Organohalide respiration in Firmicutes: energy metabolism and transcription regulation

Organohalides are a class of compounds often considered as persistent pollutants and harmful to environmental and human health. Some bacteria, among which are representatives from the Firmicutes phylum, are capable of using these compounds as terminal acceptors in a process called organohalide respiration (OHR). To do so, organohalide-respiring bacteria (OHRB) are equipped with enzymes called reductive dehalogenases which perform the terminal reduction in the OHR respiratory chain by replacing the halide by a hydrogen. OHR is not only interesting from a biological point of view but it also represents a powerful process for bioremediation purposes. The present work focuses on two different fundamental aspects of OHR which remain poorly understood.

The first part of the work focused on the transcriptional regulation of the reductive dehalogenase genes. OHRB can encode up to several dozens of different reductive dehalogenases in their genome which suggest the use of a broad number of organohalides, but this diversity remains largely unexplored. In Firmicutes OHRB, RdhK are transcriptional regulators specialised in the activation of reductive dehalogenase genes upon exposure of the cell to organohalides. Thus, the identification of RdhK binding partners (organohalide effector and DNA target sites) represents an interesting alternative to identify new OHR substrates while preventing the tedious and challenging characterisation of the complex membrane-associated and oxygen-sensitive reductive dehalogenases. Here, a strategy based on RdhK hybrid proteins was designed and optimised, and is proposed to serve to improve the efficiency of RdhK characterisation. The approach enabled a functional decoupling of the two domains of RdhK regulators targeting either the effector or the DNA target site. Therefore, it reduces the complexity of the screening procedure in the identification of RdhK binding partners. Further implementation of the hybrid strategy will increase the global comprehension of Firmicutes OHR regulatory networks and ultimately provide an indirect way to explore the reductive dehalogenase substrate diversity.

The second part of the thesis aimed to increase knowledge in the energy metabolism of Firmicutes OHRB. Quantitative comparative proteomics was applied to Desulfitobacterium hafniense strain DCB-2, which revealed the proteome adaptations to the growth on different substrates promoting either fermentation or respiration. In addition, possible roles of the complex I-like enzyme was investigated using physiological and biochemical approaches. Respiratory complex I is the entry point of electrons produced by cytoplasmic metabolic activity in the respiratory chain and is composed of three modules. Most OHRB express a version of the complex which lacks the N-module responsible for the electron uptake from NADH, suggesting the use of alternative electron donors. While a few candidate partners for the OHR complex I-like enzyme were proposed based on proteomics results, the study of the physiological role of the enzyme helped in developing different respiratory models for strain DCB-2, which mainly differ in their dependence on the complex I-like enzyme.