Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

Helgerson, Sam

doi:10.1002/jss.400050304

Cited by 27 publications

(19 citation statements)

References 45 publications

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“…Low-speed centrifugation (7,700 x g, 10 min) was found to remove the increased fluorescence from the sample, indicating that it was cell bound. The probe was also found to have undergone a shift in the emission maximum, from 440 to 410 nm, indicative of the probe being in a lipid environment (9,17).…”

Section: Resultsmentioning

confidence: 99%

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Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli

Dougherty¹,

Saukkonen²

1985

Antimicrob Agents Chemother

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Inhibition of DNA synthesis in Escherichia coli B/r by a DNA gyrase inhibitor results in cell death after a 50-min lag period. Examination of the cells under phase-contrast and electron microscopes revealed that they appeared to undergo plasmolysis coincident with the onset of cell death. The inhibited cells were also found to become susceptible to low levels of detergent at this time. With a fluorescent membrane probe, the level of membrane permeability was assessed and found to increase concurrently with the decrease in culture viability. Analysis of the cell envelope constituents revealed that, other than a shift in the protein/lipid ratio, the compositions of the cell membranes were unperturbed.There has been a renewed interest in DNA gyrase inhibitors with the recent introduction of new quinolone derivatives with increased in vitro activity and improved pharmacokinetics (3). Treatment with these DNA gyrase inhibitors ultimately leads to loss of bacterial viability. A number of studies have focused on the effects that these antibiotics have on the topology of DNA and gene expression (for review, see reference 6). Cessation of DNA synthesis' by gyrase inhibitors activates a cascade of events within the cell involving a wide variety of biochemical pathways, including cell filamentation (12). Only a few studies have addressed the physiological consequences of DNA replication inhibition on the bacterial cell envelope (21,22).During a series of studies on DNA replication inhibitors, we noted morphological changes consistent with a loss of membrane integrity and leakage of cytoplasmic constituents. In this report, the effect of a DNA replication inhibitor on the structure and function of the bacterial envelope is examined. Substantial alterations in the integrity of the membranes are documented, and their possible relationship to loss of viability is discussed. MATERIALS AND METHODSBacterial strain. Escherichia coli B/r T-, a thymine-requiring auxotroph, was derived from a prototrophic E. coli Bir strain (obtained from George Khachatourian, University of Saskatchewan, Saskatchewan, Saskatoon, Canada) by an amethopterin selection procedure (4). The strain was a lowlevel thymine requirer (thyA deoB) that grew fully on 2 ,ug of thymine per ml. Cells were treated with 20 ,ug of nalidixic acid (Nal; Sigma Chemical Co., St. Louis, Mo.; prepared in 0.5 N NaOH) per ml, which is 10 times the MIC. Chloramphenicol (Sigma) was also used at 10 times the MIC (25 ,ug/ml).Medium. The organisms were grown in M9 medium (16) without glucose or thymine). The strain was maintained on M9 medium solidified with 1.5% agar.Growth conditions. A sample (0.5 ml) of an overnight culture was inoculated into 100 ml of prewarmed M9 medium and grown with aeration at 35°C for at least five doublings before the start of an experiment. Viable counts were obtained by making serial 10-or 100-fold dilutions in M9 buffer and spreading 0.1-ml samples in triplicate on M9 agar plates with a sterile glass rod. After incubation for 48 h, the plates were cou...

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Section: Resultsmentioning

confidence: 99%

“…An interpretation consistent with the data is that the cells lose their capacity to maintain the proton motive force, possibly as a result of either a recAinduced process or the imbalance in membrane composition. Loss of the potential across the membrane is known to have profound effects on outer membrane permeability (9,25).…”

Section: Resultsmentioning

confidence: 99%

Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli

Dougherty¹,

Saukkonen²

1985

Antimicrob Agents Chemother

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“…Upon addition of CCCP to EDTA-treated cells preloaded with TMG, the cells rapidly lose all of their accumulated TMG (Fig. 4) (28), might simply allow CCCP to act as a sulfhydryl reagent on the TMG carrier protein (29,30). Yet Two recently received manuscripts bear on our findings and conclusions.…”

Section: Resultsmentioning

confidence: 99%

Reduction of membrane potential, an immediate effect of colicin K.

Weiss¹,

Luria²

1978

Proc. Natl. Acad. Sci. U.S.A.

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(2), and inhibition of several active transport systems (3-6). Uptake and respiration of glucose remain normal, as do retention of a-methyl glucoside and facilitated diffusion of flD-galactosides (2).Previous experiments have shown that some of the observed changes are secondary effects (7). The processes that seem to be directly affected are those that are presumed to be dependent on an "energized membrane state." According to Mitchell's chemiosmotic hypothesis (8,9), the energized membrane state is identified as an electrochemical gradient of protons or protonmotive force (Ap), composed of a concentration gradient (ApH) and an electrical gradient (A41). The value of Ap is given in mV by the formula Ap = A4' -6OApH at 27°. Ap is postulated to be the driving force that energizes, either directly or indirectly, a wide variety of metabolic processes, including motility and various active transport systems. The primary effect of colicin K is apparently to uncouple the energy produced by respiration or by ATP hydrolysis from energy-utilizing processes such as active transport and motility. Colicin K might act either by lowering the energized membrane state or by preventing the utilization of the energized state. This paper presents evidence that colicin K acting on E. coil cells causes rapid membrane depolarization as measured by the accumulation of the lipophilic cation triphenylmethylphosphonium (TPMP+) (10). This conclusion is consistent with previous findings of Brewer (11) and Gould and Cramer (12). From measurements of the uptake of a weak acid analyzed by the flow dialysis method of Ramos et al. (13), we conclude that colicin K has only a slight effect on the pH gradient.In the course of these experiments we noticed that both the commonly used pretreatment of bacterial cells with EDTA and the presence of the lipophilic ions tetraphenylboron and TPMP+ have unexpected effects on certain bacterial membrane functions, effects that must be considered in the interpretation of experimental results. Ramos et al. (13). A pH electrode and a reference electrode (Radiometer G222C and K4112) were inserted in the cell suspension and connected to a recording pH meter. The flow rate of the dialysis buffer was about 5.1 ml/min. Fractions were collected directly into scintillation vials and sampled for radioactivity. The fraction collector and recorder were wired so that a small blip was recorded on the pH trace at the beginning of every fifth fraction.The method used by Ramos et al. (13) to calculate the amount of weak acid taken up and hence the internal pH had to be modified because our experiments, involving lower pH gradients and using dilute cell suspensions to maintain aerobiosis, required more precise measurements of relatively small changes in substrate concentration. A computer program was therefore devised § that calculates and plots the internal and external concentrations and the pH gradient. The same program was also used for proline uptake and can be used for any substrate that is actively concentrate...

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“…This change is sensitive to the state of membrane energization of the cell. Thus, energization decreases fluorescence intensity and deenergization reverses this effect [2][3][4][5]. Recently, we have shown that the alteration in fluorescence intensity in Escherichia coli has two components [6].…”

Section: Introductionmentioning

confidence: 99%

The fluorescence intensity of the lipophilic probe N‐phenyl‐1‐naphthylamine responds to the oxidation‐reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

Sedgwick

Bragg

1988

FEBS Letters

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N-Phenyl-l-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified eytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.

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Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

Cited by 27 publications

References 45 publications

Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli

Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli

Reduction of membrane potential, an immediate effect of colicin K.

The fluorescence intensity of the lipophilic probe N‐phenyl‐1‐naphthylamine responds to the oxidation‐reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

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