ORGANOHALIDE RESPIRATION: STUDY OF THE MEMBRANE PROTEIN COMPLEX RESPONSIBLE FOR THE REDUCTION OF HALOGENATED COMPOUNDS

Organohalide respiration (OHR) is a bacterial anaerobic respiratory process in which halogenated compounds are used as terminal electron acceptors. Desulfitobacterium and Dehalobacter, paradigmatic organohalide-respiring bacteria (OHRB), harbour the pceABCT gene cluster, representing one model system in OHR. While both Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1 dechlorinate tetrachloroethene (PCE), 3,5-dichlorophenol (DCP) is dechlorinated by D. hafniense strain DCB-2. The relatively high DNA sequence identity (70%) and similar gene composition and regulation between the pceABCT and dcpABCT gene clusters triggered our interest in studying the possible co-transcription of the genes, in establishing stoichiometric relationships between the corresponding proteins, and in trying to decipher the reductive dehalogenase (RDH) protein complex from the cytoplasmic membrane of those bacteria. Gene co-transcription was investigated by a combination of RNA extraction and RT-qPCR, while the stoichiometry of the proteins is analyzed via quantitative proteomics. Clear-Native PAGE technology combined with an in-gel reductive dehalogenase activity assay are established to identify proteins from the RDH complex. The transcriptional analysis conducted on D. hafniense strain DCB-2 showed that dcp gene cluster is actively up-regulated by 3,5-DCP. At protein level, reference peptides were defined for PceA, B, C and T from D. restrictus and D. hafniense strain TCE1 and they will be applied for measuring the relative stoichiometry of the targeted proteins. Furthermore, preliminary results from CN-PAGE analysis suggest that the PCE reductive dehalogenase (PceA) catalytic subunit is part of a ~150 kDa protein complex in D. restrictus. At transcriptional level the results suggest that dcp gene cluster shows an operon structure. However, the possibility of additional promoters regulating the transcription of individual genes within the operon are under investigation. At protein level, the identification of the RDH complex allows us to address the question of the proteins that are associated with PceA.