Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were cloned and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease can be stimulated up to 11-to Bacillus subtilis is capable of secreting a wide variety of extracellular enzymes (proteases, a-amylase, levansucrase, and P-glucanases) directly into the medium (22). Several mutants that can stimulate the production of extracellular enzymes have been isolated. These mutants carry mutations in degQ and sacU (12,14). Structural genes encoding degQ and sacU have been cloned (1,8,11,34,36,45). Furthermore, the cloning of several regulatory genes from B. subtilis, B. natto, and B. stearothermophilus has been shown to enhance the production of extracellular enzymes when these genes are in a multicopy plasmid. These genes are sacV (17), degR (19, 47), senS (38), senN (39,42), and degT (33). The size of the protein products derived from these genes varies from small polypeptides (46 to 65 amino acids for the degR, sacV, senS, and senN products) to larger polypeptides (372 amino acids for the degT product). Although these gene products show no sequence homology with each other, they all stimulate the production of extracellular enzymes. The target genes encoding extracellular enzymes such as aprE (alkaline protease) (30, 41), nprE (neutral protease) (46), amyE (oa-amylase) (44), and sacB (levansucrase) (32) have also been cloned. With these mutants and the cloned genes available, the mechanism for the enhanced production of extracellular enzymes can be studied in detail. degR, senN, degQ, and sacU have been shown to exert their stimulatory effects at the transcriptional level (28,35,42). However, in the sacU minus mutant strain, no enhanced production of extracellular enzymes can be observed with cells carrying either degR or degQ on a multicopy plasmid (1,34).In this report, we describe in detail the cloning, nucleotide sequence, genetic mapping, and organization of a set of two novel genes from B. subtilis that can regulate the production of several extracellular enzymes. They were isolated by using a shotgun cloning approach and were selected by their abilities to stimulate the production of alkaline protease. The * Corresponding author. characterization of these genes and their roles in regulating gene expression are discussed. MATERIALS AND METHODSPlasmids. Plasmid pUB18 (42), a derivative of pUB110, was used for routine subcloning in B. subtilis. pPQ is a pUB18 derivative carrying degQ (40). Bluescribe plasmid from Escherichia coli was used for cloning and doublestranded DNA sequencing. Standard DNA transformation was performed by the competent cell method in B. subtilis (29) and E. coli (16).Med...

Streptokinase is one of the major blood-clot-dissolving agents used in many medical treatments. With the cloned streptokinase gene (skc) available, production of the secreted streptokinase from various Bacillus subtilis strains was studied. The use of the six-extracellular-protease-deficient strain, WB600, greatly improved the production yield of the secreted streptokinase. A modified skc which has the original skc promoter and signal sequence replaced with the B. subtilis levansucrase promoter and signal sequence was also constructed. B. subtilis carrying either the wild-type or the modified skc produces streptokinase at a comparable level. Even with WB600 as the expression host, a C-terminally-processed streptokinase was also observed. Through region-specific combinatorial mutagenesis around the C-terminal processing sites, streptokinase derivatives resistant to C-terminal degradation were engineered. One of the derivatives showed a 2.5-fold increase in specific activity and would potentially be a better thrombolytic agent.

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