We have cloned a 2.5-kilobase fragment of the Bacillus subtilis genomic DNA which caused the reduction of extracellular and cell-associated protease levels when present in high copy number. This fragment, in multicopy, was also responsible for reduced levels of a-amylase, levansucrase, alkaline phosphatase, and sporulation inhibition. The gene relevant to this pleiotropic phenotype is referred to as pai. By DNA sequencing, two open reading frames-ORF1 and ORF2, encoding polypeptides of 172 and 207 amino acid residues, respectively-were found. These open reading frames seemed to form an operon. Deletion analysis revealed that an entire region for ORFI and ORF2 was necessary for the pai phenotype. In addition, it was observed that the presence of the pai gene, in multicopy, caused overproduction of two proteins (molecular masses, 21 and 24 kilodaltons [kDa]). Analyses of the N-terminal amino acid sequences of these two proteins suggested that they were products of ORF1 and ORF2. Disruption of the pai gene at ORF1 in the genomic DNA resulted in the release of repression on protease synthesis and sporulation in glucose-enriched (2%) medium. The mutant carrying insertional disruption at ORF2 could not be constructed, suggesting that the ORF2 product, the 24-kDa protein, is essential for growth. The 21-kDa protein contains a helix-turn-helix domain observed in other DNA-binding proteins. Chromosomal mapping of pai indicated that this gene is located close to thr-5. These results suggest that the pai gene is a novel transcriptional-regulation gene involved in glucose repression.In Bacillus subtilis, the expression of genes for extracellular enzymes such as a-amylase, neutral protease, and alkaline protease is temporally linked to the initiation of sporulation. It is also known that sporulation and expression of these degradative-enzyme genes are regulated under catabolite repression (29).It has been revealed that these degradative-enzyme genes are transcriptionally controlled by several regulatory genes such as degQ, degR, and degU (11), which were formerly designated sacQ, prtR, and sacU, respectively (12). The production levels of extracellular neutral and alkaline proteases are elevated by mutations such as sacQ(Hy) and sacU(Hy) (18) located within degQ (39) and a sacU operon composed of degS and degU (12) or by the presence of degQ, degR, or sen in a multicopy state (23,(37)(38)(39)(40). On the other hand, these protease genes are negatively regulated by hpr (28) and sin (7). Recently, Tanaka and Kawata have found that the degU product has a dual function: negative and positive regulation of extracellular protease production (36). Kunst et al. (17)
