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Détection et signalisation du monoxyde de carbone chez des bactéries aérobies - Hémo-senseur RcoM-2 et réponses mycobactériennes au CO

Abstract : The toxic gas CO can act in low quantities as signaling molecule; detected by heme-based sensor proteins.The CO-dependent transcription factor RcoM-2 has a very high affinity for CO, while being insensitive to O₂. RcoM-2 binds to DNA only when CO is bound to heme. We characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2. RcoM-2 can bind CO with lower effective affinity than RcoMH-2. CO dissociates with a 20-fold higher rate than in RcoMH-2, where CO binding is almost irreversible. A small fraction of CO can escape from the protein, thus allowing RcoM-2 to act as CO sensor. The presence of the DNA binding domain influences the binding properties of CO to heme. Identification of the precise molecular origin of the dynamic properties must await the 3D structure RcoM-2.CO detection is crucial for Mtb, the infectious agent of tuberculosis that must overcome the host's defense mechanisms, including CO. The gene cor (rv1829) has been implicated in these processes. We have shown that Cor is a highly stable dimer that is able to stoichiometrically bind a heme cofactor, suggesting a potential function as direct CO sensor. A histidine residue was identified as potential heme ligand. The internal CO dynamics is very similar to other bacterial CO sensors. Cor exhibits DNA binding activity that depends on the presence of heme and CO, which is abolished in the H70A mutant. Our studies also showed that the transcriptional regulator Rv0081, induced in response to gaz changes, can bind to the predicted regulatory region of cor. The creation of a Δcor strain in the non- pathogenic model M. smegmatis continues and will be a first step towards transcriptomic analyzes.
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Mayla Salman. Détection et signalisation du monoxyde de carbone chez des bactéries aérobies - Hémo-senseur RcoM-2 et réponses mycobactériennes au CO. Biologie moléculaire. Université Paris Saclay (COmUE), 2019. Français. ⟨NNT : 2019SACLX076⟩. ⟨tel-02484860⟩

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