Genetical and physiological studies in Escherichia coli K-12 using clorobiocin, an inhibitor of DNA gyrase.

Escherichia coli, in common with other bacteria studied, contains an enzyme termed DNA gyrase which introduces negative supercolis into closed circular DNA. DNA gyrase is inhibited by novobiocin and coumermycin, two structurally related antibiotics. In this study I have clorobiocin, an antibiotic structurally similar to novobiocin, as an inhibitor of DNA gyrase. Mutants of E. coli K-12, including those exhibiting a conditional lethal phenotype, were isolated as resistant to clorobiocin and the mutation responsible was mapped at gyrB (cou), the gene coding for the b subunit of DNA gyrase. The gene order was concluded to be: dgoD, gyrB(cou), dnaA, tna, ilv. One resistant mutant was studied in detail, and was shown to have a reduced DNA concentration. This defect could not be accounted for by a reduced velocity of replication, and it was concluded that the initiation of DNA replication must be delayed in this strain. An examination of the mode of action of clorobiocin demonstrated that this antibiotic caused an immediate inhibition of DNA synthesis and cell division in exponential cultures of E. coli; RNA and protein synthesis were affected to a lesser extent. The rate of synthesis of total outer membrane protein relative to that of the inner membrane fell immediately upon clorobiocin treatment, although the rates of synthesis of individual outer membrane proteins varied widely during the course of antibiotic treatment. The role of DNA gyrase in control of DNA replication, transcription and cell division is discussed.