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The assimilation of amino-acids by bacteria. 10. Action of inhibitors on the accumulation of free glutamic acid in Staphylococcus aureus and Streptococcus faecalis

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... {c) Bacteriostasis by aryl nitro compounds (Cowles & Klotz, 1948). [e] Inhibition of the accumulation of glutamate in Staphylococcus aureus by 2:4dinitrophenol (Gale, 1951). ...
... Quantitative experiments on the penetration of weak acids into the vacuoles of large plant cells indicate that substances like hydrogen sulphide and carbonic acid enter the vacuoles by the diffusion of undissociated molecules (Davson & Danielli, 1943;Hober 1947), and it is possible that the same may be true for the penetration of /3-indoleacetic acid (cf. Albaum, Kaiser & Nestler, 1937;Sutter, 1944). ...
... Moreover, it would be necessary to know precisely how this internal pH was influenced by changes of the external pH. Such detailed information is of course not available, although changes of external pH are well known to influence the metabolic activity of cells, and in some cases at least they must have some eflect on internal pH levels (Gale, 1943;Dubos, 1949, chapter 4;Rietsema, 1949). ...
... {c) Bacteriostasis by aryl nitro compounds (Cowles & Klotz, 1948). [e] Inhibition of the accumulation of glutamate in Staphylococcus aureus by 2:4dinitrophenol (Gale, 1951). ...
... Quantitative experiments on the penetration of weak acids into the vacuoles of large plant cells indicate that substances like hydrogen sulphide and carbonic acid enter the vacuoles by the diffusion of undissociated molecules (Davson & Danielli, 1943;Hober 1947), and it is possible that the same may be true for the penetration of /3-indoleacetic acid (cf. Albaum, Kaiser & Nestler, 1937;Sutter, 1944). ...
... Moreover, it would be necessary to know precisely how this internal pH was influenced by changes of the external pH. Such detailed information is of course not available, although changes of external pH are well known to influence the metabolic activity of cells, and in some cases at least they must have some eflect on internal pH levels (Gale, 1943;Dubos, 1949, chapter 4;Rietsema, 1949). ...
... These are summarized in the schema (figure 1). Gale (5) reported that glutamate did not enter M. pyogenes var. aureusun less this organism was metabolizing glucose. ...
... In 1954 Peter Mitchell stated that "we have no information at present as to the chemical nature of the substance which carries the phosphate across the osmotic barrier of the Staphylococcus". However, the transport of glucose across the placenta (Widdas, 1952), of amino acids into Gram-positive bacteria (Gale, 1951Gale, , 1953), of phosphate into Staphylococcus aureus (Mitchell, 1954) and of lactose into E. coli (Rickenberg et al., 1956) exhibited the properties of high substrate specficity, susceptibility to protein reagents, and a high temperature coefficient typical of enzyme reactions. So Peter Mitchell then suggested "that specific permeation reactions of high specificity may occur by a process quite analogous to that of enzyme reactions and that the thermal movement by which the reactant is converted to the resultant in enzyme reactions may, in specific permeation reactions, cause a change of configuration or position of a protein carrier in the membrane such that the accessibility of its adsorbed passenger changes from one side of the membrane to the other" (Mitchell, 1956). ...
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There is a symbiotic relationship between the evolution of fundamental theory and the winning of experimentally-based knowledge. The impact of the General Chemiosmotic Theory on our understanding of the nature of membrane transport processes is described and discussed. The history of experimental studies on transport catalysed by ionophore antibiotics and the membrane proteins of mitochondria and bacteria are used to illustrate the evolution of knowledge and theory. Recent experimental approaches to understanding the lactose-H+ symport protein of Escherichia coli and other sugar porters are described to show that the lack of experimental knowledge of the three-dimensional structures of the proteins currently limits the development of theories about their molecular mechanism of translocation catalysis.
... The lipid fraction of this strain has been investigated by Macfarlane (1962b). The organism was grown in a liquid medium containing salts, glucose, arginine and Marmite (Gale, 1951) Incubation condition8. Cell suspensions, at a final density of 0-2 mg. ...
