Combined mutagenesis by UV light and nitrosomethylurea of Streptomyces hygroscopicus spores

Abstract

By means of combined treatment with UV light and nitrosomethylurea (NMU) the spores of Streptomyces hygroscopicus were mutagenized. The rate of the colony-forming survivals exhibited two phases depending on the duration of NMU treatment.The slope of the first phase is interpreted as the result of alkylation of purin bases without repair functions. The slope of the second phase is discussed on the basis of a different content of plasmid DNA within the cells.Following the mutagenic treatment the growth rate of the colonies diminished. This effect is quantitatively measured by the aid of an automatic picture analyzer. The size distribution of the colony diameters showed two peaks in the control indicating that the Streptomyces spores grew up with one or two mycelium tubes resulting in two different growth rates.With increasing duration of NMU treatment the colonies with high diameters disappeared in favour of colonies with small diameters.By mathematical comparison it could be shown that a cooperative effect exists between the action of UV light and NMU on DNA. The spores containing DNA damages as a result of cooperative effects give rise to a distinct class of colony size. With the aid of an automatic picture analyzer it will be possible to select very effectively the desired mutants.The formation of auxotrophic mutants by mutagenesis of Streptomycetes is usually very ineffective because these strains grow as mycelium and as a result the initial mutated sites within the bacterial DNA fail to segregate into separate individuals. The mutative alterations produced within one DNA strand yield mutated daughter helices after replication. However, these remain together within the mycelium and most of the mutations produced by mutagenesis are not expressed phenotypically due to the dominance of most of the wild type alleles. From this point of view, stable mutants can be expected if either two DNA lesions are produced in both strands within the same codon or anticodon respectively, or two DNA lesions produced a t both strands lie within the same structural gene, resulting in the synthesis of defective gene products by both daughter helices after replication of the mutated double-stranded DNA. I n the latter case a postreplicative repair process can give rise to prototrophic recombinants between the daughter chromosomes.The aim of the investigations presented here was the production of auxotrophic mutants of the bacterial strain Streptomyces hygroscopicus by combined treatment with a physical and a chemical mutagen and the qualitative estimation of the mutation rate with the automatic picture analyzer "Quantiniet 720 M".The mutagenesis was carried out by combined treatment with UV light and N-nitroso-N-methylurea (NMU 14'