CLONING, SEQUENCING AND EXPRESSION IN ESCHERICHIA-COLI OF 2 RHIZOBIUMSP GENES ENCODING HALOALKANOATE DEHALOGENASES OF OPPOSITE STEREOSPECIFICITY

A 6.5-kb EcoRI fragment of genomic DNA from a Rhizobium sp. cloned int o pUC19 was able to endow Escherichia coli K-12 with the novel ability to grow at the expense of 2-chloropropionic acid. Subcloning showed t hat this property was a consequence of two dehalogenases encoded on a 2.2-kb PstI fragment. Further subcloning of the PstI fragment led to t wo constructs that encoded, separately, dehalogenase activity that act ed stereospecifically on D-2-chloropropionic acid and L-2-chloropropio nic acid, respectively. The genes encoding these two stereospecific de halogenases have been sequenced and shown to be separated by 177 bp of non-coding DNA. Expression of the dehalogenase genes involved the vec tor promoter, suggesting that the anticipated Rhizobium sp, regulatory sequences were not functional in E. coli. Comparison of the deduced a mino acid sequences of the two dehalogenases (18% identity) indicated that there was no obvious evolutionary relationship between them. Nor was there any striking identity with any other 2-chloropropionic acid dehalogenase studied so far.