Detection of mutagen-induced lesions in isolated DNA using a new Bacillus subtilis transformation-based assay

Abstract

A new mutagenesis assay based on the induction of mutations in isolated transforming DNA of Bacillus subtilis is described. The assay tests the ability of chemical to induce lesions in isolated DNA which lead to reversion of the ilvB2 base-substitution or leuA169 frameshift mutations which are carried by the recipient cells that are transformed with the treated DNA. This report demonstrates the capacity of the assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl- N′-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl- N-acetoxy-2-aminofluorene (AAAF). The later 4 chemicals were reacted with the test DNAs in a dimethyl formamide-containing buffer which was used both to maintain the denatured DNA in an extended single-stranded state and to increase the solubilities of the chemicals. The relative mutagenic activities of the compounds in this buffer system were PO < MNNG < AAAF < CAA. In addition to mutagenesis, the capacity of the test compounds to inactivate transforming activity and disrupt gene linkages in the treated DNAs also was assayed. PO, although a much weaker mutagen than CAA, produced more extensive disruption of gene linkage than CAA when compared at concentrations which produced comparable levels of inactivation of transforming activity. The new mutagenesis assay offers 2 advantages over previously described transformation-based assays. (1) In contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution type mutagens. (2) The mutants generated can be detected at high cell-plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.