SUMMARY:The effect of the ionic composition of the medium on the respiration of resting-cell suspensions of two Gram-negative micro -organisms has been investigated. Potassium or rubidium salts with C1-, Br-or I-as anion were found to be necessary for the maximal rates of oxidation of glutamate or glucose by Pasteurella tularensis, and KCl or K,SO, by Escherichia coli. The pH value of the medium and the concentration of the substrate influenced the effect of the electrolytes. Under certain conditions, a synergistic effect of Naf ion was observed. The same ion mixtures which supported the respiration caused the maximal swelling of the bacterial cells.The influence of KC1 and NaCl on the respiration of halophilic bacteria was investigated by Baxter & Gibbons (1956). Krebs, Whittam & Hems (1957) showed that the oxidation of various substrates by washed cells of Alcaligenes faecalis was greatly accelerated by potassium ions. In the present communication observations are reported on the effect of various electrolytes on the rate of oxidation of glutamate and glucose by Escherichia coEi and by an osmoticallysensitive micro-organism, Pasteurella tularensis. METHODSEscherichia coli, strain B, was grown on nutrient agar (Difco). Pasteurella tularensis, strain S and D, was grown on a 3 yo (w/v) peptone (Difco) + cysteine medium (Mager, Kindler & Grossowicz, 1954). The cultures were incubated a t 37' for 24 hr. The bacteria were collected in 0.25~-sucrose and, unless otherwise stated, washed three times with this sucrose solution. The final sucrose (0.25 N) suspensions contained 50 mg. wet weight organisms/ml. Manometric experiments were carried out by the conventional Warburg technique. Spectrophotometric examinations were performed in the Beckman model DU spectrophotometer, with Corex cells of 1 cm. light path. RESULTSEffect of various cations and anions o n the oxidation of glutamate Glutamic acid, neutralized by the addition of tris (buffer), 2-amino-2-hydroxymethylpropane-1 : 3-dio1, to pH 7-4, was not oxidized by thoroughly washed Pastewella tularensis when other salts were excluded from the reaction medium. When, however, various potassium salts were added to the bacteria suspended in isotonic sucrose, it was found that KBr caused the greatest respiratory stimulation, while KC1 and K I were slightly less effective; other potassium salts had only an insignificant effect. Of the chlorides tested other than KCl only the
SUMMARYThe inter-relationship between the intracellular concentration of potassium ions and the rate of oxidation of glutamate was investigated in washed Escherichiu coli. The time-curve of glutamate oxidation by the potassiumdepleted coli showed a marked lag phase and the rate of oxygen uptake increased concurrently with the accumulation of potassium ions. After a constant intracellular potassium concentration was reached, the rate of oxidation remained constant. Carbonyl cyanide m-chlorophenylhydrazone (m-CI-CCP) and methylene blue inhibited the respiration when added to the reaction mixture during the initial phase of K+ accumulation. The extent of inhibition induced by these compounds was inversely related to the rate of oxidation prevailing a t the time of their addition. No inhibition resulted when the substances were added after the K+ accumulation and respiratory rate had reached the steady state values. Pre-incubation with glucose and KCI abolished the initial lag of glutamate oxidation as well as the inhibitory action of rn-CI-CCP. It is concluded that the intracellular concentration rather than the flux of potassium ion governs the control of respiration in E . coli. The possible relation of the mode of respiratory inhibition induced by m-C1-CCP and methylene blue to the known ability of these compounds to uncouple oxidative phosphorylation, is discussed in terms of the presumed energy requirements of the system mediating the K+ transport.
Gramicidin was seen to promote rapid release of Rb+ from cells of Streptococcus faecalis ATCC 10541 which had been previously enriched with this cation. This response was logarithmic between gramicidin concentrations of 0.25 μg/ml and 2.5 μg/ml. This efflux was measured by flame emission spectroscopy. The results presented here form the basis for a potential rapid bioassay for gramicidin.
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