The cloning and analysis of Rhizobium dehalogenase genes.
thesisposted on 19.11.2015, 09:07 by Stephen Scott. Cairns
A Rhizobium genomic library was constructed in Escherichia coli NM522 and the library was screened for the ability to grow on the halogenated compound 2-chloropropionate (2CP). Two positive clones were identified and the plasmids designated pSC1 and pSC530. Both clones allowed E. coli to grow on 2CP at growth rates slower than that seen for the Rhizobium. Investigation of the dehalogenase activities of the clones showed that extracts from cells containing pSC1 were active against D-, L- and D/L-2CP and extracts from cells containing pSC530 were also active against dichloropropionate. Restriction enzyme mapping of the clones indicated that they were unique regions of the genome that conferred the new growth ability on E. coli. Southern analysis of the Rhizobium genomic DNA using insert DNA as probes confirmed that both of the clones were from the Rhizobium and that the clones were not contiguous on the genome. Subcloning of pSC1 resulted showed that the insert encoded two stereospecific dehalogenase genes and two plasmids were constructed by subcloning that were each able to express each of the two dehalogenases. The stereospecific dehalogenases (HadD and HadL) were purified and the N-terminal amino acid sequences were determined. HadL was also purified from the Rhizobium and the N-terminal sequence was shown to be the same as the cloned HadL. The complete nucleotide sequences of the hadD and hadL genes were determined and showed little similarity to each other or to other known dehalogenases. HadD and HadL were used, both in vivo and in vitro, to resolve a racemic solution of 2CP, an analagous situation to possible industrial applications of dehalogenases.