability.

Growth Response to Cyanide by Plants Grown in the Presence or Absence of Iron

Abstract: Seedlings of Phaseolux vulgaris were grown in media with and without iron. Growth was measured in terms of leaf length and fresh weight increase. Cyanide at & concentration of 0.05 mM was found to inhibit severely plants growing in complete media but failed to inhibit growth to (he same extent in those plants grown in media lacking iron. It was established that this result was obtained whether the plants were grown in an unchanged medium or if the growth medium was replaced at intervals. A number of suggestions were made in an attempt to account for this anomalous behavior.

Convenient microscopic tests for yeast viability and sporogenic capability

Abstract: Drops of cell suspension are seeded on nutrient medium containing 5% purified agar and spread between the agar surface and a cover glass. The liquid phase is absorbed by the concentrated agar, so that the cells are held itz situ between glass and agar. After incubation nongerminated cells and microcolonies are counted microscopically. Sporogenic capability is assessed by seeding very dense cell suspensions on acetate agar and covering with Teflon F.E.P. membrane which is permeable to oxygen. Plating, slide culture, and staining techniques are commonly used for estimating the proportion of viable cells in yeast populations, though often with conflicting results (2, 3, 6, 7). A viability test has been developed which combines some of the desirable features of the plating and slide culture techniques. Agar SP (Fisher Scientific Co.) is dissolved in distilled water in 5% concentration by heating in a double boiler. Then glucose (27,), malt extract (0.3y0), peptone (0.5y0), yeast extract (0.3y0), and KH2P04 (0.17,) are added. After it is autoclaved for 5-10 min at 120°C the medium is poured in 15- to 20-ml quantities into petri dishes. The 57, agar should not be allowed to set before pouring as it is difficult to melt. The yeast sample is prepared as a suspension containing 10-20 X 106 cells per milliliter in liquid growth medium or in water. One or two loops, 2 mm, of the suspension are placed on the agar and immediately covered with a sterilized 16-mm No. 1 cover glass which is pressed down against the agar to spread the suspension over the area beneath the cover glass. Within an hour the suspending liquid is absorbed by the concentrated agar so that individual cells are held in situ between the glass and agar surfaces. After incubation the proportion of cells that have produced microcolonies can be determined by microscopic examination. The optimum incubation time varies from strain to strain, but in most instances 12 h at 27 "C or 18 h at 20 OC