Early Logarithmic Growth Phase Research Articles

Depletion of cellular glycogen during the early logarithmic growth phase of human fibroblasts

Human diploid fibroblasts deplete 50% of their cellular glycogen by day 4 after subcultivation in 100 mg% glucose medium. The glycogen content increases again as the cells approach confluency. Growth of cells in low glucose medium results in rapid glycogen depletion and indicates that stored glycogen has a limited potential as an energy source.

Changes in Malate Content and in Enzymes Involved in Dark CO2 Fixation during Growth of Acer pseudoplatanus Cells in Suspension Culture

AbstractIt has been reported in some cases that an increase in pCO2 stimulates growth in diluted cell suspension cultures. Experiments have been designed to study the pattern of dark CO2 fixation in sycamore cells grown in liquid suspension and to correlate this pattern with the culture growth phases. Comparisons were made between enzymatic activities, CO2 incorporation, malic acid content during lag, logarithmic and stationary phases of growth. Malic enzyme (NADP‐dependent) was at its maximum activity during early logarithmic growth phase, when biosynthetic capacities were at the highest. Phosphoenolpyruvate‐carboxylase activity was strongly correlated with the ability of cells to fix CO2. Malic acid content decreased soon after transfer of the cells to a new medium and increased at the onset of stationary phase. Under optimal conditions, the CO2 incorporation pattern did not change during growth, with an almost identical incorporation in the basic (amino acids) and acidic (organic acids) fractions. These observations have been discussed in relation to a possible effect of increased pCO2 in the cell environment.

Heat-stable enterotoxin from Escherichia coli: factors involved in growth and toxin production.

Six enterotoxigenic strains of Escherichia coli produced variable levels of heat-stable enterotoxin (ST) when grown under pH control at 8.5 in a simple synthetic medium containing neither amino acids nor vitamins. Bacterial growth and ST production were at levels as high as or higher than those observed in complex media. ST elaboration was detectable in the early logarithmic phase of growth and appeared to be related to disappearance of glucose in the growth medium. The results of this study did not suggest pH-dependent release of ST. Imposition of pH control in complex media resulted in increased growth rates, earlier detectable ST synthesis, and elevated levels of ST. In synthetic medium, attainment of the stationary growth phase was followed by a significant decrease in culture density and a concomitant increase in ST. Cellular autolysis experiments revealed that as much as 20% of the total ST activity was present in a cell-associated form.

Drug-induced differentiation of a rat glioma in vitro: II. The expression of S-100, a glial specific protein and steroid sulfatase

Amethopterin and 5-bromodeoxyuridine (BUdR) were used to induce morphological changes in cloned rat glioma (C6). The expression of S-100 protein, an acidic protein localized in glial cells, and steroid sulfatase, an ubiquitously distributed enzyme found in high concentration in glial cells, were followed during cell growth, from subculture to well into the stationary phase of control and drug-treated cultures. Amethopterin and BUdR differed in their effects on glioma morphology and in the expression of the biochemical parameters. Amethopterin coordinately stimulated both the production of S-100 protein and steroid sulfatase activity when cell division was inhibited during early logarithmic growth phase. BUdR stimulated steroid sulfatase activity but repressed production of S-100 protein. The results are discussed with respect to the mechanism of regulation of the differentiated state of tumor cells.

