Polysome Turnover During Amino Acid Starvation in Escherichia coli

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains.     

Adenylate Energy Charge in Escherichia coli During Growth and Starvation

The value of the adenylate energy charge, [(adenosine triphosphate) + (1/2) (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.     

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

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.     

Loss of Deoxyribonucleic Acid-Thymine During Thymine Starvation of Escherichia coli

A substantial loss of deoxyribonucleic acid-thymine occurs during thymine starvation of several thymine auxotrophs derived from Escherichia coli strains B, 15, and K-12.     

A Plasma Membrane Mutation acrA in Escherichia coli

Protein analysis and electron microscopic observation of thefreeze-fractured plane of the plasma membrane were performedwith an acriflavine-sensitive mutant carrying mutation acrA(at min 10) and with the wild type (acrA⁺) strain of Escherichiacoli K-12. The acrA mutant membrane was deficient (or much lower)in one protein when analyzed by the polyacrylamide gel electrophoresistechnique. (Received May 7, 1981; Accepted July 28, 1981)     

Metabolic Events Occurring During Recovery from Prolonged Glucose Starvation in Escherichia coli

The effects of starving Escherichia coli for glucose and required amino acids were determined. The disappearance of the majority of the ribosomal population, particularly of the ribonucleic acid (RNA) moiety, was noted. The events during recovery are detailed, with emphasis placed on the timing, requirements, and control of deoxyribonucleic acid, RNA, and protein synthesis and on ribosome reassembly. Finally, the applicability of the system to the study of temperature-sensitive mutants is documented and discussed.     

Magnesium Starvation of Aerobacter aerogenes IV. Cytochemical Changes

A number of cytochemical changes were revealed by microscopic observations of Aerobacter aerogenes populations starving for Mg(++). During the first few hours, while the synthesis of deoxyribonucleic acid (DNA) was paralleled by an increase in viable bacteria, the cells became progressively smaller. Subsequently, the number of viable cells in the culture remained constant in spite of continuing DNA synthesis, and the cells progressively elongated into filamentous forms. During this time, a second population of very small bacteria could be identified. These cells, whose number increased progressively, were inert with respect to (i) growth or reproduction when returned to a complete medium and (ii) biosynthetic activity as judged by autoradiographic estimation of uracil-H(3) incorporation into nucleic acids. When observed by electron microscopy, many thin sections from bacteria that had been starved of Mg(++) for 20 hr appeared to be almost devoid of ribosomal particles. Thionine staining indicated that the inert cells contain DNA. Furthermore, the rate of DNA synthesis in the culture corresponded to the rate of accumulation of inert cells, suggesting that their presence can account for the difference between total DNA and viable count.     

Effects of Acriflavine on the Formation of the Plasma Membrane of Escherichia coli

When cells of acriflavine-sensitive (acrA) and acriflavine-resistant(acrA⁺) Escherichia coli K-12 strains were treated with a ratherhigh concentration (100 µg ml^-1) of acriflavine in mediumthat had been adjusted to pH 8.1, distinct whirlpool-like structuresderived from the plasma membrane appeared not only in the acrAcells but also in the acrA⁺ cells. Chemical analysis was performedto determine the lipid composition of the cells by thin-layerchromatography on silica gel and gas-liquid chromatography.The amount of total fatty acids was significantly higher inthe acrA cells than in the acrA⁺ cells, when cells were culturedin the presence of acriflavine. This difference seems to becaused by the greater accumulation of unsaturated fatty acids(palmitoleic and cis-vaccenic acid) in the acrA mutant cellsthan in the acrA⁺ cells and by the acceleration of this accumulationas a result of the presence of the dye. A comparison of phospholipidcontents between the acrA and acrA⁺ cells cultured under acriflavine-freeconditions showed that the former cells contained more phosphatidylethanolamine(PE) and, in particular, more cardiolipin (CL) than the lattercells. However, the situation was reversed in the case of phosphatidylglycerol(PG). Addition of acriflavine to the medium led to a markedincrease in levels of PE and CL in both acrA and acrA⁺ cellsbut an increase in levels of PG was found only in the acrA⁺cells. (Received October 13, 1992; Accepted May 31, 1993)     

Control of Nucleotide Metabolism and Ribosomal Ribonucleic Acid Synthesis During Nitrogen Starvation of Escherichia coli

