Escherichia coli Mutants Thermosensitive for Deoxyribonucleic Acid Gyrase Subunit A: Effects on Deoxyribonucleic Acid Replication, Transcription, and Bacteriophage Growth

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Abstract
Temperature-sensitive nalA mutants of E. coli were used to investigate structure and functions of DNA gyrase. Extracts of 1 such mutant (nalA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a 2nd nalA (Ts) mutant (nalA45) had a 50-fold deficiency of gyrase subunit A activity. Residual DNA super-twisting was catalyzed by the mutant DNA gyrase rather than by a novel super-twisting enzyme. The nalA45(Ts) extract was deficient in the nalidixic acid target, which is the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Gyrase subunit A and the nalidixic acid target are the same protein, the nalA gene product. Shift of the nalA43(Ts) mutant to a non-permissive temperature resulted in precipitous decline in rate of [3H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. Rates of incorporation of [3H]uridine pulses and continuously administered [3H]uracil were quickly reduced approximately 2-fold upon temperature shift of the nalA43(Ts) mutant and some but not all transcription requires the nalA gene product. Thermosensitive growth of bacteriophages .vphi.X174 and T4 in the nalA43(Ts) host shows that these phages depend on the host nalA gene product. Phage T7 growth was strongly inhibited by nalidixic acid but essentially unaffected by nalA43(Ts) mutation. Inhibition of T7 growth by nalidixic acid was eliminated by temperature inactivation of the nalA43 gene product. Nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered and relevance to the puzzling dominance of drug sensitivity is discussed.