A Genetic Defect in Retention of Potassium by Streptococcus faecalis *

Escherichia coli membranes were isolated in the presence of 6 mM Mg++. (1, 2). These syntheses are organized in the cell to produce yields of infectious virus as high as 104 per cell, but enzyme preparations give net synthesis of infectious RNA of only 10-fold (3) or less (4, 5). The difference in the in vivo and in vitro yield of phage RNA could be explained if the synthesis were tightly ordered in the cell. Use of membrane as a site of RNA synthesis should increase the efficiency of synthesis by providing a surface for adsorption of precursors, spatial organization of sequential steps, and limitation of the possible configurations of the RNA to those that are the most favorable for synthesis and assembly. The observation that a histidine-containing MS2-specific protein is associated with a rapidly sedimenting cell fraction orginally led to the suggestion that MS2 RNA synthesis could be organized on membrane (6). Further work (7) showed that labeled uracil is first incorporated in the presence of actinomycin D into MS2-specific RNA associated with a fraction sedimenting at 30,000 X g in the presence of 6 mM Mg++. This RNA was incorporated into a virion precursor particle (7) or organized into polyribosomes (unpublished data) before its release into the "supernatant." The rapidly sedimenting fraction was thought to be membrane because it was solubilized by detergents and urea (7,8) and because it contained cell lipid (8).Electron microscopic studies confirmed that this fraction contained membrane (8). This membrane preparation differs from many in that no detergents were used and 6 mM Mg++ was continuously present. When the rapidly sedimenting cell fraction is washed with buffer lacking Mg++ (or other divalent cations) some components are released, "membrane eluate," while others still sediment, "membrane." This work describes the kinetics of labeling of the different MS2 RNA species in the three cell fractions, supernatant, membrane eluate, and membrane. MATERIALS AND METHODSBacteria and Growth Medium have been described (7). Bufers. TKA buffer is 10 mM Tris HCl (pH 8)-40 mM KCI-10 mM NaN3. TMKA buffer TKA plus 6 mM MgC12.Cell Fractions. To harvest cells, NaN3 (10 mM) and unlabeled uracil (100 ,ug/ml) were added to 200-ml aliquots of cells at a concentration of 2 X 108/ml. These were rapidly chilled by swirling in a wide-bottom flask in a dry-ice-alcohol bath. The following steps were done at 0-4'. The cells were centrifuged and suspended in 0.6 ml of TMKA buffer. The success of the lysis depends upon a high cell concentration.Lysozyme (1 mg/ml) was added, and after 3 min, the cells were rapidly frozen and thawed twice. Electrophoretically purified DNase (10 /ug/ml) was added, and at least 10 min later, the lysate was centrifuged at 30,000 X g for 20 min. The resulting sediment was resuspended twice in TMKA buffer with vigorous mixing on a Vortex mixer and centrifuged at 30,000 X g for 20 min. The resultant supernatants were combined and referred to as "supernatant." The resultant sediment was resuspend...

Section: Resultsmentioning

confidence: 98%

Two Classes of Membrane Binding of Replicative RNA of Bacteriophage MS2

Haywood¹

1973

“…In the absence of respiration, ATPasenegative mutants cannot utilize ATP from substrate level phosphorylations to drive the ATP-linked transhydrogenase (6, 7)'or the accumulation of various metabolites (3,5,8). Such anaerobic function of the ATPase is also suggested by the effects of NN'-dicyclohexylcarbodiimide (DCCD), an inhibitor of this enzyme (9,10). In E. coli, DCCD blocks both the ATP-linked transhydrogenase (7) and the accumulation of proline found under anaerobic conditions (11).…”

mentioning

confidence: 98%

“…In E. coli, DCCD blocks both the ATP-linked transhydrogenase (7) and the accumulation of proline found under anaerobic conditions (11). DCCD also inhibits active transport of metabolites in Streptococci, which lack oxidative metabolism (9,12,13).…”

