Analysis of Rhizobium sp. Dehalogenases and their regulation

The genus of the organism was confirmed as Rhizobium by partial sequencing of its 16S rRNA gene. The Rhizobium sp. DehL, DehE and DehD were produced by heterologous expression of the cloned genes in Escherichia coli K-12 and the proteins purified. All three dehalogenases were further characterised by kinetic analysis. The Km, Kcat and the Specificity Constants of these enzymes were determined. Rhizobium sp. was able to grow at 0.2mM 2,2DCP, which were 100x lower than the concentration of the substrate routinely used. Apparently, no new dehalogenases are required to allow growth on this low concentration of 2,2DCP as judged by electrophoretic mobility of dehalogenase proteins on Native-PAGE analysis and protein separation by anion-exchange column chromatography. The kinetic analysis suggested that the known dehalogenases were able to act efficiently on low concentrations of haloalkanoic acids. The amount of each dehalogenase, from cells grown on low substrate concentration was different to that seen at 20mM 2,2DCP due to complex regulatory controls, which respond to the growth environment. The cloning of the putative Rhizobium sp. regulator gene was achieved using phenotypic co-selection and the deduced amino acid sequence was compared to the databases. The putative regulatory sequence of Rhizobium sp. was highly homologous to the DehR1 of Pseudomonas putida PP3 with a sequence identify of 72% and the DhlR of Xanthobacter autotrophicus GJ10 with 48% identity. The regulatory gene was cloned into a high expression vector but the protein produced was found in inclusion bodies and presumably not active.