Enrichment of auxotrophic mutants of Aspergillus flavus by tritium suicide.

A method based on tritium suicide was developed to enrich auxotrophic mutants of Aspergillus flavus. N-methyl-N'-nitro-N-nitrosoguanidine (NG) was chosen as a mutagen, since a wide variety of mutations were induced by the action of 0.1% NG on A. flavus conidia suspended in phosphate buffer (pH 7.0). The decimal reduction time under these conditions was about 30 min, and the surviving population contained 4 to 6% auxotrophs after 1 hr of mutagenesis. This proportion was then increased by tritium suicide of wild-type cells. At a concentration of 1.3 mum, (3)H-leucine was incorporated better than (3)H-proline or (3)H-thymidine into the germinating conidia. With about 20 hr of incubation and a short treatment in a high-speed mixer to disentangle mycelia and conidia, a 5- to 20-fold decrease in the number of survivors resulted from the incorporated (3)H-leucine (5 c/mmole) after 1 week of storage at 5 C. At a 10-fold lower concentration, the uptake of radioactivity and the subsequent suicide rate were much lower. With (3)H-leucine, the proportion of auxotrophs in the surviving population rose from 5 to about 20% during 2 weeks of storage at 5 C. Mutants requiring various intermediates for protein or nucleic acid synthesis or requiring vitamins were isolated. Finally, it was noted that A. flavus shows a much higher resistance to tritium suicide than does Escherichia coli.