The formation of endospores in bacteria is generally studied in media which do not support growth: the exhausted growth medium or a solution of mineral salts into which washed growing bacteria are transferred.", 2 Under carefully adjusted cultural conditions, a reasonably synchronous sporulation can thus be obtained in the majority of the population. There seems therefore to be no need, and even no possibility, for a change in the conventional methods used to study sporulation.An entirely new approach became available in 1961, however, when it was observed that spores are constantly being formed in cultures of Bacillus rnegateriuin growing exponentially in a mineral-glucose medium.3 Similar results have recently been obtained with B. subtilis.' Relative to the total viable population, the number of spores first reaches a limit value, and then increases with time at a constant rate, equal to the growth rate of the total population.3The present work investigates the influence of various factors on the size of the sporulated fraction (hereafter called p) in growing cultures of B. subtilis. The interpretation of the results is that the probability for a cell in a growth medium to become committed to sporulate" I must be determined by the intracellular concentration of at least one nitrogen-containing catabolite repressing, directly or indirectly, the expression of all the sporulation genes. A preliminary account of this work has been included in recent reviews;6 further evidence supporting the proposed interpretation will be published elsewhere.8' I Experimental Procedure.-The wild Marburg strain of Bacillus subtilis has been used. A common mineral base for all media was prepared from the following three solutions (all figures in gm/liter); solution I: K2HP04, 10.5; KH2PO4, 3.5; solution II: MIgSO4, 7H20, 5.0; FeSO4, 7H20, 0.5; solution III: CaCl2, 5.0; MnCl2, 4H20, 0.5; solutions I and III were autoclaved, solution II sterile-filtered. The base medium is made of 100 vol of solution I for 1 vol each of solutions II and III. Various substances are then added as carbon or as nitrogen source; the complete medium is thus fully described with the mention of the nature of each source, followed, in gm/liter, by its concentration [e.g., NH4Cl(0.5)-glucose (2)].

Summary. This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.

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The formation of cytoplasmic inclusion bodies by Escherichia coli overproducing Clostridium thermocellum endoglucanase D (EGD) was investigated. EGD was found in inclusion bodies as a 68 kDa form, whereas the size of the cytoplasmic form was 65 kDa. Upon solubilization with urea followed by dialysis, the 68 kDa form was converted to the 65 kDa species. Proteolysis occurred within the COOH‐terminal, reiterated region of the 68 kDa form, which is conserved among most C. thermocellum endoglucanase, but is not required for catalytic activity. The specific activity of the enzyme embedded in inclusion bodies was close to that of the purified protein. Thus, inclusion body formation does not involve denaturation of the catalytic domain of EGD, but more likely, the participation of the reiterated, conserved region in intermolecular interactions.