Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

Cantwell, B. A.; McConnell, David J.

doi:10.1016/0378-1119(83)90053-7

Cited by 95 publications

(39 citation statements)

References 25 publications

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“…1), which had the insert in the opposite orientation. This result, which was observed in the plate assay (10) and in in vitro reactions (20), was due to the effect of the lacZ promoter, as deduced from induction experiments done with isopropyl-1-D-thiogalactopyranoside (not shown).…”

mentioning

confidence: 64%

“…Nevertheless, this is the first report of a well-demonstrated physical association between cellulase and hemicellulase genes in the genus Bacillus. Cloning and expression in E. coli of the xynD and gluB genes. E. coli transformants obtained from a B. polymyxa genomic library in plasmid pUC13 were tested for lichenase or xylanase activities by the plate assay (10), using barley ,-glucan, carboxymethyl cellulose, or xylan. Two clones, E. coli(pBPL1000) and E. coli(pBPL5800), showed lichenase activity (both clones were able to degrade barley P-glucan and lichenan but not carboxymethyl cellulose), and one clone, E. coli(pXYN1), showed xylanase activity.…”

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confidence: 99%

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Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase

Gosalbes¹,

Pérez-González²,

González³

et al. 1991

J Bacteriol

116

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Two genes, xynD and gluB, encoding a xylanase and an endo-4-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtWis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows Ot-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (0. Grepinet, M. C. Chebrou, and P. Beguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.Plant cell walls are degraded by many enzymes that fall into two groups: cellulases (endo and exoglucanases) and hemicellulases (endoxylanases, arabinofuranosidases, etc.). Many bacteria, fungi, and yeasts show such activities, and one species can exhibit several activities of the two classes of enzymes (4, 11). The biotechnological importance of these microorganisms and their activities in food, paper, and other industries is great. The industrially important genus Bacillus is generally considered to be noncellulolytic. However, some species, such as B. polymyxa, show cellulase and/or hemicellulase activities (27). One B. polymyxa endo-1-(1,4)-glucanase (2) and two ,-glucosidases (15, 16) have been described, and their genes have been cloned and sequenced. There is another report of an endo-3-(1,3)-glucanase (laminarinase) in this species (12). Also, the existence of two endoxylanases (35) and one ,-xylosidase (28) from this species has been reported, and at least one of the endoxylanase genes (35) and the P-xylosidase (28) gene have been cloned. Here, we described the cloning, sequencing, and expression in Escherichia coli and B. subtilis of an endo-,-

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confidence: 64%

mentioning

confidence: 99%

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase

Gosalbes¹,

Pérez-González²,

González³

et al. 1991

J Bacteriol

116

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“…Besides the degradation of extracellular proteins by the alkaline protease, the degradation of five other polymeric substrates was increased in the presence of pPR41 ( Fig. 2 (5,9). Degradation of xylan and starch is probably due to xylanase (20) and a-amylase (34), respectively.…”

Section: Resultsmentioning

confidence: 99%

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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“…The bgZS gene has been cloned (Cantwell & McConnell, 1983) and sequenced (Murphy et al, 1984). Here we show that the temporal activation of P-glucanase synthesis is a result of a sharp decrease in the intracellular GTP concentration after a nutrient limitation.…”

Section: Introductionmentioning

confidence: 81%

Temporal activation of -glucanase synthesis in Bacillus subtilis is mediated by the GTP pool

Stülke¹,

Hanschke²,

Hecker³

1993

Journal of General Microbiology

214

180

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Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

Cited by 95 publications

References 25 publications

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Temporal activation of -glucanase synthesis in Bacillus subtilis is mediated by the GTP pool

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