Cloning, sequencing, and some properties of a novel Bacillus amyloliquefaciens gene involved in the increase of extracellular protease activities

Tomioka, Noboru; Honjo, Masaru; Funahashi, Kei; Manabe, Kazuaki; Akaoka, Akiko; Mita, I.; Furutani, Yoshio

doi:10.1016/0168-1656(85)90009-4

Cited by 27 publications

(14 citation statements)

References 15 publications

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“…Our results confirm recent reports describing the independent cloning of sacQ genes from B. subtilis and B. amyloliquefaciens carried out by Yang et al (35) and Tomioka et al (30). The 46-residue polypeptide from B. licheniformis identified here is different from the 60-aminoacid protein from B. natto identified as responsible for the increased secretion of alkaline protease, neutral protease, and levansucrase (17).…”

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confidence: 79%

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Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Amory¹,

Kunst²,

Aubert³

et al. 1987

J Bacteriol

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The sacQ gene from Bacillus An interesting property of Bacillus subtilis is its ability to secrete a great number of enzymes (21), including some industrially useful ones like ax-amylase, proteases, or glticanases. However, the amount of proteins secreted by B. subtilis has been found to be a serious limitation compared to Bacillus strains used in industry, like B. licheniformis, B. amyloliquefaciens, or B. stearothermophilus, which are capable of producing much larger amounts of secreted enzymes.A number of mutations of B. subtilis 168 have been isolated that increase the production of secreted enzymes. sdcU-hyperproducing [sacU(Hy)] mutants (12), which are identical to pap (3, 37) and amyB (25) mutants, were characterized by a pleiotropic phenotype including hyperproduction of levansucrase, protease, and ax-amylase, absence of flagella, and low transformation levels. A similar phenotype was given by the sacQ(Hy) mutation, which was mapped at a different locus (13,14).In this report, we describe the identification of a plasmid containing a DNA fragment from B. licheniformis that confers to the recipient B. subtilis strain a hypersecretion phenotype similar to the one given by the sacQ(Hy) or sacU(Hy) mutation. From subcloning experiments, we obtained evidence that a 46-residue polypeptide is involved in the expression of the phenotype. A series of deletions was camed out in the cloned fragment, and the phenotype was lost when the deletions affected the polypeptide coding sequence. Moreover, hypersecretion was given by a 228-base-pair (bp) Moreover, we clearly show in this report that the sacQ gene isolated from a B. licheniformis high protease producer appears to encode a sacQ protein which is more efficient than the B. subtilis sacQ polypeptide in conferring the hypersecretion phenotype. The natural promoter of the sacQ gene was indeed replaced by an inducible promoter (spac), and the hypersecretion of protease and levansucrase was specifically obtained under conditions of full induction of the promoter. This is strong evidence that the increased expression of both enzymes is due to an increase of sacQ protein synthesis.Nothing, however, is known about the mechanism by which the increased expression of the target genes is produced by sacQ. The six different target genes identified in this study seem to be submitted to different growth phase regulation. For example, levansucrase is only synthesized during exponential growth, whereas alkaline protease is produced during the stationary phase.An attractive hypothesis is to suggest that sacQ regulates the synthesis of an essential component of the secretion machinery, since all of the target genes identified in this report encode a secreted enzyme. However, our results suggest that this hypothesis is unlikely. This is shown from a detailed study of one target of sacQ regulation, the levansucrase gene. We show that the target site is included in a 440-bp fragment located upstream from the coding sequence and ribosome-binding site of levansucrase, which sugges...

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confidence: 79%

“…However, Tomioka et al have shown that the sacQ gene from B. amyloliquefaciens, when present in multiple copies in B. subtilis, has no effect on a-amylase secretion (30), contrasting with the present work. This difference might be due to the growth medium used in the ox-amylase test.…”

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confidence: 56%

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Amory¹,

Kunst²,

Aubert³

et al. 1987

J Bacteriol

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“…How these mutations act to increase transcription of the sacB gene is unknown. Deletion analysis of the sacB promoter suggested that a region near -100 was necessary for complete stimulation of sacB transcription by the sa,cU32(Hy) and sacQ36 (Hy) (1,14,20,21). It is not known whether these polypeptides act directly on their target genes or indirectly by stimulating the production of other regulatory factors.…”

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confidence: 99%

“…The prtR and sacQ genes have been isolated and shown to encode 60-and 46-amino-acid polypeptides, respectively (14,20). They have no obvious similarity to other known polypeptides and only very limited similarity to each other.…”

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confidence: 99%

Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter

Henner¹,

Ferrari²,

Perego³

et al. 1988

J Bacteriol

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A number of mutations have been described with pleiotropic effects on the expression of genes for degradative enzymes in Bacillus subtlis. The sacU32(Hy) and sacQ36 (Hy) In Bacillus subtilis, the expression of a variety of degradative enzymes can be increased by mutations at a number of loci. These loci are unlinked to the structural genes for the affected enzymes. Mutations at the sacU and sacQ loci can increase the expression of levansucrase, alkaline protease, neutral protease, xylanase, ,-glucanase, a-amylase, and intracellular senine protease (1, 2, 10-13). Other mutations of the sacU locus decrease the expression of these enzymes.Another locus, designated hpr, has been defined which has a more restricted phenotype. Mutations at this locus lead to increased expression of alkaline and neutral proteases but do not affect the expression of levansucrase or a-amylase (8). Mutation at the hpr locus may have a small effect on the expression of intracellular serine protease (M. Ruppen, personal communication). Another gene, designated prtR, has recently been isolated whose overexpression on a plasmid can stimulate levansucrase and alkaline and neutral protease production (14,21). No chromosomal mutation of this gene has been described.The mechanism by which the sacU32(Hy) and sacQ36 (Hy) mutations act to increase the expression of the levansucrase (sacB) gene has been the focus of a number of recent reports (2, 17). Mutations at both loci can increase the amount of sacB mRNA, probably by increasing the rate of transcription initiation. The mRNA start site was identical in strains that have sacU32(Hy), sacQ36(Hy), or wild-type alleles at these loci (17). How these mutations act to increase transcription of the sacB gene is unknown. Deletion analysis of the sacB promoter suggested that a region near -100 was necessary for complete stimulation of sacB transcription by the sa,cU32(Hy) and sacQ36 (Hy) (1,14,20,21). It is not known whether these polypeptides act directly on their target genes or indirectly by stimulating the production of other regulatory factors. The nature of the sacU gene is unknown, and an initial report of its isolation has subsequently been retracted (G. Rapoport, personal communication). The hpr gene has been recently isolated, and the nature of stimulatory mutations at this site should soon be known (M. Perego and J. A. Hoch, unpublished data).In these studies, the aprE promoter was analyzed to determine the site at which the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations act to stimulate its transcription. We determined that there are at least two regions involved in the stimulation of this promoter. The target site for hpr-97 stimulation is separable from that of sacU32(Hy) and sacQ36(Hy) stimulation, and both target sites lie rather far upstream of the transcription start site. We also demonstrated that the hpr-97 and sacU32(Hy) mutations act by increasing the amount of mRNA initiated at the same start point as in wild-type strains. The generation and characterization of deletions are descri...

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“…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.…”

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confidence: 99%

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

1991

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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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Cloning, sequencing, and some properties of a novel Bacillus amyloliquefaciens gene involved in the increase of extracellular protease activities

Cited by 27 publications

References 15 publications

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter

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

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