Germination properties as marker events characterizing later stages of Bacillus subtilis spore formation

At various stages during spore formation sporangia were shocked by cold treatment or with toluene, and the germination requirements of the prespores were examined. Up to 5 h after induction of sporulation (t5) germination was spontaneous; i.e., it occurred without any added germinants. After t5, during stages V and VI, the capacity for spontaneous germination diminished progressively, and the spores acquired a need for externally added germinants. At t6 this need was satisfied by either L-alanine or a mixture of KCl, glucose, and fructose. By t8, the latter response had disappeared. The spores germinated only with L-alanine, and the response was much slower. Experiments with chloramphenicol showed that the germination properties of the spores appearing between t6 and t8 were the expression of events in protein synthesis that had occurred before t5. Although the germination requirements developed at about the same time as heat resistance, they could be dissociated from heat resistance in wild-type and mutant cells. The germination properties of the developing spores are additional marker events characterizing the later stages of sporulation, as follows: (i) spontaneous germination (up to the end of stage IV); (ii) germination requirements that are satisfied by KCl-glucose-fructose or L-alanine (stage V); and (iii) slow germination response with L-alanine only (stage VI).