Open AccessCloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene
Author(s) -
Dick B. Janssen,
Frens Pries,
Jan van der Ploeg,
Bert Kazemier,
Peter Terpstra,
Bernard Witholt
Publication year - 1989
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.171.12.6791-6799.1989
Subject(s) - biology , gene , escherichia coli , microbiology and biotechnology , dehalogenase , biochemistry , peptide sequence , ribosomal binding site , ribosome , rna
A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.