Abstract :
[en] Copper, which is prevalent in the environment, is an essential trace element required by all cellular organisms. Although this metal fulfills many biological functions, copper is also extremely toxic due to its ability to cycle between stable oxidized Cu(II) and unstable reduced Cu(I) states, which can lead to cellular damage. The dual role of copper compels microorganisms, such as bacteria, to develop resistance mechanisms to tightly control copper homeostasis. Cupriavidus metallidurans strain CH34 is a β-proteobacterium able to resist and to survive in the presence of many metal ions found in anthropogenic biotopes. Consequently, C. metallidurans CH34 is an important model for studying resistance to numerous metals.
Copper resistance in C. metallidurans CH34 essentially involves the cop cluster, which is present on the plasmid pMOL30, copVTMKNSRABCDIJGFOLQHEW. Plasmid-encoded Cop proteins include proteins involved in periplasmic copper detoxification, such as CopSRABCD; proteins involved in cytoplasmic copper detoxification, such as CopF; and proteins whose exact functions remain still unknown. The plasmid-encoded CopB protein has gained attention due to its unique N-terminal extremity, which is rich in methionine residues. The functional study of this protein is the subject of this thesis. Interestingly, the plasmid-encoded CopB protein has two distinct structural domains, as shown experimentally: an unstructured N-terminal extremity, wherein a sequential methionine-rich motif (MQGMDHSKMQGMDQGS) is repeated ten times, and a structural C-terminal domain, which contains many β-strands and few α-helices. Studies of these domains using synthetic peptides and recombinant proteins demonstrated an interaction between the sequential methionine-rich motif and Cu(I) ions and, to a lesser extent, Cu(II) ions. Subcellular fractionation and immunogold labeling of C. metallidurans CH34 in the presence of copper indicated that the CopB protein was outer-membrane-associated and might be folded into a β-barrel conformation, as shown by circular dichroism and transmembrane protein topology prediction. Chemical cross-linking experiments strengthened this study and identified the CopA protein, which is a putative multicopper oxidase, as one of the CopB-interacting partners involved in this copper resistance mechanism.
Moreover, our investigations also highlighted a vesiculation phenomenon that had not previously been described in C. metallidurans CH34 and appears to be specific to copper exposure and the presence of metal-resistance genes harbored by plasmids (e.g., pMOL28 and pMOL30). The high abundance of copper ions complexed by the periplasmic Cop proteins inside these outer membrane vesicles suggested that this vesiculation production may serve as a new copper resistance mechanism in C. metallidurans CH34. This study paves the way towards a better understanding of the fate of copper in this microorganism.
