Expression of the major intracellular serine protease (ISP-1) gene of Bacillus subtilis was studied by using a translational fusion plasmid in which the isp promoter regon was fused to the lacZ gene. ,-Galactosidase activity, used to measure transcription from the isp promoter, was produced immediately after the end of exponential growth, whereas intracellular protease activity was not detected until 4 h later. These results are consistent with a previous suggestion that ISP-1 initially accumulates in the cell in an enzymatically inactive form. ISP-1 activity was detected in all of the sporulation-deficient strains examined, and the amount of protease activity always corresponded to the amount of ,B-galactosidase activity. These results indicate that the activation of ISP-1 is not dependent on a sporulation-specific gene product. Expression of ISP-1 is regulated by a number of mutations known to affect the expression of extracellular enzymes. In sacU(h) and sacQ(h) mutants, the expression of ISP-1 was 10-fold higher than in the wild-type strain. In catA, hpr, and scoC strains, expression of ISP was stimulated two-to threefold, whereas in sacU mutants the expression of ISP-1 was reduced to less than 10% of the wild-type level. The temporal expression and activation of ISP-1 was not affected by any of these mutations. This is the first evidence that the expression of a native intracellular protein is affected by these hyperproduction mutations.Soon after the initiation of sporulation, Bacillus subtilis synthesizes an intracellular serine protease (ISP-1) (3,16). The isp gene has been cloned, and studies have shown that ISP-1 is not essential for normal sporulation (2, 10). The exact role of this enzyme in sporulation has not been established, and little is known about the regulation of ISP-1 expression and control of protease activity. It has been suggested that ISP-1 activity might be regulated by interaction with a proteinaceous inhibitor, calcium availability, or activation by membrane-bound proteases (12,13,19). A recent report presented immunological evidence that ISP-1 was initially synthesized as an enzymatically inactive protein that was subsequently converted to an active protease (3).Several mutations affecting the production of extracellular enzymes in B. subtilis have been described. Mutations in the sacU and sacQ genes are pleiotropic in nature, affecting the prpduction of many extracellular enzymes and some sporulation-related events (11,22). Other mutations (catA, hpr, ScoC), although from independently isolated mutants, may be mutations of the same gene. These mutations affect production of the extracellular proteases and catabolite repression of sporulation eyents (5,8,9). The only common feature of the target genes of these hyperproduction mutations seems to be their participation in adapting to starvation conditions requiring the utilization of a complex carbon or nitrogen source. Since one of the postulated roles of ISP-1 is to provide amino acids by degrading proteins during sporulation, it wa...