Molecular and biochemical aspects of the chloroethene dehalorespiration process

Tetrachloroethene (PCE) is a major groundwater pollutant as a result of extensive use and spillage since 80 years. Several obligate anaerobic bacteria have the ability of using chlorinated aliphatic compounds as terminal electron acceptor in a process called dehalorespiration. Lines of investigation such as the maturation of the reductive dehalogenase and the identification of the respiratory chain components are currently being studied with Desulfitobacterium hafniense strain TCE1 as model organism. The pce gene cluster containing the gene pceA encoding the key enzyme in dehalorespiration, PCE reductive dehalogenase, is embedded in a composite transposon. Additional genes present are pceB, pceT and pceC as well as tatA1, a putative gene of the Twin-Arginine Translocation (Tat) system. PceA is the actual catalytic subunit containing a corrinoid cofactor and two iron-sulfur clusters, and PceB is possibly functioning as membrane anchor for PceA. PceA is synthesized in an immature form containing a twin-arginine signal peptide with the conserved motif RRxFLK that is found in a class of complex redox proteins exported to or across the cytoplasmic membrane by the Tat system. Overexpressed PceA was used for the preparation of anti-PceA antibodies and a combination of cell fractionation, proteinase K mediated proteolysis, and Western blot analyses, was applied for the localization of PceA. The gene pceT is encoding a putative trigger factor possibly involved in correct folding of PceA. However, PceT is missing the typical N-terminal domain constituting the ribosome-binding domain of trigger factor. Experiments with mutant strains of Escherichia coli lacking the trigger factor will be carried out in order to get experimental evidence on the proposed function of pceT. Additional missing links of the dehalorespiratory chain were investigated by two-dimensional proteomic analysis. Membrane proteins were extracted from cells cultivated on lactate or hydrogen as electron donor in combination with fumarate or PCE as terminal electron acceptor. Preliminary results revealed the presence of PceA as the most induced protein when strain TCE1 grows on PCE. Moreover proteins predicted to function as ABC-type transporters, a CO dehydrogenase and especially a sulfur transferase (rhodanese-like protein) are also strongly induced, but their dedicated role in PCE dehalorespiration remains to be elucidated.