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Biochemistry. 1984 Jan 3;23(1):166-76.

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.

PMID:
6197994
DOI:
10.1021/bi00296a027
[Indexed for MEDLINE]

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