Interaction between tetanolysin and mycoplasma cell membrane

1. 1. A partially purified tetanolysin preparation lysed the sterol-requiring Mycoplasma capricolum cells but had no effect on M. capricolum cells adapted to grow with no or very little cholesterol. The sterol-non-requiring Acholeplasma laidlawii cells grown either in a cholesterol-rich or a cholesterol-poor medium were unaffected by the tetanolysin preparation. 2. 2. The lysis of M. capricolum cells by the tetanolysin preparation was temperature dependent, inhibited by cholesterol, sublytic concentrations of lucensomycin, and Mg 2+. The sensitivity to lysis was greatly affected by the age of the culture, being highest in cells from the early logarithmic phase of growth and declining sharply thereafter. 3. 3. Isolated M. capricolum membranes were capable of binding large amounts of the tetanolysin activity (up to 30 hemolytic units per μg membrane protein), 20 times as much as membranes of the adapted strain. The binding of tetanolysin activity to membranes was almost the same at 4, 22, or 37°C, and was very little affected by the age of the culture. The binding capacity of the membranes was not affected by the removal of 60–70% of membrane proteins by pronase digestion but markedly decreased with the removal of membrane lipids. 4. 4. Of the five polypeptide bands detected in electrophorograms of the partially purified tetanolysin preparation, two bands (mol. wt. 44 000 and 42 000) were found to bind to the cholesterol-containing mycoplasma membrane preparation. EPR spectrometry revealed that the freedom of motion of fatty acid spin labels in the tetanolysin-treated membranes was markedly higher than that in untreated membranes. 5. 5. The concept that tetanolysin interacts specifically with membrane cholesterol resulting in the shielding of cholesterol from its interaction with membrane phospholipids is discussed.

On the regulation of adenosine 3′,5′-monophosphate synthesis in bacteria I. Effect of carbon source variation on cyclic AMP synthesis in escherichia coli B/r

1. 1. The effect of carbon source variation in bacterial growth media on their growth rate, inducible enzymes and cyclic AMP synthesis was examined: an inverse relationship between the culture's growth rate and its differential rate of inducible enzyme (tryptophanase and β-galactosidase), and cyclic AMP synthesis was found. 2. 2. The effect of the culture's growth phase on its sensitivity or resistance to glucose catabolite repression was determined in the wild type and a catabolite insensitive mutant (ABDROI): the wild type's sensitivity to glucose repression was not affected, whereas the insensitivity of the mutant was found to be limited to its early logarithmic phase of growth. At late log, or stationary phase, the mutant was found to be sensitive to glucose repression. 3. 3. Examination of the kinetics of glucose uptake by the mutant, using α-[ 14C] methyl-glucoside showed evidence for two transport systems each with a different affinity to glucose. A low affinity transport system (apparent K m of 3.4 · 10 −5 M) which appears mostly at the early logarithmic phase of growth. A high affinity transport system (apparent K m of 1.2 · 10 −5 M) which appears mostly at the late log and stationary phases of growth. 4. 4. The effect of the culture density variation on its sensitivity to glucose repression showed that sensitivity to glucose catabolic repression is primarily a reflection of the formation of an allosteric effector molecule between glucose and its specific transport molecule which in turn regulates the activity of the adenylate cyclase.

Morphological Characterization of Mycobacteriophage R1

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

Ecological Application of Antibiotics as Respiratory Inhibitors of Bacterial Populations1

Two terregenous and four marine bacterial isolates were treated with six antibiotics and antibiotic combinations. Comparisons made between responses of cells in early and late logarithmic and stationary growth phases indicated variable sensitivity to the agents. Bacteria in stationary and late log-phase cultures exhibited the greatest resistance, whereas the early log-phase cells exhibited greatest antibiotic susceptibility. We conclude that the tested antibiotics cannot be used for ecological purposes to delineate bacterial respiration in mixed microbial communities.

Role of zinc in the production of Clostridium perfringens alpha toxin.

Clostridium perfringens was found to produce alpha toxin in a synthetic medium containing zinc; in medium containing no zinc, a little toxin was detected in the early logarithmic phase of growth and it disappeared rapidly. No intracellular accumulation of alpha toxin protein occurred whether or not zinc was present in the medium. In zinc-deficient medium, the organisms produced and released into the surrounding medium the protein specifically precipitable with alpha antitoxin in an amount comparable to that of alpha toxin produced in the zinc-containing medium. The protein combined rapidly in some unknown way with zinc to form the active and stable alpha toxin.

Biotin deficiency in Arthrobactger globiformis: comparative cell ultrastructure and nonreplacement of the vitamin by structurally unrelated compounds.

