Characterization of an Intracellular Protease Isolated from <i>Bacillus thuringiensis</i> Sporulating Cells and Able to Modify Homologous RNA Polymerase

Abstract

The level of intracellular proteolytic activities was measured in Bacillus thuringiensis sporulating cells and it is shown that the maximum is reached after 4 h from onset of sporulation ( t 4 ). A purification procedure using cells harvested at t 5 is reported, which leads to the isolation of an intracellular protease totally different from the extracellular enzymes and from another intracellular fraction detected in crude extracts. The homogeneity of the purified enzyme was verified using immunological techniques, electrophoretic migration and kinetics of thermodenaturation. Its molecular weight was estimated to be about 23000. This enzyme was characterized as a seryl protease, totally inhibited by phenylmethylsulfonyl fluoride or EDTA and requiring Ca 2+ ions for activity. From the esterolytic activity of the enzyme we deduce a chymotrypsin‐like specificity. The pure protease was able to cleave specifically the β' subunit of the form II of B. thuringiensis RNA polymerase purified from t 5 sporulating cells. The vegetative holoenzyme and core enzyme appear much less susceptible to the action of the protease. Moreover the σ and α subunits do not seem to be altered by similar treatments. The existence of a mechanism regulating the level of intracellular proteases during the sporulation phase is postulated.