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.

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A comparison was made of morphological changes and successive, mainly biochemical, marker events for sporulation in 14 asporogenous mutants. The morphological and biochemical sequences are linked so that arrested development in one is accompanied by corresponding effects in the other. Thus mutants that fail to produce both protease and antibiotic do not progress beyond stage 0, formation of alkaline phosphatase appears to be associated with the transition from stage II to stage III and glucose dehydrogenase with that from stage III to stage IV. Stage II mutants may produce ;pygmy' cells or other bizarre cell-division forms. The biochemical sequence is dependent in the sense that if the occurrence of any one event is blocked that of all the succeeding events is also blocked. This has implications for biochemical models that have been proposed to explain the temporal sequence observed in spore development.

A spollA : : lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. P-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known sp00 loci but on none of the 'later' loci tested. The P-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage I1 mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spoOJ, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.

Analysis of mature spores or their integuments by extraction with sodium dodecyl sulphate/dithioerythritol followed by electrophoresis shows that the coat contains four major proteins and about ten others. Nine offie 14 proteins begin to be synthesized in stage I1 or stage I11 and their synthesis must be controlled by stage I1 or stage I11 operons. Some of these proteins are incorporated into the spore structure from about t, onwards (i.e. 4 h after induction of sporulation). Their deposition in the coat between t, and t , is not stopped by chloramphenicol, with the exception of one protein (mol. wt 36000) which begins to be synthesized only at t,. Spores were isolated at various stages from about t, onwards, and the surface proteins were labelled with 1251. The labelling patterns show that proteins which are exposed on the surface at t5.3 are successively overlaid as the spores mature. It appears that the coat of the mature spore contains at least three layers. The outermost layer is mainly composed of an alkali-soluble protein (mol. wt 12000), which is synthesized early (t2), and the 36000 mol. wt protein, which is synthesized very late. Deposition of the former seems to require processing by proteolytic action, and both proteins are apparently necessary for the acquisition of resistance to lysozyme, though not for resistance to heat or organic solvents. The results are discussed in relation to the classification of sporulation events and the nomenclature of the genetic loci controlling sporulation.? Present address: is described. The results suggest that the times of synthesis of the coat proteins are determined by an order of gene expression beginning at stage I1 but that their incorporation into the structure of the spore does not necessarily follow the same order. M E T H O D SChemicals. Acrylamide (purified) and N,N'-methylenebisacrylamide were obtained from BDH. Radioactively labelled compounds were purchased from The Radiochemical Centre, Amersham. All other chemicals were from Sigma unless otherwise specified.Bacteria. Bacillus subtilis 168 trpC2, which requires indole or tryptophan and sporulates normally, was used. Stocks were kept at 4 "C as dilute spore suspensions in distilled water.induction of sporulation. Cells in the exponential phase of growth in a hydrolysed casein medium at 37 OC were harvested by centrifugation when the culture contained about 0.25 mg dry wt bacteria m1-I. The pellet was resuspended to the same density in warm resuspension medium containing glutamate, inorganic salts and 20 pg tryptophan ml-I (Sterlini & Mandelstam. 1969) and incubated with shaking. About 80% of the cells contained refractile spores at 8 h after resuspension. The time of resuspension is denoted to and subsequent times (h) as t , , t,, etc.Determination of spore incidence. Spores were counted by phase-contrast microscopy and expressed as a percentage of the total number of cells. The term 'phase-bright' describes all gradations from dull white to bright spores; earlier visible sporal inclusions are termed da...