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The composition and application of a single, chemically defined medium for growth and sporulation of Bacillus subtilis is described. At 37°C cells grew with a doubling time of about 40 min; cultures attained nearmaximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 x 109 to 2 x 109 heat-resistent free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.

Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium iondependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose-and Ca2+-dependent manner and stimulated NAD kinase from peas in a dosedependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzymelinked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pl of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.

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