Molecular analysis of halorespiration in Desulfitobacterium spp. : catalysis and transcriptional regulation

Keywords: iron-sulphur proteins, protein-DNA interaction, allosteric regulation, redox regulation, gene redundancy, chlorophenols, bioremediationSoil and ground water contamination by halogenated organic compounds mainly used as biocides in agriculture or solvents and cleaning agents in industry has been a long-standing problem. The main barrier in the chemical degradation of organohalides is the presence of the halogen group in the molecule, which also contributes to the toxic nature of these compounds. However, a remarkable group of microorganisms, the halorespiring bacteria, are able to reductively dehalogenate organohalides under anaerobic conditions and use the energy generated via a proton gradient pump for bacterial growth (halorespiration). Due to their versatile dehalogenating capacity, halorespiring bacteria have a large potential in the clean-up of contaminated sites (bioremediation). The work described in this thesis aimed to gain knowledge on two aspects of the molecular basis of halorespiration: (i) the reaction mechanism of dehalogenation, catalyzed by the B 12 /iron-sulphur containing reductive dehalogenases, and (ii) the regulatory mechanism which enables transcriptional activation of genes involved in halorespiration. As model organisms, two Desulfitobacterium spp. were chosen, capable of ortho and/ormetadechlorination of chlorophenols and hydroxylated polychlorinated biphenyls.Unravelling of the novel reaction mechanism of reductive dehalogenases was so far hampered by difficulties in functional overproduction of these enzymes. We developed a protocol which involves co-expression of molecular chaperons to aid functional synthesis of the metalloenzymes in Escherichia coli . Next, multiple potential transcriptional activators were characterised from D. hafniense by promoter fusions and in vitro DNA-binding assays. We found that CprK1 and CprK2 activated transcription in the presence of an ortho -chlorophenol (CHPA) but not with its dechlorinated derivative (HPA), while meta -chlorophenols proved to be effectors for CprK4. All CprK paralogues recognized a conserved motif (dehalobox) in halorespiration-inducible promoters. Site-directed mutagenesis of CprK1 provided further insight on the role of conserved residues in the DNA-recognitiona-helix and on redox regulation of the protein.Crystalstructures of the CHPA-bound CprK1 and the effector-free form of the closely related CprK of D. dehalogenans revealed a possible mechanism by which the regulators distinguish between CHPA and the non-effector HPA ("pK a interrogation" theory). Native mass spectrometry of protein-DNA complexes confirmed these results and-together with limited proteolysis experiments-also contributed to the fundamental knowledge of ligand-induced changes in the conformation and dynamics of CprK-related transcriptional regulations