A stage 4 sporulation mutant of a strain of Bacillus cereus var. alesti fails to synthesize a cortex although all other structural components appear normal. With terminal lysis the spore core as well as the sporangium is lysed. Both the uptake of 45 Ca and the synthesis of dipicolinic acid (DPA) are similar to these activities in the parent strain, but these components (DPA and Ca) are lost to the medium with the drastic lysis. The first stage of diaminopimelic acid incorporation, that into germ cell wall mucopeptide, is intact in the mutant; the second stage, that into cortical mucopeptide, is absent. These biochemical studies as well as phospholipid metabolism and freeze-etch analysis suggest the lesion lies in the outer forespore membrane.

The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate. These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label. Mannitol-1-phosphate occurs in E. coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase. The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s. These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.

Previous studies on this cortexless mutant of Bacillus cereus var. alesti indicated that the forespore membrane was the site of the biochemical lesion. This hypothesis is supported by the results presented here: fatty acid composition of sporulating cells of themutant is altered, while in vegetative cells it is comparable to the parent; soluble precursors of peptidoglycan synthesis are accumulated in the mutant, at the time of cortex formation; homogenates of the mutant prepared at the time of cortex formation are unable to incorporate tritiated diaminopimelic acid into peptidoglycan, while homogenates of cells forming germ cell wall do so to an extent comparable to that of the parent; lipid-linked intermediates are formed by the mutant as in the parent. Apparently the mutant is unable either to transfer disaccharide penta-peptide units from the carrier lipid to the growing peptidoglycan acceptor, or to transport lipid-linked intermediates across the forespore membrane.

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)I, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. 4"Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of "Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.

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