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Régulation in labo et in situ de l'expression de génomes de souches bactériennes méthylotrophes dégradant le dichlorométhane

Abstract : Dichloromethane (DCM; CH2Cl2) is a toxic chlorinated pollutant mainly emitted in the environment through industrial activities. Some methylotrophic bacteria that utilize reduced one carbon compounds as sole source of carbon and energy can degrade DCM. The four dcm genes found in the catabolic dcm transposon are highly conserved among DCM-degrading bacteria, including in the Proteobacterium Methylorubrum extorquens DM4. The dcmA gene encodes a DCM dehalogenase of the glutathione-S transferase family which is essential for growth with DCM. The transcriptional factor DcmR regulates the expression of dcmA and its own gene, two genes which are divergently oriented. DcmR consists of a helix-turn-helix domain for DNA fixation and of a domain, called MEDS for ‘methanogen / methylotroph, DcmR sensory’, proposed to bind a hydrocarbon ligand. The aim of my PhD was to address the following questions: i) What is the expression level of dcm transcripts and their corresponding proteins compared to other genes and proteins whose abundance is modulated in response to growth with DCM? ii) How does DcmR regulate the expression of the dcm genes? iii) How variable is dcmR and its genetic environment in situ? Global transcriptomic and proteomic approaches were followed to inventory transcripts and proteins whose abundance varied in the wild-type DM4 strain grown with DCM compared to with methanol, the reference substrate for methylotrophy. The dcm genes were among the most expressed in cultures grown with DCM, confirming their regulation in response to DCM. The identification of transcription start sites (TSS-Seq) and translation starts (N-terminome) allowed the subsequent search for regulation motifs in the promoter and 5’-untranslated terminal regions (5’UTR) of regulated genes in response to DCM utilization. The role of dcmR was studied using growth phenotypes, promoter activities in transcription fusion assays, dcm transcript and corresponding protein product quantification, comparing the wild-type strain with strains mutated only in dcmR, or also in other dcm genes. In absence of DCM, DcmR inhibited the transcription of its own gene and of dcmA. In addition to DcmR, repression requires the expression of one of the other dcm genes by a mechanism that does not involve previously predicted DcmR-binding sites (a 12 bp conserved box shared by dcmR and dcmA promoters). Mutants with impaired dcmR show slower growth with DCM, although no difference in transcript or protein abundance encoded by the dcm transposon was observed. The complete dcmR-dcmA intergenic region was shown to be required to activate dcmA expression. This suggests that regulatory sites present in the intergenic region may be involved in DcmR-independent transcription activation. Taken together, these data allowed to propose a new model of dcm gene regulation. The dcmR gene was detected and quantified in similar amounts as dcmA in DCM-contaminated environmental samples, despite the fact that bioinformatics analysis of sequence databases suggests that dcmR-like genes are not necessary associated with the catabolic dcm transposon. Thus, DcmR may play a role in other contexts. This may provide new leads for future investigations of potential ligands of the MEDS domain.
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Submitted on : Saturday, January 25, 2020 - 1:01:45 AM
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Bruno Maucourt. Régulation in labo et in situ de l'expression de génomes de souches bactériennes méthylotrophes dégradant le dichlorométhane. Génomique, Transcriptomique et Protéomique [q-bio.GN]. Université de Strasbourg, 2019. Français. ⟨NNT : 2019STRAJ003⟩. ⟨tel-02454963⟩



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