Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis

Abstract

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...

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