Cloning and Sequencing of the Major Intracellular Serine Protease Gene of Bacillus Subtilis

Abstract

A Bacillus subtlis 2.7-kilobase DNA fragment containing an intracellular protease gene was cloned into Escherichia coli. The transformants produced an intracellular protease of approximately 35,000 Mr whose activity was inhibited by both phenylmethylsulfonyl fluoride and EDTA. Introduction of the fragment on a multicopy vector, pUBllO, into B. subtilis caused a marked increase in the level of the intracellular protease. The nucleotide sequence of the cloned fragment showed the presence of an open reading frame for a possible proenzyme of the major intracellular serine protease (ISP-I) of B. subtilis with an NH2-terminal 17-or 20-amino-acid extension. The total amino acid sequence of the protease deduced from the nucleotide sequence showed considerable homology with that of an extracellular serine protease, subtilisin. The transcriptional initiation site of the ISP-I gene was identified by nuclease Si mapping. No typical conserved sequence for promoters was found upstream of the open reading frame. An ISP-I-negative mutant of B. subtilis was constructed by integration of artificially deleted gene into the chromosome. The mutant sporulated normally in a nutritionally rich medium but showed decreased sporulation in a synthetic medium. The chloramphenicol resistance determinant of a plasmid integrated at the ISP-I locus was mapped by PBS1 transduction and was found to be closely linked to metC (99.5%).Bacillus subtilis produces a variety of extracellular and intracellular proteases. An alkaline serine protease (subtilisin), a neutral metalloprotease, and an esterase are secreted into media, whereas at least two intracellular serine proteases are produced within B. subtilis cells (7,22,24,34,35). The major intracellular serine protease here designated as ISP-I is characterized by its sensitivity to EDTA due to an absolute requirement for Ca2+ for stability and activity (35). The partial NH2-terminal amino acid sequence of ISP-I shows considerable homology with that of subtilisin (35). A minor serine protease (ISP-II), possessing trypsin-like substrate specificity, has also been found in B. subtilis cells (29,34).Essential roles have been postulated for these intracellular proteases during sporulation, possibly through the turnover of intracellular proteins (13, 33), the processing of spore coat protein precursors (15,16,29), and the inactivation of ornithine transcarbamylase and several other enzymes (17, 21). Involvement of ISP-I in sporulation was suggested from observations that a thermosensitive sporulation mutant of B. subtilis lost both its intracellular protease activity and sporulating ability at the restrictive temperature (10). Hageman and Carlton also isolated an ISP-I-deficient mutant which failed to sporulate normally (5). On the other hand, a mutant with temperature-sensitive ISP-II was shown to have several altered functions at the postexponential growth phase and to produce spores deficient in a major lowmolecular-weight coat polypeptide at the restrictive temperature (29).In this paper we describe th...