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1. Incubation of washed cells of Staphylococcus aureus with [1-(14)C]glycerol results in the incorporation of glycerol into the lipid fraction of the cells. The rate of incorporation is increased by the presence of glucose and amino acids. The presence of amino acids increases incorporation into the fraction containing O-amino acid esters of phosphatidylglycerol. 2. Glycerol, incorporated into washed cells by incubation with glycerol, glucose and amino acids, is rapidly released from the lipid fraction when cells are incubated at low suspension densities in buffer. 3. Of nine amino acids tested, only lysine is significantly incorporated into the lipid fraction. The incorporation is increased by the presence of glycerol, glucose and other amino acids, especially aspartate and glutamate. 4. The incorporation of lysine is increased by the addition of puromycin at concentrations that inhibit protein synthesis. Chloramphenicol does not increase the incorporation of lysine but abolishes the enhancing effect of puromycin. 5. The enhancing effect of puromycin is accompanied by a similar increase in the incorporation of lysine into the fraction soluble in hot trichloroacetic acid. 6. Lysine is incorporated into the lipid fraction that contains O-amino acid esters of phosphatidylglycerol and corresponds in properties to phosphatidylglyceryl-lysine. 7. Lysine is rapidly released from the lipid of cells incubated in buffer only at low suspension densities. 8. Incubation of cells with the phosphatidylglyceryl-lysine fraction does not lead to the appearance of free lysine or to incorporation into the fraction insoluble in hot trichloroacetic acid.
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This chapter discusses the assimilation of amino acids by gram-positive bacteria. The growth of the bacterial cell involves, and is the result of, synthesis of all its components. Many bacteria can synthesize protein from ammonia and a carbon source, such as glucose. “Biochemical mutants” of molds and bacteria have indicated the probable biosynthetic pathways and individual steps in pathways that have been studied with cell-free extracts of microorganisms. Species of bacteria that are unable to synthesize amino acids, assimilates the preformed substances, to form suitable material for the study. Experimental correlations among the various processes are observed, and certain stages in the processes are found to be sensitive to antibiotics and growth inhibitors.
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Wood, N. P. (A. & M. College of Texas, College Station), and D. J. O'Kane. Formate-pyruvate exchange reaction in Streptococcus faecalis. I. Factor requirement for whole cells. J. Bacteriol. 87 97–103. 1964.—A factor present in plant and animal sources was found necessary for the incorporation of formate-C¹⁴ into pyruvate by Streptococcus faecalis 10Cl. Yeast extract produced a response linear in the range between 10 and 30 mg/ml of reaction mixture. Soy peptone, beef peptone, and Brain Heart Infusion replaced yeast extract, but various intermediates, cofactors, amino acids, purines, pyrimidines, and peptides did not stimulate the reaction. A lag occurred in the rate of formate incorporation that was not influenced by anaerobic conditions or growth of cells in a medium containing pyruvate and formate. Phosphate or maleate buffer permitted rapid exchange velocities but tris(hydroxymethyl)aminomethane or collidine buffer was inhibitory. Heating yeast extract at 121 C for 15 min in 3 n H2SO4 produced 66% inactivation of the factor(s), whereas treatment with 3 n KOH produced 97% inactivation. The factor(s) was insoluble in butanol, benzene, ethyl acetate, or chloroform. The material adsorbed on Dowex-1 (OH⁻) and Amberlite IR-120 (H⁺) but not on Amberlite IR-4B (OH⁻). The active component(s) was highly polar, nonvolatile, dialyzable, and had amphoteric properties.
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Repair ofinjury induced byfreeze-drying Salmonella anatuminnonfat milk solids occurred rapidly after rehydration. Injury insurviving cells wasdefined asthe inability toformcolonies on aplating mediumcontaining deoxycholate. Death was defined asinability toformcolonies inthesame mediumwithout thisselective agent. Therateofrepair ofinjury was reduced bylowering thetemperature from 35C to10C andwas extremely lowat1C.Repair wasindependent ofinfluence ofpHbetween 6.0and7.0. Repair didnotrequire synthesis ofprotein, ribonucleic acid, or cellwallmucopeptide, butdidrequire energyintheformofadenosine triphosphate (ATP)synthesized through oxidative phosphorylation. Therequire- mentforATP was based on dinitrophenol or cyanide interference withrepair. Dinitrophenol activity waspH-dependent; no repair occurred atpH 6.0andsome repair was observed atpH6.5andabove. Injured cellswereextremely sensitive to lowconcentrations ofethylenedinitril otetraacetate. Thisindicated thatfreeze-
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The requirement for Na+ for the uptake of α-aminoisobutyric acid (AIB) by cells of a marine pseudomonad was found to be quantitatively similar to the requirement for Na+ to prevent the release of AIB from previously loaded cells of the organism. At concentrations of Na+ different from the optimum, the viability of cells decreased in proportion to the extent of loss of AIB from previously loaded cells. AIB loss, however, preceded the loss of viability. The uptake of AIB required Na+ specifically while retention could be at least partially effected by other ions, particularly Li+. As the Na+ concentration in the suspending medium increased from 0 to 50 mm, the Km for transport decreased 12-fold while Vmax remained essentially constant. Between 50 and 200 mm Na+, Km remained constant at 8.33 x 10-5 m while Vmax continued to increase. Up to 200 mm Na+ the rate of uptake of AIB and the capacity of the cells for AIB were a direct function of the Na+ concentration or of the Li+ concentration if Na+ was present. Above 200mm Na+ the capacity of the cells for AIB decreased. Na+ could not be replaced by Li+ or K+ in effecting exchange diffusion of unlabeled AIB in the suspending medium with AIB-14C in the cells. Cells suspended at pH 7.2 in a medium containing NaCl or LiCl at a concentration optimum for retention of AIB lost AIB in the presence of NaCl but not LiCl when treated with 2,4-dinitrophenol or KCN. At pH 5.3 dinitrophenol caused the loss of AIB from the cells in the presence of either salt. The results suggest that there are two functions of Na+ in these cells, one concerned with uptake of compounds and the other involved in the prevention of their release.
