Drug-induced changes in lipid composition of E. coli and of mammalian cells in culture: ethanol, pentobarbital, and chlorpromazine.
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Cell Line↗
Biomedical subjects
L O Ingram
Publications and source records attributed to L O Ingram.
Purification of lipoteichoic acids by using phosphatidyl choline vesicles.
Lipoteichoic acid (LTA) is a component of nearly all gram-positive membranes and recently has been found to be excreted into growth media by certain lactic acid bacteria. Cell-free extracts of LTA are usually contaminated with proteins, polysaccharides, and nucleic acids, thus causing problems to investigators studying the true biological function(s) of LTA. This report describes the preparation of purified extracellular LTA of Streptococcus mutans BHT and intracellular LTA of S. mutans AHT by three techniques: gel filtration, hydrophobic interaction chromatography, and adsorption to phospholipid vesicles. Gel filtration, the most commonly employed method for LTA purification, was found to remove nucleic acids, teichoic acids, and much polysaccharide while greatly concentrating LTA. But gross amounts of antigenic carbohydrate and protein remained associated with the LTA preparation. Hydrophobic interaction chromatography employing octyl Sepharose-4B allowed the separation of protein but not polysaccharide from partially purified BHT LTA preparations. By means of a new technique described in this paper, synthetic membranes (vesicles) were found to effectively separate all contaminants from the intracellular (AHT) and extracellular (BHT) LTA of S. mutans. This rapid method, on a comparative basis, proved to be the most effective approach for the purification of LTA from two widely differing sources.
Adsorption↗
Low-temperature conditional cell division mutants of Escherichia coli.
Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.
Cell Division↗
Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food additives.
Cells of Escherichia coli contain an altered fatty acid and phospholipid composition when grown in the presence of sublethal concentrations of a variety of organic solvents and food additives. The diversity of compounds examined which caused these changes indicates that no single catabolic pathway is involved. Many of the observed changes are consistent with the hypothesis that cells adapt their membrane lipids to compensate for the presence of these compounds in the environment. Both sodium benzoate and calcium propionate caused the synthesis of unusual fatty acids.
Escherichia coli↗
Propionate-induced synthesis of odd-chain-length fatty acids by Escherichia coli.
Exogenous propionate is incorporated in vivo by Escherichia coli as a primer to produce lipids with fatty acids of odd chain lengths. This provides a method for the specific labeling of the three terminal carbons in the fatty acyl chains of phospholipids.
Carboxylic Acids↗
Preferential inhibition of phosphatidyl ethanolamine synthesis in E. coli by alcohols.
Growth of E. coli in the presence of alcohols of chain lengths 1 through 8 results in an increase in the relative abundance of phosphatidyl glycerol. This results primarily from the preferential inhibition of phosphatidyl ethanolamine synthesis. This inhibition appears to be unrelated to membrane fluidity or to changes in fatty acid composition caused by alcohols. Alcohol-induced changes in total fatty acid composition are reflected in all phospholipid classes. Phosphatidyl serine synthetase is proposed as the most likely site for the effects of alcohols on phospholipid synthesis.
Alcohols↗
Potassium requirement for cell division in Anacystis nidulans.
A potassium requirement for growth can be readily demonstrated in the autotrophic blue-green bacterium Anacystis nidulans strain TX20 equivalent to 0.7% of the cellular dry weight. Starvation of this organism for potassium partially dissociates growth from cell division, thereby inducing 50% of the population to form filaments.
Cell Division↗
Adaptation of membrane lipids to alcohols.
The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined. My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols. These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane. Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs.
Acetaldehyde↗
Peptidoglycan synthesis and turnover in cell division mutants of Agmenellum.
The synthesis and turnover of peptidoglycan in Agmenellum quadruplicatum was investigated using D-[U-14C]alanine followed by proteolytic digestion. The rate of turnover of alanine in the peptide portion of the peptidoglycan was measured in strain BG-1 and in two division mutants of this strain: one was blocked in cell separation; and the other was a low-temperature, conditional cell division mutant. The peptide portion of peptidoglycan turned over in all three strains tested, but no correlation was observed between septum formation or cell separation and the rate of turnover. Peptidoglycan synthesis was measured during induced division in snake forms of strain SN-29. A stimulation of peptidoglycan synthesis was observed during the period of cross-wall formation, even in the absence of new protein synthesis. Thus in A. quadruplicatum, cross-wall synthesis is accompanied by a stimulation of peptidoglycan synthesis.
Alanine↗
Cell division in Agmenellium quadruplicatum: evidence for the negative control by a protein.
A high temperature conditional snake mutant, strain D1, of Agmenellum quadruplicatum was isolated which immediately stopped dividing following a shift to 41 degrees C following treatment with nitrosoguanidine. This mutant was stimulated to divide at 41 degrees C by the addition of inhibitors of RNA or protein synthesis: rifampicin, streptomycin, puromycin and chloramphenicol. Each of these inhibitors exhibited a discrete concentration optimum. The optimal concentration of chloramphenicol for cell division corresponded to the minimal concentrations necessary for the rapid inhibition of protein synthesis. The ability of chloramphenicol and other inhibitors to induce cell division in filaments decayed rapidly upon shifting to 30 degrees C. These results are interpreted as evidence for a protein acting as a negative regulator late in the cell cycle. At 41 degrees C, DNA was found distributed as a continuous zone throughout the length of the filaments. The addition of inhibitors of protein or RNA synthesis resulted in a rapid condensation of this nuclear material into multiple discrete nuclear regions suggesting that the negative control may be at the level of nuclear compartmentalization.
