Development of an in vivo Model to Investigate the Role of the Heavy Metal Efflux System Sil from cupriavidus Metallidurans Ch34 in Resistance to Silver and Copper Ions

Abstract

The RND-type (Resistance Nodulation and cell Division) efflux systems play an important role in the ability of bacteria to survive in the presence of a broad range of toxic compounds. Two major subfamilies are represented by the systems involved in Heavy Metal Efflux (HME-RND) or Hydrophobic and Amphiphilic compounds (e.g. antibiotics) Efflux (HAE-RND). These tripartite protein complexes are composed of an inner membrane (RND) and an outer membrane (member of the Outer Membrane Factor family - OMF) component linked together by a periplasmic adaptator protein (member of the Membrane Fusion Protein family - MFP). The RND protein is a cation/proton antiporter and is responsible for the substrate specificity. SilABC is an uncharacterized HME-RND system from C. metallidurans CH34. Using a proteomic approach, we have previously demonstrated the induction of Sil proteins in response to the presence of silver or copper in the culture medium. We report here on the development of an in vivo model to investigate the role of the SilABC proteins in the active transport of these heavy metal ions. The metal-sensitive E. coli strain GR17 was transformed with the silABC genes. Silver and copper tolerance of transformed bacteria was evaluated by the determination of minimal inhibitory concentration (MIC) values in a minimal Tris-glucose medium. A twofold increase in MIC values was observed when the three proteins were expressed demonstrating that the SilABC system transports efficiently silver and copper ions in vivo. In addition, we have demonstrated that the RND protein SilA alone is able to mediate partial resistance which is compatible with its cation/proton antiporter function.