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 Comparison with unpublished data from Desulfitobacterium sp.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 the former enzyme and the presence of one [3Fe-4S] and one [4Fe-4S] cluster in the latter one. Structure breaking residues such as glycine and proline are present at the two extremities of the ten amino acid stretch separating the first and second ironbinding cysteine residues of the second motif in PceA, but not in CprA. This primary structure probably allows the formation of a loop in the tertiary structure and the participation of the first cyteine as a ligand in a [4Fe-4S] cluster. In both new sequences, the presence of a short gene (pceB) encoding a hydrophobic protein with three conserved trans-membrane α-helices was confirmed, indicating a possible role in anchoring the catalytic unit of the reductive dehalogenase into the membrane. The complete sequence identity observed in the newly isolated reductive dehalogenases raised the question of a possible horizontal gene transfer between Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1. Therefore, the flanking regions of the reductive dehalogenase genes (pceAB) in Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus were investigated. This study revealed the presence of a composite transposon (named Tn-Dha1) in strain TCE1 bordered with two identical insertion sequences (ISDha1, including the transposase gene tnpA1) and containing six open reading frames: the already characterized pceAB, two genes (pceCT) related to members of the o-chlorophenol reductive dehalogenase gene cluster of Desulfitobacterium dehalogenans, and two possibly truncated genes with homology to another transposase (tnpA2) and to a subunit of the Tat machinery (tatA), respectively. In contrast, only the pceABCT gene cluster (i.e. without the transposon structure and the other two genes) was present in Dehalobacter restrictus, indicating that the genes encoding the key enzymes for the dechlorination activity are stably integrated into the genome. A detailed investigation of Tn-Dha1 by PCR and Southern blot analysis indicated that Tn-Dha1 may form various circular molecules, an indication for an active mobile genetic element. A model for the transposition of Tn-Dha1 was proposed, in which the transposon may excise from the chromosome and circularize, forming an unstable structure with two abutted ISDha1. The strong promoter formed by the junction of both IS would lead to high expression of the transposase, which in turn reacts with the circular element by either re-integrating it in the chromosome or excising one or both ISDha1 from that element. The resulting structures would be single IS, IS tandems and circular molecules with one or no remaining IS, both latter structures being dead-end products of the transposition event. The hypothesis of mobile reductive dehalogenase genes was also investigated using a genomic approach in preliminary sequence data (released by The Institute for Genome Research, TIGR) of the genome of Dehalococcoides ethenogenes, a dehalorespiring bacterium capable to completely dechlorinate PCE to ethene. The genome was shown to contain the extraordinary number of eighteen different copies of reductive dehalogenase genes, including the well characterized tceA. A genomic signature of D. ethenogenes was obtained by calculating the frequency of 4-letter DNA words along the genome and was graphically represented. Local disruptions of the genomic signature in certain segments of the genome were highlighted, corresponding to DNA, which may have been acquired by horizontal gene transfer, so-called original regions. It revealed that from the eighteen putative reductive dehalogenase genes present in the genome of D. ethenogenes, fifteen were located in original regions. Moreover, several genes encoding for recombinases (transposase, integrase) were found within these original regions, strongly indicating that these may have been acquired horizontally. The complete electron transport chain leading the electrons to the reductive dehalogenase has not yet been characterized for any of dehalorespiring bacteria and the direct electron-donor has not yet been elucidated for any of reductive dehalogenases. Therefore, the presence of cytochromes in cells of Desulfitobacterium hafniense strain TCE1 was investigated with regard to the presence or absence of PCE in the growth medium. Detection of cytochromes using a sensitive detection method based on chemiluminescence revealed a strongly enhanced signal in the membrane fractions of strain TCE1 cells grown on PCE instead of fumarate as terminal electron acceptor. Western blot analysis revealed the presence of a 45 kDa protein in membrane fraction, corresponding most probably to a c-type cytochrome. UV-visible spectroscopy confirmed the presence of c-type cytochromes in membrane fractions. This study, although further investigations are needed, indicated that a c-type cytochrome may be involved in the direct electron transfer to the PCE reductive dehalogenase of D. hafniense strain TCE1. At present, numerous sequences of reductive dehalogenase genes have been reported and deposited on sequence databases, revealing the great interest shown for this new anaerobic respiration pathway. While several degenerate PCR approaches have led to the isolation of 22 mostly partial genes, analysis of preliminary genome sequence data from Dehalococcoides ethenogenes and Desulfitobacterium hafniense strain DCB-2 has revealed 18 and 6 sequences, respectively. Sequence alignment and homology analysis of the 66 reductive dehalogenases genes available in August 2004 revealed four main clusters, two corresponding to chlorophenol and chloroethene reductive dehalogenases found in the phylum Firmicutes, one with sequences mostly isolated from ε-Proteobacteria, and one containing most of the genes isolated from the genus Dehalococcoides. Hence, the reductive dehalogenases appear to be rather conserved wihtin phylogenetic groups, indicating a relatively ancient enzyme class. Reductive dehalogenases show some features such as the presence of a Tat signal peptide and iron-sulfur clusters that are common to most of terminal reductases. However, the presence of a corrinoid at the catalytic center and of several specific conserved amino acid stretches makes them a new class of anaerobic reductases. Finally, the strong variation in the topology of the dehalorespiration chain and the variable presence and involvement of different electron transferring components such as quinones and cytochromes in dehalorespiring bacteria indicate that reductive dehalogenases may have been integrated into existing respiration chains rather than that dehalorespiration has evolved as a whole.