Inhibition of in vivo transcription by actinomycin D treatment of ethylenediaminetetraacetic acid-permeabilized Escherichia coli: effects on bacteriophage proliferation.
Rifampicin, which is able to block DNA-dependent RNA synthesis, has been widely used to selectively inhibit host protein synthesis in RNA bacteriophage-infected Escherichia coli without affecting the viral specific protein synthesis. However, in many cases it is necessary to increase rifampicin levels to 200 micrograms/ml in order to obtain an almost complete suppression of bacterial protein synthesis, and these high antibiotic concentrations cause at the same time a strong inhibition of phage proliferation resulting in a 50- to 100-fold reduction of phage yields. We have partially avoided this difficulty by using actinomycin D after permeabilization of bacteria by a brief incubation with EDTA. To optimize the method the effects of changing EDTA and actinomycin concentrations as well as the duration of the permeabilization period have been studied. With this procedure it has been possible to shut off bacterial RNA and protein synthesis with phage yields about 10 times higher than those observed in the presence of high levels of rifampicin. The usefulness of the described method is particularly evident when working with rifampicin-resistant strains of E. coli.
Fastame
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Instituto de Investigaciones Bioquímicas Fundación Campomar, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Algranati
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ID
D003090:Coliphages / Q000187:drug effects* / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects*; D012293:Rifampin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects
D003609:Dactinomycin; D004492:Edetic Acid; D012293:Rifampin