Effect of mutation, electric membrane potential, and metabolic inhibitors on the accessibility of nucleic acids to ethidium bromide in Escherichia coli cells

Abstract

The uptake of ethidium bromide by Escherichia coli K 12 cells has been studied by using 14C-labeled ethidium and spectrofluorometry on three E. coli strains: the first one (AB1157) has an ethidium-resistant phenotype; the second one derives from the first one after a single mutation (at 10 min on the E. coli genetic map) and has an ethidium-sensitive (Ebs) phenotype; the third one is the acrA strain which appeared to have the same phenotype as the Ebs strain. When the cells are in exponential growth, no ethidium enters wild-type cells, and a very limited amount of ethidium enters Ebs and acrA cells. Massive quantities of ethidium enter AB1157, Ebs, and acrA cells treated by uncouplers and respiring Ebs cells treated by the membrane ATPase-inhibitor dicyclohexylcarbodiimide. A small amount of ethidium enters cells treated in M9 succinate medium by metabolic inhibitors such as KCN or cells starved with oxygen in the same M9 medium. The amount of ethidium and ethidium dimer retained at equilibrium by either type of cell, and by cells infected by T5 phage, as well as the kinetics of influx and efflux, has been measured under a variety of situations (membrane energized or not, and/or membrane ATPase inhibited or not). Furthermore, it was shown that ethidium binds to both RNA and DNA when it enters CCCP-treated wild-type E. coli cells, whereas it binds mainly to DNA when it enters Ebs and acrA cells in exponential growth. As it will be discussed, it is difficult to account for the EthBr uptake by invoking only membrane functions and active transport. Therefore, it is proposed that the variations of the nucleic acid accessibility in E. coli cells might play a role in the control of this uptake. Accordingly, in ethidium-sensitive cells, the mutation would have caused a significant part of the chromosomal DNA (10-20%) to become accessible to ethidium. Hansen [Hansen M. T. (1982) Mutat. Res. 106, 209-216], after a study of the photobinding of psoralen to nucleic acids in the acrA mutant, also suggested that DNA environment was modified in acrA cells.

Bioenergetics of the Staphylococcal Multidrug Export Protein QacA

“…Verapamil and TMA-DPH were solubilized in 50% and 100% methanol, respectively. CCCP was prepared as a solution in 1 mg/ml ethanol and then diluted to 0.1 mg/ml in 10 mM NaOH as described previously (22). All other chemicals were prepared as stock solutions of 1 or 10 mg/ml in water, depending on solubility, immediately prior to use.…”

“…These substrates bind to specific cellular components and in so doing undergo a change in fluorescence that can be used to indicate how much of the substrate is retained by the cell, e.g. ethidium and DAPI bind to doublestranded DNA resulting in a substantial increase in fluorescence (22,23), DiOC 3 , when bound to the cytoplasmic side of hyperpolarized membranes, is highly fluorescent (24), and pyronin Y binds to RNA resulting in fluorescence quenching (25). The S. aureus strain SK982, carrying the qacA-encoding plasmid pSK1 or the qacB-encoding plasmid pSK23, was grown for 16 h in 10 ml of LB (with selection) from which 1 ml was subcultured into 10 ml of fresh LB and grown to OD 650 ϭ 0.85 (ϳ2.5 h).…”

“…To determine if QacA and QacB interact specifically with structurally dissimilar substrates, kinetic analyses of transport were performed using various fluorescent substrates. These kinetic analyses, undertaken in whole cells, were based on the assumptions that after loading the cells in the presence of CCCP, the intracellular substrate concentration is effectively equivalent to the extracellular concentration, and that 14 C]ethidium produced identical results (22). Consistent with these assumptions was the observation that the amount of the fluorescent substrate in loaded cells was proportional to the concentration used.…”

Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis

“…Ethidium displays approximately a 10-fold increase in fluorescence quantum yield upon binding to DNA or RNA (19). This property was used to monitor indirectly the intracellular ethidium concentration (18,24). A washed cell suspension (0.2 mg of protein per ml) was incubated for 10 min with 10 ,uM ethidium bromide at 30°C to allow the ethidium to diffuse across the membrane.…”

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

“…We were not able to reliably assay downhill transport of MPP ϩ in proteoliposomes because of the relatively high passive permeability of the substrate. We therefore adapted a whole-cell ethidium uptake system used previously with other transporters (40,45). This assay enabled us to monitor both downhill passive transport driven by the ethidium gradient as well as uphill active efflux driven by the proton gradient.…”