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(1) Substrates capable of activating mitochondrial electron transfer and oxidative phosphorylation, namely, pyruvate, acetate, propionaldehyde and butanol, stimulated the concentrative uptake (transport and accumulation) of L-[14-C]leucine by Saccharomyces cerevisiae (wild type strain 207, starved cells). Under adequate experimental conditions, the L-[14-C]leucine uptake versus the oxygen uptake ratio was almost the same with either pyruvate, acetate or D-glucose as energy sources. Substrate oxidation also increased L-[14-C]leucine incorporation into the cell protein. (2) With S. cerevisiae D261 and D247-2 and propionaldehyde as an energy source, or with strain 207 and glucose as energy source, 2,4-dinitrophenol (50 muM) inhibited L-[14-C]leucine uptake, the inhibition being accompanied by stimulation of respiration. With S. cerevisiae 207 and propionaldehyde as energy source, 2,4-dinitrophenol inhibited both respiration and L-[14-C]leucine uptake, but with respiration being less affected than uptake. Displacement of accumulated L-[14-C]leucine was also inhibited by 2,4-dinitrophenol. (3) In the presence of glucose, and for relatively brief incubation periods, anaerobically grown cells of S. cerevisiae 207 and of a p-minus "petite" mutant of this strain incorporated L-[14-C]leucine with less efficiency than the original wild type strain 207, grown aerobically. With D-glucose as energy source, 2,4-dinitrophenol and iodoacetate inhibited alike L-[14-C]leucine uptake by the respiration competent cells. (4) It is postulated that in respiration-competent yeasts, the mitochondrion contributes to 6-[14-C]leucine uptake by supplying high-energy compounds required for amino acid transport and accumulation. Conversely, the promitochondrion in the anaerobically grown yeast, or the modified mitochondrion in the respiratory deficient mutant, competes for high energy compounds generated by glycolysis in the cytosol.
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Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.
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Iandolo, John J. (University of Illinois, Urbana), and Z. John Ordal. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91 134–142. 1966.—Exposure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance.
Article
Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.
Article
to assimilate glutamic acid and lysine from the external medium and to concentrate these amino-acids in the internal environment, while eleven Gram-negative organisms were unable to do so. It has been shown (Gale, 1947) that Streptococcus faecalis cells possess a high concentration of certain amino-acids existing in a free state within the cells, the internal concentration of lysine and glutamic acid being much greater than that in the external environment in equilibrium with the internal environ- ment. Lysine is able to diffuse into the cell under certain conditions, while the migration of glutamic acid across the cell wall appears to be a process requiring energy, obtainable from fermentation processes. The gradient in concentration of lysine and glutamic acid across the cell wall appears to be maintained by properties of the cell wall itself, since rupture of the cell wall with tyrocidin, etc. results in the release of the internal amino-acids (Gale & Taylor, 1947). A biological consequence of this gradient across the cell wall is that the cell is able to select and concentrate certain amino-acids from a deficient medium. This mechanism should be of greater importance to organisms which are nutritionally exacting with regard to amino-acids than to organisms which can synthesize their amino-acid requirements (Gale, 1947). The present com- munication deals with the distribution amongst various bacterial genera and species of this cell wall gradient effect, as judged by the presence of free amino- acids in the internal environment of the cells.
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Gale, E. F. & Rodwell, A. W. (1949). J. gen. Microbiol. 3,127. Gale, E. F. & Taylor, E. S. (1947). J. gen. Microbiol. 1, 77. Loomis, W. F. & Lipmann, F. (1948). J. biol. Chem. 173, 807. Najjar, V. A. & Gale, E. F. (1950). Biochem. J. 40, 91. Needham, D. M. & Pillai, R. K. (1937). Biochem. J. 31, 1837. Spiegelman, S., Kamen, M. D. & Sussman, M. (1948). Arch. Biochem. 18, 409.