Ribosomal ribonucleic acid (RNA) synthesis and ribonucleoside triphosphate metabolism were studied in cultures of Escherichia coli subjected to starvation for inorganic nitrogen. In a strain that was under stringent control, a 50-fold reduction in the formation of both 16S and 23S RNA was accompanied by a severe restriction on nucleotide biosynthesis. These inhibitions were relieved in part by incubating the starved cells with amino acids. This result suggests that regulation by the functional RNA control (RC) gene is involved in the effect. This suggestion was confirmed by showing that the effector of the stringent response, guanosine-5'-diphosphate-2'- or 3'-diphosphate ((pp)G(pp)), accumulated at the onset of starvation and disappeared immediately when the amino acids were added. Ribosomal RNA synthesis was severely restricted and the same nucleotide, (pp)G(pp), accumulated at the onset of nitrogen starvation of a relaxed mutant too. These findings suggest that a control mechanism other than the one provided by the functional rel gene might operate to regulate RNA synthesis and that this mechanism is expressed through the synthesis of (pp)G(pp).     

Changes in the Ultraviolet Sensitivity of Escherichia coli During Growth in Batch Cultures

The ultraviolet (UV) sensitivity of Escherichia coli B/r harvested at various times during growth in batch cultures was measured. The results showed a period of increased UV sensitivity in late log phase, just before the cultures entered stationary phase. This increase in sensitivity was associated with a decreased shoulder in the UV survival curves. The postirradiation division delay of survivors was shortest for cells harvested during the period of maximal sensitivity. This period of increased UV sensitivity during late log phase was not found in the radiation-sensitive, repair-deficient mutant B(s-1) (a strain which is unable to excise pyrimidine dimers from UV-damaged deoxyribonucleic acid). These results suggest that the variation in UV sensitivity of E. coli B/r as a function of time of harvesting of the cells from batch cultures is related to the varying capacities of these populations to repair UV-damaged deoxyribonucleic acid. Further experiments designed to elucidate the mechanism underlying this variation in UV sensitivity indicated that it arises from the partial depletion of nutrients in the medium during late log phase. We suggest that growth in such depleted media leads to a depression in the intercellular concentration or activity of one or more of the repair enzymes concerned with the repair of damaged deoxyribonucleic acid.     

Polarized TIRFM Reveals Changes in Plasma Membrane Topology Before and During Granule Fusion

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr²⁺ is used instead of Ca²⁺ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.     

Membrane malfunctions in freeze-dried Escherichia coli

Freeze drying and exposure to oxygen of E. coli causes damage to the bacterial cytoplasmic membrane. Freeze-drying itself produces an injury to the transport system for ONPG and potassium, so as to make the membrane leaky to these compounds. This damage is partially repaired upon incubation of the reconstituted bacteria in nutrient medium. When, however, freeze dried bacteria are not held in vacuo before reconstitution, but exposed to oxygen, this damage to the bacterial membrane becomes more extensive and irreversible.     

dGTP Starvation in Escherichia coli Provides New Insights into the Thymineless-Death Phenomenon

Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death.     

Starvation as an inducer of error-free DNA repair in Escherichia coli

Previous work in this laboratory has shown that heat shock or vitamin B1 deprivation induces an error-free DNA-repair process in Escherichia coli. The system is absolutely dependent on excision repair, while its induction is delayed in lon- or recA- cells. We have now shown that starvation of E. coli for amino acids, glucose or phosphate, conditions known to induce the stringent response or the glu and pho regulons, respectively, leads to a similar uvrA-dependent increase in UV resistance and decrease in UV-induced mutation frequency. These results support the hypothesis that the effect is a general response to non-mutagenic stress that may play an important role in the survival of cells exposed to harsh environments.     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Control of Cell Division in Escherichia coli: Experiments with Thymine Starvation

This paper describes the kinetics of cell division in populations of cells which have been grown first under conditions which specifically inhibit deoxyribonucleic acid (DNA) synthesis (in the absence of thymine or the presence of nalidixic acid) and subsequently under conditions which allow DNA synthesis to recommence. Cell division does not take place during inhibition of DNA synthesis. There is a delay between recommencement of DNA synthesis and recommencement of cell division. The length of this delay increases as a function of the length of the preceding period of inhibition of DNA synthesis. The first division after this delay is partly synchronous, but all subsequent division is asynchronous. These observations are explained in terms of a model which supposes that the formation of initiator of chromosome replication during a period when DNA synthesis is inhibited results in a block to cell division. Division does not then occur until this "extra" round of DNA synthesis is completed.     

