Organohalide respiration (OHR) is a bacterial anaerobic respiratory metabolism that makes use of halogenated organic compounds as terminal electron acceptors. While organohalogens have been initially thought to be mainly from anthropogenic origin, thousands of them are naturally produced [1]. Organohalide-respiring bacteria (OHRB) have been isolated for the first time 30 years ago and revealed to be a key player in the bioremediation of environments polluted with organohalogens. Among OHRB, Dehalobacter restrictus and Desulfitobacterium hafniense represent two model bacteria that belong to the Firmicutes. While the former is an obligate OHRB, the latter displays a very versatile energy metabolism. In OHR, reductive dehalogenases (RdhA, RDases) are the terminal reductases and are encoded in a large variety of rdh gene clusters across OHRB. Nevertheless, common features have been recognised: RDases are Tat-dependent redox enzymes, harbour a corrinoid cofactor and two iron-sulphur clusters, and are associated with the cytoplasmic membrane in OHRB [2]. In our laboratory, we focus on the functional characterisation of rdhABCT(K) gene clusters commonly found in D. restrictus and D. hafniense. First, we developed a hybrid protein strategy that will help identifying the targets of new RdhK transcriptional activators [3,4]. The highly conserved pceABCT gene cluster found in D. restrictus strain PER-K23 and D. hafniense strain TCE1, responsible for reductive dehalogenation of tetrachloroethene (PCE), was then investigated for the stoichiometry of its gene products both at RNA and protein levels. While different results were obtained for each of the strains at transcriptional level, quantitative proteomics revealed similar amounts of PceA and PceB proteins in the membrane fraction of both strains, confirming the presence of a membrane-bound reductive dehalogenase complex (RDHC). In contrast, PceC, an FMN-binding membrane protein, was detected at a significantly lower level, likely ruling out its proposed function as direct electron donor to PceA in RDHC [5]. The elucidation of the PceA-containing RDHC is investigated by a combination of mild membrane protein extraction, Clear-Native PAGE and the development of an in-gel RDase assay. So far, a ~180 kDa protein complex could be identified, the composition of which is now under scrutiny. The versatile energy metabolism of D. hafniense was the subject of a comparative physiological and proteomic approach to identify adjustments to the OHR metabolism, highlighting the possible involvement of additional protein key players. [1] Atashgahi, S., Häggblom, M.M., and Smidt, H. (2018) Organohalide respiration in pristine environments: implications for the natural halogen cycle. Environ. Microbiol. 20, 934-948. [2] Fincker, M., and Spormann, A.M. (2017) Biochemistry of catabolic reductive dehalogenation. Annu. Rev. Biochem. 86, 357-386. [3] Willemin, M.S., Vingerhoets, M., Holliger, C., and Maillard, J. (2020) Hybrid transcriptional regulators for the screening of target DNA motifs in organohalide-respiring bacteria. Front. Microbiol. 11, 310. [4] Maillard, J., and Willemin, M.S. (2019) Regulation of organohalide respiration. Adv. Microb. Physiol. 74, 191-238. [5] Buttet, G.F., Willemin, M.S., Hamelin, R., Rupakula, A., and Maillard, J. (2018) The membrane-bound C subunit of reductive dehalogenases: topology analysis and reconstitution of the FMN-binding domain of PceC. Front. Microbiol. 9, 755.