Characterization of the hydrogenase activity coupled to the reductive dechlorination of tetrachloroethene by Dehalobacter restrictus

• Unité de recherche: COST
• Numéro de projet: C96.0066
• Titre du projet anglais: Characterization of the hydrogenase activity coupled to the reductive dechlorination of tetrachloroethene by Dehalobacter restrictus

Mots-clé (Anglais)

tetrachloroethene; biodegradation; envrionmental pollutant; anaerobic respiration

Programme de recherche (Anglais)

COST-Action 818 - Hydrogenas and biological redox events in environmental research and biotechnology

Description succincte (Anglais)

See abstract

Autres indications (Anglais)

Full name of research-institution/enterprise: EPF Lausanne ENAC-ISTE / Laboratoire de biotechnologie environnementale (LBE)

Partenaires et organisations internationales (Anglais)

B, CH, D, E, F, GR, H, I, NL, P, S, UK

Résumé des résultats (Abstract) (Anglais)

Tetrachloroethene and trichloroethene, two compounds belonging to the most abundant organic environmental pollutants, are reductively dechlorinated by the bacterium Dehalobacter restrictus to cis-1,2-dichloroethene, a product that can be further dechlorinated by other anaerobic bacteria or mineralized by aerobic bacteria. D. restrictus, a strictly anaerobic bacterium, oxidizes molecular hydrogen and couples this reaction to the reduction of tetrachloroethene in an anaerobic respiration. The respiration chain is quite simple and consists of a hydrogenase that is located on the outside of the cytoplasmic membrane and a tetrachloroethene reductase that is located on the inside. The electrons derived from hydrogen oxidation are transferred via different electron mediators to the tetrachloroethene reductase. It has been shown that menaquinone and cytochrome b are involved in this electron transfer. The hydrogenase activity was found for 56-85% in the soluble fraction while activities in the membrane fraction ranged from 15-44% suggesting that the enzyme is loosely associated with the membrane. The activity was highest at 35°C and pH 8.1. Soluble fractions and cell extracts applied to native gels both revealed three bands of hydrogenase activity while a single band was detected in the membrane fraction. This indicated that the hydrogenase is composed of several subunits one serving as membrane anchor protein, similar to results obtained with Wolinella succinogenes. Two molecular approaches including PCR and dot blot hybridization were applied. PCR using genomic DNA of D. restrictus and specific primers for NiFe-hydrogenases resulted in three amplification products of 650 bp, 833 bp, and 1.0 kb. Additionally, a PCR product obtained with specific primers for a cytochrome b gene was examined. These PCR products were sequenced and analyzed but did not show any sequence homology with already known sequences of NiFe-hydrogenases and b-type cytochromes. In a second approach, genetic probes for Fe-only- and NiFeSe-hydrogenases were used to hybridize genomic DNA of D. restrictus. A weak signal of D. restrictus DNA was seen in dot blots hybridized with the Fe-only hydrogenase probe. This result remains to be confirmed by additional experiments.

Références bases de données (Anglais)

Swiss Database: COST-DB of the State Secretariat for Education and Research Hallwylstrasse 4 CH-3003 Berne, Switzerland Tel. [phone] Swiss Project-Number: C96.0066