Supercritical CO2 Induces Marked Changes in Membrane Phospholipids Composition in Escherichia coli K12

Supercritical carbon dioxide (SC-CO₂) treatment is one of the most promising alternative techniques for pasteurization of both liquid and solid food products. The inhibitory effect of SC-CO₂ on bacterial growth has been investigated in different species, but the precise mechanism of action remains unknown. Membrane permeabilization has been proposed to be the first event in SC-CO₂-mediated inactivation. Flow cytometry, high performance liquid chromatography–electrospray ionization–mass spectrometry and NMR analyses were performed to investigate the effect of SC-CO₂ treatment on membrane lipid profile and membrane permeability in Escherichia coli K12. After 15 min of SC-CO₂ treatment at 120 bar and 35 °C, the majority of bacterial cells dissipated their membrane potential (95 %) and lost membrane integrity, as 81 % become partially permeabilized and 18 % fully permeabilized. Membrane permeabilization was associated with a 20 % decrease in bacterial biovolume and to a strong (>50 %) reduction in phosphatidylglycerol (PG) membrane lipids, without altering the fatty acid composition and the degree of unsaturation of acyl chains. PGs are thought to play an important role in membrane stability, by reducing motion of phosphatidylethanolamine (PE) along the membrane bilayer, therefore promoting the formation of inter-lipid hydrogen bonds. In addition, the decrease in intracellular pH induced by SC-CO₂ likely alters the chemical properties of phospholipids and the PE/PG ratio. Biophysical effects of SC-CO₂ thus cause a strong perturbation of membrane architecture in E. coli, and such alterations are likely associated with its strong inactivation effect.     

Membrane localization of small proteins in Escherichia coli

Escherichia coli synthesize over 60 poorly understood small proteins of less than 50 amino acids. A striking feature of these proteins is that 65% contain a predicted α-helical transmembrane (TM) domain. This prompted us to examine the localization, topology, and membrane insertion of the small proteins. Biochemical fractionation showed that, consistent with the predicted TM helix, the small proteins generally are most abundant in the inner membrane fraction. Examples of both N(in)-C(out) and N(out)-C(in) orientations were found in assays of topology-reporter fusions to representative small TM proteins. Interestingly, however, three of nine tested proteins display dual topology. Positive residues close to the transmembrane domains are conserved, and mutational analysis of one small protein, YohP, showed that the positive inside rule applies for single transmembrane domain proteins as has been observed for larger proteins. Finally, fractionation analysis of small protein localization in strains depleted of the Sec or YidC membrane insertion pathways uncovered differential requirements. Some small proteins appear to be affected by both Sec and YidC depletion, others showed more dependence on one or the other insertion pathway, whereas one protein was not affected by depletion of either Sec or YidC. Thus, despite their diminutive size, small proteins display considerable diversity in topology, biochemical features, and insertion pathways.     

Plasma Membrane Alterations and Cytoskeletal Changes in Apoptosis

During apoptosis, one of the first membrane changes that can be detected is exposure of phosphatidylserine residues at the outer plasma membrane leaflet, while early apoptosis is also accompanied by changes in the cytoskeletal organization. In this study we investigated the relationship between these two phenomena during olomoucine- and roscovitin-induced apoptosis in human lung cancer and neuroblastoma cell lines. Loss of membrane asymmetry was detected by biotin-labeled or FITC-labeled annexin V binding to negatively charged phosphatidylserine, while cytoskeletal components were visualized by immunocytochemistry. The apoptotic, annexin V-positive, cells were analyzed by flow cytometry, confocal scanning laser microscopy, and Western blotting. We report that cytokeratin and vimentin aggregation in early apoptosis occurs simultaneously with phosphatidylserine exposure and chromatin condensation. In contrast to these intermediate filament proteins, which were disassembled and proteolytically cleaved in early apoptosis, microfilaments and microtubuli were not proteolytically degraded but were found to be present as aggregated filaments in the apoptotic bodies. We also show that loss of membrane asymmetry and cytokeratin aggregation are independent processes, sinceN-ethylmaleimide-induced phosphatidylserine exposure does not cause cytokeratin disassembly. Vice versa, phorbol 12-myristate 13-acetate-induced cytokeratin filament aggregation does not result in phosphatidylserine exposure.