Organohalide respiration: breakthrough towards the elucidation of the electron-accepting moiety of the respiratory chain

Background Organohalide respiration (OHR) is a bacterial anaerobic process in which chlorinated compound, e.g. tetrachloroethene (PCE), is used as terminal electron acceptors. Dehalobacter restrictus PER-K23, a paradigmatic organohalide-respiring bacterium (OHRB), harbours the pceABCT gene cluster, representing one 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. PceB and PceC are predicted integral membrane proteins, however no evidence has been obtained for their physiological function. The present work aims to investigate the stoichiometric relationships between the proteins encoded in the pceABCT gene cluster and to elucidate the membrane-bound reductive dehalogenase (RDH) protein complex. Methods The stoichiometry of the pceABCT gene products was assessed at RNA and protein level by RT-qPCR and quantitative proteomics, respectively. Clear-Native PAGE combined with an in-gel RDH activity assay was applied to identify the RDH complex from membrane extracts of D. restrictus. Results At RNA level, pceA, pceB, pceC and pceT genes appeared with relative stoichiometry (normalized for pceA) of approximately 1.0:3.0:0.1:0.1. At proteomic level, the cell-free extract of D. restrictus displayed a stoichiometry of 1.0:0.5:0.02:0.2 for the proteins PceA, PceB, PceC and PceT, respectively. CN-PAGE and in-gel enzymatic assay revealed an active RDH complex with a molecular mass of ~180 kDa identified in the membrane extract, while no in-gel activity could be detected in the soluble fraction. Conclusions Taken together, the results suggest that PceA and PceB form a complex in the membrane, while PceC might act as an accessory protein. Furthermore, the in-gel RDH activity assay allowed for the first time to visualise an active ~180 kDa PceA-containing complex from the membrane. The protein composition of the active RDH complex is currently underway and shall be presented at the conference.