title: The direct repeat sequence upstream of Bacillus chitinase genes is cis-acting elements that negatively regulate heterologous expression in E. coli.
abstract: To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, PR China.
authors.forename: Liang
authors.initials: L
authors.lastname: Xiao
authors.forename: Chuan
authors.initials: C
authors.lastname: Liu
authors.forename: Chi-chu
authors.initials: CC
authors.lastname: Xie
authors.forename: Jun
authors.initials: J
authors.lastname: Cai
authors.forename: Yue-hua
authors.initials: YH
authors.lastname: Chen
mesh_terms: D001407:Bacillus / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; D002688:Chitinases