Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Organohalide respiration (OHR) is a bacterial anaerobic process that use halogenated compounds, e.g. tetrachloroethene (PCE), as terminal electron acceptors. D. restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and D. hafniense strain TCE1, a bacterium with a versatile metabolism, harbour the pceABCT gene cluster, which represents our model system for the study of PCE respiration. To date, the function of PceA, the key enzyme in the process, and PceT, the molecular chaperone for PceA maturation, are well defined. However, the role of PceB and PceC are still not elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis that PceB and PceC may play a role in electron transfer in the metabolism of organohalide respiration is tempting but the question remains largely unanswered. In the present Ph.D. thesis, a multilevel study aiming at characterizing the electron-accepting part of the respiratory chain of OHRB will be presented. The investigation was assessed at molecular level by the deciphering of the stoichiometry of pceABCT individual gene products, and at physiological and biochemical levels, where the characterization of the membrane PceA-containing protein complex of both obligate and facultative OHRB was the main focus. The stoichiometry analysis at RNA level revealed that the pce gene cluster is an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, while low abundance of PceC in comparison to the other two proteins was observed. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. Furthermore, a combination of Clear-Native PAGE gel and a in-gel PceA enzymatic activity assay were applied to identify the RDH complex from the membrane of D. restrictus and D. hafniense strain TCE1. The results revealed an active RDH complex in the membrane extract of both organisms with an estimated molecular mass of 180 kDa, while no RDH activity could be detected in the soluble fraction. Furthermore, immunoblotting for PceA on CN-PAGE gel revealed the presence of a second, however inactive, PceA-containing complex with a molecular mass of 670 kDa, which was confirmed by MS analysis to be largely dominated by the molecular chaperone GroEL alongside with PceA and likely representing the GroEL maturation complex. The RDH complex was purified from the membrane extract by chromatography. The obtained fractions were analysed by LC-MS/MS showing unambiguously the presence of PceA and PceB. Furthermore, preliminary cryo-EM analysis led to propose a 3D reconstruction of the RDH complex, revealing the likely presence of a Pce(AB)2 complex. In summary, this study represents a road-map for the identification of RDH complexes from OHRB. The identification of PceA associated with the GroEL maturation complex raised new questions about the biosynthetic pathway of PceA and invites to consider the involvement of GroEL into the maturation of the key enzyme in organohalide respiration. Finally the preliminary results obtained via cryo-EM analysis set the basis for further investigation on the structure of the RDH complex and open new horizons towards the elucidation of the electron transfer chain involved in the reduction of PCE.