Open AccessCloning, Sequencing and Expression in Escherichia coli of Two Rhizobium sp. Genes Encoding Haloalkanoate Dehalogenases of Opposite Stereospecificity
Author(s) -
Cairns Stephen S.,
Cornish Alex,
Cooper Ronald A.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.t01-1-00744.x
Subject(s) - subcloning , biology , escherichia coli , molecular cloning , gene , microbiology and biotechnology , ecori , operon , genetics , peptide sequence , plasmid
A 6.5‐kb EcoRI fragment of genomic DNA from a Rhizobium sp. cloned into pUC19 was able to endow Escherichia coli K‐12 with the novel ability to grow at the expense of 2‐chloropropionic acid. Subcloning showed that this property was a consequence of two dehalogenases encoded on a 2.2‐kb Pst I fragment. Further subcloning of the PstI fragment led to two constructs that encoded, separately, dehalogenase activity that acted stereospecifically on D‐2‐chloropropionic acid and L‐2‐chlompropionic acid, respectively. The genes encoding these two stereospecific dehalogenases have been sequenced and shown to be separated by 177 bp of non‐coding DNA. Expression of the dehalogenase genes involved the vector promoter, suggesting that the anticipated Rhizobium sp. regulatory sequences were not functional in E. coli. Comparison of the deduced amino acid sequences of the two dehalogenases (18% identity) indicated that there was no obvious evolutionary relationship between (hem. Nor was there any striking identity with any other 2‐chlompropionic acid dehalogenase studied so far.