1. Experiments to determine the point of commitment to sporulation were carried out by restoring nutrients at different times to suspensions of sporulating Bacillus subtilis. 2. No single point of commitment to the process as a whole was found. Instead, the cells became committed in turn to the following successive events connected with sporulation: formation of alkaline phosphatase, development of refractility, synthesis of dipicolinic acid and development of heat-resistance. 3. Each point of commitment was followed within about 30min. by a period in which the event concerned ceased to be inhibited by actinomycin D. 4. The implication of these results is that each point of commitment is probably due to the formation of a species of long-lived messenger RNA and that, in any case, sporulation is regulated at the level of both transcription and translation. 5. It is also shown that sporulation and growth are perhaps not mutually exclusive functions and that histidase, an enzyme typical of the vegetative state, can be induced in sporulating suspensions.
Thymine-requiring mutants of Bacillus subtilis and mutants that are temperature-sensitive for initiation of chromosome replication have been used to study the relationship between sporulation and chromosome formation. The DNA synthesis that normally occurs when cells are transferred to sporulation medium is essential for spore induction. This is shown by the fact that thymine-starved cells are unable to form spores and are unable to perform even the earlier steps of sporulation, such as septum formation or synthesis of alkaline phosphatase. The nature of the medium in which the cells are growing while the DNA is being completed is also important because it determines both the shape and the position of the daughter chromosomes. If the cells are in a rich medium, the newly synthesized chromosomes are discrete and compact bodies: the cells are primed for growth, and sporulation cannot be induced by transferring them at this stage to a spore-inducing medium. If DNA synthesis was completed with the cells in a poor medium the daughter chromosomes, by the time DNA synthesis has ceased, are spread in a single filamentous band and the cells are morphologically already in stage I of sporulation.
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