The development of aberrant cell forms of Arthrobacter globiformis 425 due to biotin deficiency was followed by electron microscopy of ultrathin sections. Upon comparison with normal cell growth, aberrant cells developed in the early logarithmic growth phase. Several membrane-bound bodies were embedded in a thick matrix, showing that cell division was impaired. Mesosomes and cytoplasmic membranes were still present in the abnormal cell although the normal cell wall was absent. This condition persisted throughout the growth cycle. This pattern of morphological development was correlated with changes in macromolecular composition of the cells. Various structurally unrelated compounds were tested for their ability to replace biotin. These included aspartic acid, oxalacetic acid, coenzyme A, oleic acid, linoleic acid, linolenic acid, and Tween 80. Only Tween 80 was able to spare biotin to a limited degree. However, this sparing action was eliminated in the presence of avidin.

Characterization and Synthesis of Anacystis Ribonucleic Acids

Photoautotroph cultures of the blue-green alga Anacystis nidulans show a pronounced maximum of 10 −7 µ g total RNA/cell in the early logarithmic growth phase and a subsequent 50% decrease during the stationary phase. The alteration of the total RNA content influences all RNA fractions in the same way. Two high-molecular weight ribosomal RNAs of 1.1 and 0.56 × 10 6 daltons, respectively, are obtained if Mg 2+ (5–10 mM) is present during the RNA extraction. Absence of Mg 2+ during the RNA extraction at 0° or heating the isolated total RNA for 1 min at 60°C in the presence of Mg 2+ results in an irreversible conversion of the 1.1 × 10 6 rRNA to 0.9 plus 0.2 × 10 6 rRNA. The 0.56 × 10 6 rRNA is found to be a very stable molecule After less than 1 min pulse-labelling of A. nidulans cells with Na 2 H 32 PO 4 and separation of the RNA fractions by MAK chromatography the radioactivity is eluted with the first part of the DNA fraction and in front of the small ribosomal RNA. Extension of the pulse-labelling to a few minutes yields radioactivity in all MAK fractions. In pulse-chase experiments the small rRNA is shown to have the most rapid turnover compared to the other RNA fractions. DNA-RNA hybridization studies demonstrate that all 3 rRNA fractions contain rapidly-labelled RNA of the non-ribosomal type, however, as competition experiments indicate, in different proportions to the amount of ribosomal precursor RNAs. Thus, the 0.9 × 10 6 fraction does not contain rapidly labelled ribosomal RNA.

Correlation of M protein production with those factors found to influence growth and substrate utilization of Streptococcus pyogenes.

In the absence of proteinase formation, factors reported to influence the growth or fermentation by streptococci have been evaluated to determine their quantitative effect upon the production of M protein during the growth of Streptococcus pyogenes. Buffers, amino acids, peptides, gross organic additions, and carbohydrate substrates were tested under a variety of cultural conditions. The M protein content was remarkably constant throughout the late logarithmic period of growth, i.e., when the cell population doubled, the M protein doubled. However, several factors affected the M protein content per milligram of cells (dry weight). When types 1, 12, and 22 were grown aerobically in a semidefined medium, the M protein content of the cell population essentially doubled; in Todd-Hewitt broth, this aerobic effect on M protein synthesis was not observed. When cells grown on Todd-Hewitt broth were transferred to medium containing 0.1% starch and no added glucose, the M protein content per milligram of cells (dry weight) increased as much as fourfold. When growth was initiated in glucose, the rate of M protein formation was at a maximum in the early logarithmic phase of growth and was comparatively greater than the rate of cellular multiplication. When the amount of substrate fermented was greater than 0.2%, increased M protein was not observed. An evaluation of the effects of medium or conditions of growth showed the units of M per milligram of cells (dry weight) were not influenced by a shift in the stoichiometry of either the anaerobic or aerobic fermentation, substrate used, or adenosine triphosphate utilized for growth. These results show that M protein synthesis is subject to limited glucose repression or substrate catabolite repression.

