Use of constructed double mutants for determining the temporal order of expression of sporulation genes in Bacillus subtilis.

Double mutants containing two Spo mutations concerned with different stages of sporulation were constructed. In these, the phenotype that is exhibited is that of the earlier sporulation block. The same procedure was applied to sporulation mutants damaged in the same stage of development. The results provide a basis for placing in a temporal order different mutations concerned in stage II and stage IV of spore development. In general, the order indicated by the phenotypes of the double mutants is in agreement with the order derived on biochemical grounds. Double oligosporogenous mutants have also been constructed. Their sporulation incidence is roughly equal to the product of the incidences of the parent strains, idicating that separate factors are involved in overcoming each oligosporogenous block. The number of dependent sequential steps in sporulation is estimated as not less than about 12.