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Derepression of Anthranilate Synthase in Purified Minicells of Escherichia coli Containing the Col-trp Plasmid

^a Biology Division, Oak Ridge National Laboratory and University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830

ABSTRACT

Purified minicells of Escherichia coli K-12 containing the plasmid Col-trp⁺ or Col-trpA2 could be derepressed for the synthesis of anthranilate synthase, the first enzyme encoded in the trp operon. Non-plasmid-containing, deoxyribonucleic acid-deficient minicells could not be derepressed. Derepressed enzyme synthesis was initiated by L-tryptophan starvation. The kinetics of derepression were studied with minicells containing the Col-trpA2 plasmid. The derepression curves were biphasic with a rapid initial rate of enzyme synthesis followed by a slower rate of synthesis. The presence of L-tryptophan (20 to 50 µg/ml) or chloramphenicol (200 µg/ml) abolished enzyme synthesis. The presence of rifamycin SV (280 µg/ml) partially inhibited enzyme synthesis after at least 3.5 min of exposure. The ratio of minicell-to-cell synthetic capacity was 1:2.4 when compared on the basis of derepressed enzyme activity per unit cell volume. This work demonstrates that plasmid-containing minicells are capable of considerable functional protein and messenger ribonucleic acid synthesis and that the regulation of at least the trp operon is similar in minicells to that observed in cells.

¹ Present address: Department of Microbiology, University of Alabama in Birmingham, University Station, Birmingham, Ala. 35294.

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