Studies on the activation of extracellular proteases in Bacillus subtilis

Abstract

The gram-positive, spore-forming bacterium, Bacillus subtilis is known to secrete at least eight extracellular proteases at the end of the exponential phase of growth. Expression of these proteolytic enzymes seems to be tightly regulated and their major function is thought to be the supply of nutrients by degrading extracellular proteins. Most bacterial proteases are synthesized as inactive precursors, or zymogens, to avoid unwanted protein degradation and to regulate proteolytic activity. Every extracellular protease in B. subtilis is synthesized as a preproenzyme in the cytoplasm and is processed to a mature enzyme in the extracellular milieu. The zymogen form of subtilisin is converted to an active form by auto-processing. The processing mechanisms of the most extracellular proteases in B. subtilis have been uncovered. Extracellular metalloprotease (Mpr) protein might be not able to activate itself. Reportedly, Mpr has very specific substrate specificity, and recognizes a glutamic acid residue as a P1 cleavage site. However a glutamic acid (Glu) residue is not present in the Mpr propeptide cleavage site. For the elucidation of the mechanism of Mpr activation, I tried to construct SB300 (nprE aprE epr) and PB300 (nprE aprE bpr) strains by disruption of epr or bpr gene in B. subtilis DB104 (nprE aprE) each, and overexpressed mpr gene. I observed that when Mpr is overexpressed, its activity is detectable in B. subtilis DB104 (aprE nprE), which lacks subtilisin and neutral protease, but is absent in strains lacking additional proteases. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blot analysis revealed that the extracellular protease Bpr is required for Mpr processing. Pro-Mpr remained as a precursor form in bpr-deficient strains, and glutarnic acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Moreover, I confirmed that Bpr activates Mpr by the propeptide processing ...