ハマチ類結節症の原因細菌の分類学的研究

Morphological and biochemical characteristics were examined on four strains of the bacteria causative of “pseudotuberculosis” among yellowtail (Seriola quinqueradiata), and on a strain of Pasteurella piscicida, Janssen et Surgalla. The observation that the pseudotuberculosis organisms were identical with P. piscicida was confirmed. These organisms were all Gram-intermediate, non-motile rods. A peculiar morphological feature of the organisms is the transformation from rod-shaped cells in the early logarithmic growth phase to small coccoids or cocci in the stationary phase. The organisms gave positive Kovacs' oxidase reaction; fermented glucose in Hugh-Leifson's medium, and produced acid but no gas from a few other carbohydrates such as fructose, galactose, and mannose. They were sentistive to most of the antibiotics and antimicrobial drugs, including penicillin and polymyxin B; were halophilic; and did not grow at pH lower than 6.1. Although the cells of these organisms did not show any typical coryneform appearance, nevertheless, the above characters suggest that they have a close relationship with coryneform bacteria and genus Arthrobacter seemed to be the most suitable genus to which they belong.

Application of Enzyme Production Properties in Subtyping of Group A Streptococci According to T Type

The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADG) and the cell-bound lipoproteinase (serum opacity reaction, SOR) by strains of different serological types of group A streptococci, in relation to the T typing, was studied. The production of both NADG and SOR, or only one of them, was found to be characteristic of serotypes, as determined by M and T antigen. No difference in the production of these enzymes was found in relation to M-positive and M-negative variants. Investigation into NADG and SOR production as related to the T type enabled the division of a single agglutination pattern into four main groups, each of which corresponds to one specific M type or more. Of the 370 strains belonging to 12 different T-agglutination patterns, 21% produced both enzymes and 42.5% failed to produce any of them, whereas the remaining 36.5% produced only one out of the two enzymes. Five streptococcal types which did not produce NADG and SOR also failed to synthesize streptolysin S at the early logarithmic phase of growth, indicating that streptolysin S production by young cultures may be also related to serotype. No correlation was found between the production of NADG-SOR as related to serotype and the production of streptolysin O, acid phosphotase, esterase, N -acetylglucosaminidase, hyaluronidase, streptokinase, and the cell-sensitizing factor. The practical and potential usefulness of NADG and SOR production in epidemiological studies is discussed.

Application of Enzyme Production Properties in Subtyping of Group A Streptococci According to T Type

The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADG) and the cell-bound lipoproteinase (serum opacity reaction, SOR) by strains of different serological types of group A streptococci, in relation to the T typing, was studied. The production of both NADG and SOR, or only one of them, was found to be characteristic of serotypes, as determined by M and T antigen. No difference in the production of these enzymes was found in relation to M-positive and M-negative variants. Investigation into NADG and SOR production as related to the T type enabled the division of a single agglutination pattern into four main groups, each of which corresponds to one specific M type or more. Of the 370 strains belonging to 12 different T-agglutination patterns, 21% produced both enzymes and 42.5% failed to produce any of them, whereas the remaining 36.5% produced only one out of the two enzymes. Five streptococcal types which did not produce NADG and SOR also failed to synthesize streptolysin S at the early logarithmic phase of growth, indicating that streptolysin S production by young cultures may be also related to serotype. No correlation was found between the production of NADG-SOR as related to serotype and the production of streptolysin O, acid phosphotase, esterase, N-acetylglucosaminidase, hyaluronidase, streptokinase, and the cell-sensitizing factor. The practical and potential usefulness of NADG and SOR production in epidemiological studies is discussed.

Electron transport and oxidative phosphorylation in the blue-green alga Anabaena variabilis.

It is shown that secretion of bacteriolytic complex enzymes in a bacterium of the family Pseudomonadaceae begins in the latent and early logarithmic culture growth phases. A maximum of specific bacteriolytic activity falls in the same time interval with a maximum of specific activity of lytic proteinase L2 and muramidase. The activity of concomitant enzymes--non-specific proteinase and neutral phosphatase--peaks in the middle of the logarithmic growth phase. Secretion of exopolysaccharide into the external medium begins in the second half of the logarithmic growth phase and, thus, is not coordinated with secretion of bacteriolytic enzymes. It is shown that relative content of enzymes and polysaccharides depends upon the composition of growth medium for the producer.