Bacteria↗
Isolation of a small-cell mutant in the blue-green bacterium Agmenellum quadruplicatum.
A new type of high-temperature conditional cell division mutant has been isolated in Agmenellum quadruplicatum strain BG1 in which the process of cell division is uncoupled from that of growth at 39 C. This mutant produces abnormally small cells under conditions of nutrient limitation and forms multinucleoid filaments under normal growth conditions.
Bacteria↗
Effects of temperature and nutritional changes on the fatty acids of agmenellum quadruplicatum.
The fatty acid composition of the blue-green bacterium Agmenellum quadruplicatum was examined under a wide variety of growth conditions. The fatty acid composition was found to undergo significant changes with variations in temperature, media composition, and growth phase (log versus stationary). With increasing growth temperature (20 to 43 C) log-phase cells exhibited an increase in saturated fatty acids (38.4% at 20 C to 63.6% at 43 C). Striking changes were seen with some of the individual fatty acids such as 18.3, which made up 16.0% of the total fatty acid at 20 C but was not neasurable at 43 C. Fatty acid 12:0 was not measurable at 20 C but made up 16.3% of the total fatty acids at 43 C. Cell lipids were separated into neutral lipid, glycolipid, and very polar liquid fractions. The neutral lipid fraction was composed almost entirely of 12 carbon fatty acids (12:0, 12:1). Glycolipid and very polar lipids were more similar in their fatty acid composition when compared to the total cellular fatty acids, although they did lack 12 carbon fatty acids. The total of 12 carbon fatty acids in the cell can be used as an indicator of the amount of neutral lipid present.
Culture Media↗
Comparison of lipopolysaccharides from Agmenellum quadruplicatum to Escherichia coli and Salmonella typhimurium by using thin-layer chromatography.
Lipopolysaccharide (LPS) was isolated from the unicellular blue-green bacterium Agmenellum quadruplicatum using the procedure of Westphal and Jann (1965). It was composed of a lipid A and polysaccharide region suggesting a similarity to other gram-negative LPSs. Chemical analyses demonstrated the presence of glucose, rhamnose, mannose, and xylose in the polysaccharide region, as well as 2-keto-3-deoxyoctonate, glucosamine, and phosphorous in the lipid A. Studies on the lipid composition revealed the presence of palmitic, behenic, and three beta-hydroxy fatty acids. A new procedure for thin-layer chromatography of bacterial LPSs was used to compare LPS from A. quadruplicatum to other gram-negative organisms. The method is capable of distinguishing between LPSs of different bacteria as well as between the wild-type organism and mutated forms unable to synthesize complete LPS. A comparison of LPS from A. quadruplicatum to Escherichia coli and Salmonella typhimurium demonstrated that, although the blue-green LPS was rather similar to that of the Enterobacteriaceae, distinct differences also existed. However, when several cell division mutants of A. quadruplicatum were compared chromatographically to the parent strain BG-1, no differences were observed. This suggests that cell division mutations in A. quadruplicatum are not associated with changes in the LPS.
Carbohydrates↗
Cell division in blue-green bacteria: stimulation of localized regions of peptidoglycan metabolism by ethanol.
D-Cycloserine was used to localize regions of active peptidoglycan metabolism as bulged, weakened areas in the lateral wall. Strain SN29 is a non-septate filamentous mutant of Agmenellum quadruplicatum and produces one to two such bulges per filament during exponential growth in the presence of D-cycloserine. The addition of ethanol to D-cycloserine inhibited filaments results in the production of many additional bulges in the lateral wall and is interpreted as evidence for the localized activation of peptidoglycan metabolism by ethanol. In the parent organism, D-cycloserine selectively inhibits cell division resulting in the formation of enlarged spindle-shaped cells. Cell separation was also affected. Chain formation was greatly reduced in septate mutants of A. quadruplicatum by growth in the presence of D-cycloserine. This was apparently due to the increase fragility of cells within the filaments.
Cell Division↗
Cell separation in blue-green bacteria.
Autolysin-like enzymes appear to be responsible for cell separation in Agmenellum quadruplicatum. Mutants that are impaired in cell separation and grow as chains exhibit reduced cell lytic activity. Lysozyme, extracted autolysin, and antibiotics that affect peptidoglycan synthesis phenotypically suppress chain formation. Various aspects of the regulation of the cell separation process were also examined. Studies involving antibiotic inhibitors of macromolecular synthesis and general growth inhibitors provided no evidence for the active regulation of the cell separation process during the latter portion of the division cycle. Evidence was obtained, however, for the partial restriction of peptidogly-can hydrolysis by unknown secondary modifications. The thin electron-dense layer of peptidoglycan along the sides of cells was much more resistant to hydrolysis by egg-white lysozyme than was the septum between daughter cells. The middle portion of the septum was more sensitive than was the layer immediately adjacent to the cytoplasmic membrane. Under conditions that would not osmotically stabilize spheroplasts, lysozyme facilitates rapid cell separation in chain-forming mutants with little leakage of cellular protein or loss of viability.
Anti-Bacterial Agents↗
Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum.
The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine, causing toxicity by some unknown mechanism. It is concluded that phenylpyruvate, potentially formed by transamination of l-phenylalanine, is an unlikely cause of growth inhibition. Although several significant questions remain unanswered, our results suggest that single-effector control of DAHP synthetase, the first regulatory enzyme activity of a branched pathway, may be more appropriate than it would seem a priori.
Amino Acid Isomerases↗
Growth inhibition by L-phenylalanine in Agmenellum quadruplicatum. A clue to some amino acid interrelationships.
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Alanine↗
Stimulation of cell division by membrane-active agents.
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Alcohols↗