Prof. Christof Holliger, Ben Zen-Ruffinen
COST Action 841 on Hydrogenases
January 2001 – June 2004
Prof. W. Hagen, TU Delft, The Netherlands
Tetrachloroethene, one of the most often encountered groundwater pollutants, can be used by several anaerobic bacteria as terminal electron acceptor in a novel kind of respiration. Most of the tetrachloroethene-dehalorespiring organisms use hydrogen as electron donor. They have a high affinity for hydrogen and can outcompete methanogens and homoacetogens for this substrate. Despite the importance of this substrate in a relevant process for the bioremediation of aquifers, little is known about the enzymes involved in the hydrogen consumption by these bacteria. The objectives of this study were to investigate the diversity of putative hydrogenase genes of dehalorespiring bacteria and to elucidate for one of theses bacteria which hydrogenase genes are expressed with hydrogen as electron donor and what the biochemical properties of these hydrogenases are.
By using degenerate PCR approaches and genome sequences available on databases, putative Ni-Fe hydrogenases as well as putative iron-only hydrogenases of different chloroethene-respiring bacteria were identified. An alignment with other Ni-Fe-hydrogenases showed that the Ni-Fe hydrogenase sequences of the dehalorespiring bacteria formed a distinct cluster within this enzyme family. Reverse transcriptase PCR on total RNA extracts from cells of Desulfitobacterium hafniense strain TCE1 grown under different conditions using primers designed for two Ni-Fe hydrogenase genes and other genes involved in this dehalorespiration process indicated that one of the two Ni-Fe hydrogenase was specifically expressed when cultivated with molecular hydrogen as electron donor. Enzyme activity measurements with whole cells and crude extracts indicated that the hydrogenase activity had a rather low half-saturation constant and is located at the outside of the cytoplasmic membrane.