Extracellular Ribonuclease Formation in Bacillus Subtilis and Its Stimulation by Actinomycin D.

1. Extracellular ribonuclease is produced linearly for at least 3hr. by washed post-logarithmic-phase cells of Bacillus subtilis suspended in a medium containing maltose (1%) and casein hydrolysate (0.5%). 2. Low concentrations of actinomycin D (less than 2mug./ml.) stimulate ribonuclease formation, the maximum effect being observed with a concentration of 1mug./ml. Concentrations greater than 2mug./ml. are inhibitory. There is no parallel stimulation of alpha-amylase formed under the same conditions, and [(14)C]uracil incorporation into a perchloric acid-insoluble form is inhibited. 3. The actinomycin D-induced stimulation is not due to the presence of an activator, nor is the inhibition due to the release of an inhibitor by the cells. The effect is on the amount of ribonuclease produced in the medium. 4. Extracellular ribonuclease formation is partially inhibited by anaerobiosis, 2,4-dinitrophenol, sodium azide and by chloramphenicol and puromycin. 5. High concentrations of antibiotic do not completely inhibit ribonuclease formation, but a basal amount of enzyme representing 20min. synthesis in an uninhibited system is always produced. This ;antibiotic-insensitive' enzyme could possibly represent preformed enzyme ;in the pipe-line' en route to secretion. 6. The stimulated appearance of ribonuclease in the presence of 1mug. of actinomycin D/ml. is shown to be dependent on enzyme synthesis. The mechanism of this effect is discussed.