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PMID: 2307659 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans.

Journal of bacteriology ·Vol. 172 ·No. 3 ·1990-03-00 ·Pages 1576-86

Baird SD, Johnson DA, Seligy VL

Abstract

Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding.

MeSH Terms
Amino Acid Sequence Bacillus/enzymology,genetics Base Sequence Cellulase/genetics Cloning, Molecular/methods Coliphages/genetics DNA, Bacterial/genetics,isolation & purification Gene Expression Genes, Bacterial Genotype Molecular Sequence Data Sequence Homology, Nucleic Acid Species Specificity Transcription, Genetic
Chemicals
DNA, Bacterial Cellulase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Baird S D
Department of Biology, University of Ottawa, Ontario, Canada.
Johnson D A
Seligy V L
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.172.3.1576-1586.1990
Published
1990-03-00
Pages
1576-86
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC208635
Subset
IM
Databases
GENBANK
M33791
External Links
PubMed source DOI: 10.1128/jb.172.3.1576-1586.1990 PMC Full Text View journal details
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