Release of protoplasts in the yeast phase of Histoplasma capsulatum without added enzyme.

Cells of the yeast phase of the dimorphic systemic fungus pathogen Histoplasma capsulatum readily released large numbers of intact protoplasts without degradation of their cell walls by snail or microbial enzymes, previously regarded as a requirement for all yeast and mycelial fungal forms. Over 90 percent of "B" type yeast cells in the early logarithmic phase of growth released living protoplasts when incubated at 37 degrees C with 2 molar magnesium sulfate, whereas "A" type yeast cells required prior exposure for 24 hours to 2-deoxy-D-glucose before incubation in the 2 molar magnesium sulfate.

Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope.

Razin, Shmuel (University of Connecticut, Storrs), and Benjamin J. Cosenza. Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope. J. Bacteriol. 91:858-869. 1966-Growth of 11 Mycoplasma strains in liquid media was followed by phase-contrast microscopy. A similar pattern of development was common to all strains. Branching filaments, 0.3 to 0.4 mu thick, characterized the early logarithmic phase of growth. The length of the filaments varied according to the strain tested and the growth medium. Addition of oleic acid to the medium induced the formation of very long filaments by M. laidlawii strain B. Upon aging, the filaments were found to break up into chains of coccoid elements. These chains further fragmented to yield shorter chains and single coccoid elements, which characterized the stationary and decline phases of growth. The size of the coccoid elements increased from 0.3 to 0.4 mu, when formed in the filaments, to 0.6 to 0.8 mu after being released from the chains. Further increase in the size of the cells took place at the decline phase of growth, leading to the formation of very large cells reaching a diameter of 10 to 20 mu. However, these large cells had the appearance of empty vesicles and were apparently nonviable as indicated by viable-count experiments.

DIFFERENTIATION BETWEEN RADIATION SENSITIVE AND -RESISTANT MUTANTS OF ESCHERICHIA COLI, STRAIN B.

Morphological heterogeneity in cell sizes of Escherichia coli, strains B, B/r, and BSprevents estimation of the viable cell count by the indirect turbidimetric method. The average dry weights of E. coli B, B/r, and BS, when grown in acetate synthetic medium, are 0.16, 0.10, and 0.13 picograms per cell respectively. Increased radiation sensitivity was correlated with those bacterial populations showing decreased intracellular glycogen content, decreased lipopolysaccharide–lipoprotein content, R-colony forms, and cellular elongation. The radiation-sensitive strains contained more DNA, RNA, and protein per cell than the radiation-resistant mutant; however, no significant differences were evident in the DNA, RNA, and protein contents on a dry weight basis. No qualitative differences in the monosaccharides or their derivatives were found. Intracellular glucose synthesis and accumulation preceded DNA, RNA, and protein synthesis throughout the lag and early logarithmic growth phases for all three strains. Multiple forms of intracellular glycogen complexes were present in all three strains. In addition to unbound glycogen and protein-bound glycogen, a bound form of glycogen was consistently associated with crude fractions which contained the DNA, RNA, and lipopolysaccharide constituents. Increased lability of the cold TCA-insoluble bound glycogen to hot TCA and alpha-amylase hydrolysis was correlated with increased radiosensitivities of the organisms.

NUCLEASES OF MYCOPLASMA.

SUMMARY: Nuclease activity was observed in several saprophytic and parasitic Mycoplasma organisms; the nucleases of Mycoplasma laidlawii were studied in detail. Nuclease activity of this organism was highest at the early logarithmic phase of growth, and was found mainly the soluble fraction of the organisms. Anion-exchange chromatography of the proteins of the soluble fraction separated ribonuclease from deoxyribonuclease activity. Each enzyme had an alkaline pH optimum, required magnesium ions for activity, and degraded native and heat-denatured nucleic acids. M. laidlawii RNase degraded RNA-core. RNA inhibited the degradation of DNA by M. laidlawii deoxyribonuclease. The implications of these findings with respect to the effects of RNA and DNA on growth of M. laidlawii are discussed.