Postlogarithmic Phase Metabolism of Sporulating Microorganisms I. PROTEASE OF BACILLUS LICHli’NIFORMIX*

Extracts and culture supernatant solutions of Bacillus Zicheniformis contain considerable quantit.ies of a protease, shown by Bernlohr and Novelli (I) to appear in the extracellular medium only in the postlogarithmic phase of growth. From genetic studies on sporulation in Bacilli, Spizizen, Reilly, and Dahl (2) report a strong association between a protease, formed in the postlogarithmic growth phase, and the sporulation process. Asporogenous mutants of Bacillus subtilis, 168, unable to synthesize protease, could be transformed with deoxyribonucleic acid isolated from sporogenous strains with the production of sporogenous organisms cont,aining the protease gene. The purpose of this investigation was to characterize the B. licheniformis enzyme to determine whether its activity is consistent with a possible funct,ion in the sporulative events of the cell that produces it. The pH and temperature optima were determined, and the influence of some inorganic ions on the activity of the protease was tested. Finally, the products of the enzyme reaction were briefly examined by measuring t.he quantity of free amino acid released by its action. An antiserum to the purified protease was prepared. Although a precipitation complex was first formed between the enzyme and the antiserum, the protease readily hydrolyzed the serum component of the complex within minut,es, resulting in a rapid solubilization and release of the protease activity. However, on agar diffusion plates the complex was more stable, allowing the determination of the time in the life cycle of B. Zicheniformis when the molecule is actually synthesized. This was of interest because t,he enzyme appears in the culture supernatant solution during the postlogarithmic phase of growth.