Abstract
Copper (Cu) is an essential micronutrient for all living organisms as this metal is required as a cofactor for crucial enzymes. However, the excess of Cu is toxic. The main Cu-induced toxicity mechanisms in bacteria rely on (i) its high redox potential resulting in deleterious oxidative stress and (ii) the displacement of the iron cofactor in proteic iron-sulfur clusters. Consequently, bacteria have evolved various mechanisms to maintain proper intracellular Cu concentrations. One of the most interesting bacterial model systems to investigate a wide range of topics including Cu tolerance is the asymmetric alpha-proteobacterium Caulobacter crescentus. In the laboratory, it was previously demonstrated that C. crescentus is able to establish a rapid fight-or-flight response upon Cu stress. While the stalk cell fights Cu using detoxification and efflux mechanisms, the swarmer cell flees away from it via negative chemotaxis.To investigate novel mechanisms involved in C. crescentus Cu tolerance, 2 different approaches were applied: a Tn-based genetic screening and RNA-seq analysis. One of the best candidates that was found in both approaches is an operon consisting of 3 genes coding for a TonB-Dependent receptor (TBDR), a small RNA (R97), and a 2-oxoglutarate iron-dependent oxygenase (OxcT), respectively. While the characterization of the TBDR is ongoing in the lab, the roles of both R97 sRNA and OxcT oxygenase remain to be studied. Hence, we aimed to investigate the involvement of the product from these 2 genes in Cu tolerance. The R97 knock-out strain indicated that the R97 sRNA does not seem essential in Cu tolerance.
Conversely, the deletion of oxcT gene induced an increased sensitivity of C. crescentus to Cu, indicating that OxcT protein is important for Cu tolerance. Therefore, the characterization of OxcT mechanism underlying Cu tolerance was initiated during this project. Secondary structure prediction of OxcT indicated the presence of a conserved double strands beta-helix (DSBH), while the Fe binding site consists of conserved residues: 2 histidines and 1 aspartic acid. The measurement of intracellular Cu concentration revealed a higher Cu concentration in the ΔoxcT mutant strain compared to the WT suggesting some cooperation of OxcT with a Cu efflux system. Finally, partner proteins of OxcT were identified using AP-MS and some interesting function hypotheses were addressed based on the best candidates.
| Date of Award | 21 Jan 2021 |
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| Original language | English |
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| Supervisor | Jean-Yves Matroule (Supervisor) |