Purification & characterization of PceC protein involved in dehalorespiration by desulfitobacterium & dehalobacter

Dehalorespiration is a process during which microbes derive energy by the reductive dehalogenation of chlorinated organic pollutants, by using them as electron acceptors. Characterization of the full dehalorespiration pathway in dehalogenating organisms is of great importance, as their physiological properties and substrate range will determine the extent of the bioremediation process and the conditions that should be used. Various Desulfitobacterium strains and Dehalobacter restrictus harbor the pceABCT gene cluster responsible for tetrachloroethene (PCE) anaerobic respiration. Our lab has previously focused on isolation & characterization of the proteins PceA and PceT, the reductive dehalogenase and its specific maturation factor, respectively. PceB is expected to play the role of the membrane anchor for PceA. We are now currently investigating the role of PceC in dehalorespiration. Sequence analysis revealed that PceC is homologous to CprC encoded in the chlorophenol reductive dehalogenase operon which has been postulated to be a membrane-bound FMN-binding transcription regulator of the NosR /NirI family. The goal of the work presented here is the heterologous production and purification of the predicted soluble FMN-binding domain in E. coli in order to raise PceC antibodies and allow an accurate detection and localization of PceC in membranes of Desulfitobacterium and Dehalobacter cells. This should also help in providing stoichiometric analysis of PceC in relation to PceA. For further biochemical characterization reconstitution of the active FMN-binding domain will be attempted.