The four replicons of Cupriavidus metallidurans CH34 contain two gene clusters putatively encoding periplasmic resistance to copper. One of these copSRABCD clusters was located on the 2.6 Mb megaplasmid (MPL) while the second was found on the pMOL30 (234 kb) plasmid as part of a larger group of genes involved in copper resistance spanning 17,857 bp in total. In this region, 19 ORFS (copVTMKNSRABCDIJGFLQHE) could be identified based on the sequencing and by using transcriptomic and proteomic data. When introduced in plasmid-cured derivatives of C. metallidurans CH34, the cop locus was able to restore the wild-type MIC (Minimum Inhibitory Concentration). Quantitative-PCR data show that the 19 ORFs were from 2 to 1159 fold induced when cells were challenged with elevated Cu(II) concentrations. Microarray data showed that the most induced genes after a Cu(II) challenge of 0.1 mM belong to the pMOL30 cop cluster. Proteomic data allowed the direct observation on bidimensional gel electrophoresis (2-DE) and via mass spectrometry. Individual cop gene expression depends on both applied Cu(II) concentration and exposure time, suggesting a sequential scheme in the resistance process. The applied concentration of 0.4 mM Cu(II) is the highest to induce maximal expression of most cop genes.
|State||Published - Jun 2006|