Enrichment of Auxotrophic Mutants of Aspergillus flavus by Tritium Suicide

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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 μm, 3H-leucine was incorporated better than 3H-proline or 3H-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 3H-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 3H-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.

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