Induction at high frequency of a unique phenotypic class of Bacillus subtilis mutants by methylxanthines

Caffeine and theophylline are mutagenic at high concentration in the B. subtilis multigene sporulation test for mutagens; caffeine is a stronger mutagen than theophylline in this test. An unusually high fraction of the mutant colonies appear to be phenotypically identical, as judged by colonial morphology and microscopic appearance of the vegetative cells. These mutants do not bring about the pH increase normally associated with sporulation of B. subtilis; such behavior is frequently associated with lack of a functional tricarboxylic acid (TCA) cycle, essential for normal sporulation of this species. Similar mutants have not been noted in the course of screening a variety of well-known mutagens, including acriflavine. Caffeine is maximally effective in inducing these mutants about 10 min after germination commences. Adenosine greatly reduces the ability of caffeine to induce these mutants.