Detection of mutagen-induced lesions in isolated DNA by marker rescue of Bacillus subtilis phage φ105

Abstract

A new mutagenesis assay is described which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative from DNA of the temperate Bacillus subtilis phage φ105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. There is a cluster of such genes tightly linked to the φ105 genetic marker Jsus11 which restricts the host range of the phage to cells capable of suppressing sus mutations. In the actual assay chemically treated DNA, from wild-type J + phage, is added to competent cells which are infected with φ105 Jsus11. Wild-type phage, capable of producing plaques on cells which are nonpermissive for φ105 Jsus11, are produced by recombination between the added chemically-treated DNA and infecting φ105 Jsus11 DNA. If the added DNA also carried mutagenic lesions in any of the genes controlling lysogeny, clear plaque mutants are produced which are readily distinguishable from the turbid plaquing wild-type phage. This report demonstrates the capacity of this marker rescue-based assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl- N′-nitro- N-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl- N-acetoxy-2-amino-fluorene (AAAF). The effect of using a host cell, defective for excision repair, on the sensitivity with which the assay detected the mutagenic activities of CAA, PO and AAAF also was examined. The new mutagenesis assay offers 2 advantages over several other 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 and (2) the mutants generated can be detected at high 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.