Forward mutation assay for screening carcinogens by alkaline phosphatase constitutive mutations in Escherichia coli K-12.

A new forward mutation assay was developed with Escherichia coli using alkaline phosphatase (APase) constitutive mutations as a genetic marker. Mutation in any one of the three regulator genes (phoR, T and S) is known to make the cell constitutive for APase synthesis and enable the mutants to form larger colonies on beta-glycerophosphate plate under the condition of excess inorganic phosphate. This property was used for qualitative and quantitative assay of chemical mutagens. Attempts were made to construct suitable strains for this assay by introduction of various genetic traits that might increase the sensitivity of mutation. Three known chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), and 4-nitroquinoline-1-oxide (NQNO)) were employed as reference compounds in the quantitative assay. Among the strains constructed, a tester strain with genetic markers tif-1, uvrA and pKM101 was the most sensitive to these compounds, judging from tests on concentration-dependent mutagenic activity. The merits and limitations of the present system are discussed.