Groundwater contamination by organochloric compounds became a major concern for public health during the last century. Chloroethenes, commonly used since the 1920's as degreasing and cleaning agents and as intermediates in chemical synthesis, accumulated in the environment due to inappropriate handling and storage. Among those, tetrachloroethene (PCE) and trichloroethene (TCE) are known to be persistence in the environment and to be toxic, carcinogenic for living organisms. These components are hardly degraded under aerobic conditions and were firstly thought to be biorecalcitrant. Field studies on PCE contaminated sites revealed naturally occurring degradation by an anaerobic respiratory process called dehalorespiration. To date over 20 microorganisms and several enrichment cultures have been described showing degradation ability by this anaerobic energy-conserving respiration. Hydrogen is generally considered as the ultimate electron donor for dechlorination. However, exceptions using more complex electron donors such as acetate and possibly formate are known in Sulfurospirillum multivorans or in Desulfuromonas chloroethenica. Studies showed that the H2-thresholds in dechlorinating cultures were lower than in cultures where acetogenesis, methanogenesis and sulphate reduction occured, raising the interest of the role of hydrogenases in this process. The aim of this study was to search for hydrogenase genes in the dechlorinating bacterium Desulfitobacterium hafniense strain TCE1, to determine the effect of hydrogen on the expression pattern of the hydrogenases most closely related to other hydrogenases in dechlorinating organisms (Hyn1 and Hyn2) and to characterize the enzymatic proprieties in whole cells and crude extracts in order to gain a better understanding of the dehalorespiration process. We started to design degenerate primers on the sequence information of the large subunit of the NiFe hydrogenase from Desulfitobacterium dehalogenans and similar putative hydrogenases found in databases from Dehalococcoides ethenogenes and Desulfitobacterium hafniense strain DCB2. As a result, we amplified and sequenced a partial sequence from Dehalobacter restrictus and two partial sequences from Desulfitobacterium hafniense strain TCE1. Subsequent analysis of these partial sequences by a combination of inverse and direct PCR in Desulfitobacterium hafniense strain TCE1 provided two genomic sequences containing small and large subunit, as well as a cytochrome. In one case, a maturation protein-like sequence was found also. In addition a sequence coding for a Fe-only hydrogenase was obtained. At the same time, new updates of the contig database of Desulfitobacterium hafniense strain DCB2 suggested the presence of at least three more hydrogenases in Desulfitobacterium hafniense strain TCE1. Primers designed based on this information allowed the sequencing of three more putative NiFe-hydrogenase operons in Desulfitobacterium hafniense strain TCE1 (Hya, Hup and Hyc). Comparison of the small and large subunits of these putative hydrogenases in Desulfitobacterium hafniense strain TCE1 with other known NiFe hydrogenases showed that Hyn1 and Hyn2 clustered together with other hydrogenases from dechlorinating organisms, forming a subgroup within the membrane-bound NiFe uptake hydrogenases. The third hydrogenase, Hya, clustered close to the E. coli isoenzyme 2, which is implicated in anaerobic respiration in this organism. The fourth one, Hup, is related to archeal hydrogenases. The last NiFe-hydrogenase merges into the membrane bound H2 evolving hydrogenases as Hyc from E. coli, which is part of the formate-hydrogen lyase complex. Influence of hydrogen on the expression pattern of hynL1, hynL2 and pceA was studied qualitatively by reverse-transcriptase PCR (RT-PCR). The 16S rRNA gene was used as internal standard. A transcript for hynL2 was amplified for cells grown under hydrogen and PCE, and, in a lesser extend, under hydrogen and fumaric acid. The hynL1 transcript was only detected in hydrogen-PCE grown cells. The 16S rRNA gene and pceA were detected under all culture conditions, indicating a certain regulation of the expression of two hydrogenases. Quantitative Real-Time RT-PCR detected transcripts for all genes under all culture conditions. However, expression was delayed in cells grown in absence of hydrogen In both cases, the regulation of the hydrogenase activity is dependent on the presence of hydrogen. Enzymatic assays with the artificial electron acceptor benzyl viologen showed no activity for hydrogenases in cells grown without hydrogen whereas activity was found in cells grown in presence of hydrogen. Kinetic studies on whole cells and crude extracts showed a Km values (65 and 25nM respectively) similar to other NiFe uptake hydrogenases published in other studies. These low values are consistent with the low H2-thresholds found for dechlorinating organisms and cultures. The complete electron transport chain involved in dehalorespiration has not yet been characterized for any dehalorespiring bacteria so far. Biochemical studies hinted towards membrane-bound outside facing hydrogenase and inside facing PCE-reductase. Presence of b-type cytochromes and menaquinones in the membrane was also shown. Based on these findings a tentative model for dehalorespiration in Desulfitobacterium hafniense strain TCE1 is proposed.
EPFL2006
