Comparison of butyric type of fermentation in sporogenic and asporogenic mutants of Clostridium botulinum.

Glucose-adapted cells of a sporogenic mutant. MSp(+), and an asporogenic mutant, RSpoIIIa, of Clostridium botulinum type E rapidly fermented glucose, fructose, maltose, and sucrose, resulting in cytoplasmic granulation, heavy growth, a pH of <6.0, and sporulation of the MSp(+) mutant ranging from 60 to 80%. In Trypticase peptone glucose broth, the MSp(+) mutant formed >80% refractile endospores in 25 h, whereas the RSpoIIIa mutant which was blocked at early forespore stage had commenced to lyse. Both mutants accumulated acetate and intracellular granules, reaching maximal levels at early stationary phase of growth. In MSp(+), as the levels of acetokinase, phosphotransacetylase, and butyryl-coenzyme A dehydrogenase reached a maximum, butyrate accumulation continued concurrently with an increase of endospore formation, whereas the levels of poly-beta-hydroxybutyrate decreased simultaneously with its precursor, acetate. Butyrate biosynthesis was blocked in the asporogenic mutant. As shown by isotopic assays, butyrate and acetate serve as precursors of spore lipids. beta-Phenethyl alcohol, fluoroacetic acid, and 2-picolinic acid inhibited anaerobic sporogenesis almost completely, butyrate biosynthesis by >87%, and acetate accumulation by 50 to 62%, showing a direct relationship between butyric type of fermentation and anaerobic sporulation.