mentioning

confidence: 99%

A Protonmotive Force Drives ATP Synthesis in Bacteria

Maloney

Kashket

Wilson

1974

149

103

When cells of Streptococcus lactis or Escherichia coli were suspended in-a potassium-free medium, a membrane potential (negative inside) could be artificially generated by the addition of-the potassium ionophore, valinomycin. In 'response to this inward directed protonmotive force, ATP synthesis catalyzed by the mnembrane-bound'ATPase (EC 3.6.1.3) was observed. (Fig. 1A). Thus, the electrochemical potential of protons (the protonmotive force) provides the driving force for ATP synthesis. The alternative (anaerobic) function of the ATPase is required when protons cannot be extruded by the respiratory chain. Under these conditions, the ATPase couples the hydrolysis of ATP to the electrogenic movement of protons out of the cell (Fig. 11B). The protonmotive force generated by ATP hydrolysis is then utilized by energy-dependent reactions such as the "ATP-linked" transhydrogenase, or the active transport of metabolites.Evidence in support of this anaerobic function of the ATPase has been presented by Harold and his collaborators, who have studied the anaerobe S. fecalis (faecium). They showed that glycolyzing cells establish both a pH gradient (interior alkaline) and a membrane potential (interior negative), and that DCCD inhibits the formation of each of these components of the protonmotive force (17)(18)(19) 1% (w/v)

“…Membranes whose permeability to potassium is altered by valinomycin include bacterial (3)(4)(5), erythrocyte (6)(7)(8), mitochondrial (9), and artificial black lipid membranes (10). The change is highly specific: only permeability to potassium and the related alkaline cations rubidium and cesium is affected (7,10).…”

mentioning

confidence: 99%

Valinomycin-Induced Uptake of Potassium in Membrane Vesicles from Escherichia coli

Bhattacharyya

Epstein

Silver

1971

Osmotically shocked Escherichia coli and membrane vesicle ghosts from E. coli cells have lost the ability to accumulate potassium by active transport. The addition of valinomycin to the membrane ghosts restores the capacity to accumulate radioactive 42K and 86Rb by a temperature-and energy-dependent process. Membrane vesicles prepared from mutants of E. coli altered in potassium transport show defects in the valinomycin-stimulated accumulation of 42K that are related to the defects in the intact cells.Valinomycin is a cyclic depsipeptide antibiotic (1, 2) that acts by greatly increasing the permeability of membranes, specifically to potassium ions (3). Membranes whose permeability to potassium is altered by valinomycin include bacterial (3-5), erythrocyte (6-8), mitochondrial (9), and artificial black lipid membranes (10). The change is highly specific: only permeability to potassium and the related alkaline cations rubidium and cesium is affected (7, 10). The membranes remain impermeable to protons, sodium, lithium, and other ions in the presence of valinomycin. Evidence from relatively thick artificial membranes shows that valinomycin acts not by forming channels or pores through which potassium can travel, but by forming one-to-one complexes with K+ ions (8) which can then diffuse across the hydrophobic lipid membranes, with the K+ sheltered from the membrane within the cyclic folds of the valinomycin molecule (2). With mitochondria, at least, valinomycin facilitates the net accumulation of potassium (9) and this finding has led to models of valinomycin as a prototype for natural carriers of potassium in cell membranes. In this paper we present a similar finding with membranes prepared from cells of Escherichia coli-that is, valinomycin-facilitated uptake of potassium in an energy-dependent and apparently concentrative manner.E. coli has a number of advantages in the study of potassium transport because this organism possesses a transport system with high affinity for K (11,12), is able to maintain very large concentration gradients for K (13), and most of all because of the extensive knowledge of the genetics and metabolism of this organism. Lubin (14) first isolated and characterized specific potassium transport mutants in E. coli strain B. Later other potassium mutants were isolated in other E. coli strains (14-18) and Bacillus subtilis (14,19), and a number of mutants of E. coli K-12 with alterations in potassium transport have recently been isolated. A total of eight genes affecting potassium transport have been identified. One set of four closely linked kdp genes ("K+-dependent"; 15) and two other genes (trkA, trkD; "transport of K+") affect primarily uptake of potassium, while mutations in the trkB and trkC genes result in a defect in the retention of potassium (Epstein, in preparation). All these mutational defects appear to be specific for potassium because the mutants are normal in the ability to transport ,3-galactosides and proline (unpublished data). If we are to pursue the model from mit...