title: Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis.
abstract: The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - sigma H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53 degrees C.
authors.affiliation: Department of Genetics, Center of Biological Sciences, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Kiel
authors.forename: J M
authors.initials: JM
authors.lastname: Boels
authors.forename: G
authors.initials: G
authors.lastname: Beldman
authors.forename: G
authors.initials: G
authors.lastname: Venema
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D015061:1,4-alpha-Glucan Branching Enzyme; D001616:beta-Galactosidase