Molecular characterization of anaerobic dehalogenation by Desulfitobacterium dehalogenans

Haloorganics such as chlorophenols and chlorinated ethenes are among the most abundant pollutants in soil, sediments and groundwater, mainly caused by past and present industrial and agricultural activities. Due to bioaccumulation and toxicity, these compounds threaten the integrity of the environment, and human and animal health. A recently discovered, phylogenetically diverse, group of anaerobic so-called halorespiring bacteria is able to couple the reductive dehalogenation of various haloorganic compounds to energy conservation and hence to growth, significantly contributing to in situ dehalogenation processes in anoxic environments. The observed persistence of halogenated pollutants in untreated ecosystems and the accumulation of degradation intermediates during bioremediation, however, indicated the need for engineering of process conditions and/or augmentation with efficient degraders. This thesis describes genetic approaches towards a thorough understanding of the molecular basis of anaerobic reductive dehalogenation in order to enable the further optimization of clean up procedures for contaminated anoxic environments.The Gram-positive Desulfitobacterium dehalogenans , capable of degrading o -chlorophenols, PCE and hydroxylated PCB's, was used as model organism throughout a major part of this study. The key enzyme o -chlorophenol reductive dehalogenase (CPR) was isolated and characterized at the biochemical and molecular levels, and comparison with known chlorophenol- and chloroalkene-reductive dehalogenases indicated that these enzymes constitute a yet unknown, but evolutionary ancient family of corrinoid-containing iron-sulfur proteins, which in addition share a twin-arginine signal sequence. Transcriptional analysis of the CPR-encoding gene cluster revealed that dehalogenation activity is strongly regulated at the transcriptional level. Efficient gene cloning, and random and specific gene inactivation systems were developed to enable (i) the elucidation of additional components involved in the anaerobic dehalogenation process and (ii) the study of their structure and function within the respiratory network. Halorespiration-deficient mutants were isolated following random chromosomal integration, and their characterization lead to the identification of genes encoding proteins possibly involved in structure, maturation and regulation of respiratory complexes. Reductive dehalogenase-encoding gene targeted (RT-)PCR-based molecular approaches were developed that were useful for the detection of reductive dehalogenation potential and activity in pure cultures of potentially halorespiring microorganisms.The results obtained in this study provide valuable knowledge on the molecular basis of anaerobic reductive dehalogenation and might serve as a sound basis for the further exploitation of halorespiring bacteria as dedicated degraders in biological remediation processes.