Directed mutation in Streptomyces lipmanii

Streptomyces lipmanii produces two β-lactam antibiotics, penicillin N and 7-(5-carboxyvaleramido)-7-methoxycephalosporanic acid. A method has been developed whereby mutation can be directed toward preselected markers, including some affecting antibiotic production. DNA synthesis was synchronized by amino acid starvation of a leucine auxotroph (LA 473). Double-isotope techniques using [14C]- and [3H]-thymidine provided a means of monitoring DNA synthesis. Aliquots of the synchronized culture were treated with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and plated on selective media. Mutation frequencies for each marker varied as much as 100-fold. The data were consistent with the hypothesis that NTG preferentially mutates the replicating region of DNA.