Replacement sporulation of Bacillus subtilis 168 in a chemically defined medium

A replacement sporulation technique (i.e., the sporulation of vegetative cells upon suspension in an appropriate medium) has been developed for Bacillus subtilis 168 (a transformable Marburg strain of B. subtilis). The replacement sporulation medium used is composed of inorganic salts and 10 mm ammonium lactate or glutamate. The requirement for ammonium lactate or glutamate could also be satisfied by other compounds that are metabolized via the tricarboxylic acid cycle. Sporulation of the suspended vegetative cells was completed by 8 to 10 hr after suspension, and the resulting spores were indistinguishable from spores produced in a conventional growth and sporulation medium. Various physiological changes previously reported to be associated with sporulation (e.g., increase in the level of tricarboxylic acid cycle enzymes and changes in the rates of synthesis of deoxyribonucleic acid, ribonucleic acid, and protein) could also be demonstrated during replacement sporulation.