Molecular characterization of key enzymes involved in dehalorespiration with tetrachloroethene

Chloroethenes, and most particularly tetra- (PCE) and trichloroethene (TCE) are major groundwater pollutants due to their extensive industrial use as solvents since the 1920s. The strong electronegativity of the chlorines renders them very stable under aerobic conditions. However, biodegradation of chloroethenes under anaerobic conditions has been shown to be a promising strategy for remediation of chloroethene-contaminated sites. To date around fifteen bacterial strains have been isolated with the property of using chloroethenes as terminal electron acceptor in a process called dehalorespiration. Anaerobic dehalorespiring bacteria show an unequal chloroethene substrate range and an unequal extent of dechlorination. While most of the dehalorespiring bacteria dechlorinate PCE and TCE to cis-1,2-dichloroethene cis-1,2-DCE) and belong to the phyla Firmicutes, δ- and ε-Proteobacteria, a few strains of the genus Dehalococcoides affiliated with the phylum Chloroflexi and are able to dechlorinate cis-1,2-DCE and vinyl chloride (VC) to the non-toxic ethene. Dehalobacter restrictus and Dehalococcoides isolates were found to be completely restricted to dehalorespiration which gave rise to some basic evolutionary questions. Identification of the key enzyme in the dechlorination reaction, the reductive dehalogenase, has revealed a new class of enzymes containing a corrinoid and two ironsulfur clusters as cofactors. At the beginning of this thesis, nine chloroethene reductive dehalogenases have been characterized on biochemical level, while only little information was available on molecular level. Therefore, the overall goal of this thesis was to characterize on a molecular level the reductive dehalogenases involved in tetrachloroethene dehalorespiration and to get some indications on the evolution of this novel anaerobic respiration process. Starting from the N-terminal sequence of the PCE reductive dehalogenase (PceA) of Dehalobacter restrictus and from a conserved amino acid stretch found in two already sequenced reductive dehalogenases, a degenerate PCR approach allowed the isolation of the gene encoding PceA. Comparison with unpublished data from Desulfitobacterium sp. strain PCE-S showed 100% sequence identity. The full sequence of the pceAB gene of strain PCE-S helped to isolate the corresponding gene cluster from D. restrictus and Desulfitobacterium hafniense strain TCE1, which has also been shown to contain an identical N-terminal sequence. Sequence analysis confirmed the presence of a Twin-Arginine Translocation (Tat) signal peptide, which is involved in the incorporation of the reductive dehalogense into the cytoplasmic membrane. Detailed analysis of the iron-sulfur cluster binding motifs present in PceA of D. restrictus and the chlorophenol reductive dehalogenase (CprA) of Desulfitobacterium dehalogenans revealed differences in the second motif, which may explain results obtained by EPR spectroscopy, namely the presence of two [4Fe-4S] clusters in