Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.
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.
Janssen
D B
DB
Department of Biochemistry, University of Groningen, The Netherlands.
Pries
F
F
van der Ploeg
J
J
Kazemier
B
B
Terpstra
P
P
Witholt
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005025:Ethylene Dichlorides / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014974:Xanthomonas / Q000235:genetics
D005025:Ethylene Dichlorides; D006843:Hydrocarbons, Chlorinated; C024565:ethylene dichloride; D006867:Hydrolases; C046137:haloalkane dehalogenase