• Citer

Towards a better understanding of bacterial resistance to heavy metal ions: the case of the Sil and Zne systems from Cupriavidus metallidurans CH34

par Ngonlong Ekende, Elisabeth
Président du jury Verbruggen, Nathalie
Promoteur Goormaghtigh, Erik ;Vandenbussche, Guy
Publication Non publié, 2012-06-18

Thèse de doctorat

• Accès en ligne
• Détails
• Contenu
• Statistiques

Résumé :

Cupriavidus metallidurans CH34 is a Gram-negative -proteobacterium which has an outstanding ability of thriving in the presence of milli-molar range concentrations of over 20 different heavy metal ions like copper, mercury, zinc, nickel, silver, cobalt, cadmium, lead … Therefore, C. metallidurans CH34 has been considered as a model organism for the study of microorganism resistance to heavy metal ions. The metal detoxification mainly occurs via ion efflux. Among the efflux systems found in C. metallidurans CH34, there are 12 potential HME-RND-driven systems (Heavy Metal Efflux - Resistance Nodulation and cell Division) encoded in the bacterial genome. However, many of these systems have not been yet characterized phenotypically and their function remains undetermined. Such HME-RND-type complexes are composed of three proteins spanning the complete cell envelope and mediating cation/proton antiporter efflux using a chemiosmotic gradient from the intracellular space into the exterior of the cell. Our group focuses on studying the HME-RND-driven systems SilCBA and ZneBAC, particularly in topological, functional and structural points of view. The membrane proteome of C. metallidurans CH34 was previously studied in our laboratory. This previous study showed an induction of the Sil proteins in the presence of copper or silver. In the present work, using a functional assay in vivo, we associated a specific copper and silver ions transport activity with the Sil system. We also report that SilA is able to mediate a transport of the metal ions from the cytoplasmic space to the periplasm. Based on multiple sequence alignments, we identified a cytoplasmic domain located between the transmembrane helices four and five and comprising residues involved in metal coordination sites (e.g. histidine, glutamate). We investigated the role of this domain by mutagenesis and we showed that it could be potentially involved in copper binding and transport. Moreover, the conformational changes undergone by SilA upon substrate binding have been studied in vitro by fluorescence and infrared spectroscopy after reconstitution of the membrane protein in a lipidic environment. Regarding the ZneBAC system, our group has previously demonstrated that ZneB was able to bind zinc and to co-crystallize in the presence of this metal ion. In order to gain complementary structural data on the ZneBAC proteins, we studied the inner membrane protein ZneA by X-ray crystallography. We co-crystallized ZneA in the presence of zinc ions using two detergents (α- or β-DDM). The structures revealed that ZneA is assembled as a homotrimer and showed sequential conformational changes during the efflux cycle. Differences have been observed between the monomers in both the spatial arrangement of two proximal substrate binding sites, as well as the occupancies of the Zn2+ ion bound to each site. Therefore, we suggest that each monomer conformation of ZneA represents an intermediate state in a transport cycle occurring from the periplasm to the outer membrane channel ZneC and finally to the outside of the cell. The role of these sites was studied by site directed mutagenesis and by an in vitro assay for Zn2+ transport.