Unraveling the members of a DNA-binding complex of a bacterial haloacid operon

Abstract

Haloacetates are structurally simple organic acids that can be found in our environment. They are generated incidentally during disinfection of water and are mutagenic. A soil bacterium, Burkholderia caribensis strain MBA4, produces an inducible dehalogenase that transforms these compounds to utilizable substrates. Previous studies using electrophoretic mobility shift assay has identified at least two retardation complexes that bind to the upstream non-coding region of the dehalogenase gene. These complexes were detected in extracts prepared from glycolate-grown cells and not in haloacid-grown cultures. In this presentation, oligonucleotide-conjugated metal beads were used to capture these DNA-binding proteins. Candidates were subjected to tandem mass spectrometry assays and the gene encoding for a candidate was disrupted. When extracts prepared from this mutant was used, the formation of one of the retardation complexes was disabled. This candidate protein was heterologously expressed, purified and used for bandshift assay. Two retardation complexes, different from those identified in wildtype, were detected. When this purified recombinant protein was added to the extract of the mutant and used in bandshift assay, the missing complex was re-established. This candidate, tentatively named as Peg8620 has been identified as a member of TetR family of regulators. This suggested that the retardation complex being considered is formed by the interaction of Peg8620 and another protein.