Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34

Abstract

The four replicons of Cupriavidus metallidurans CH34 (the genome sequence was provided by the US Department of Energy–University of California Joint Genome Institute) contain two gene clusters putatively encoding periplasmic resistance to copper, with an arrangement of genes resembling that of the copSRABCD locus on the 2.1 Mb megaplasmid (MPL) of Ralstonia solanacearum, a closely related plant pathogen. One of the copSRABCD clusters was located on the 2.6 Mb 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) were identified based on the sequencing of a fragment cloned in an IncW vector, on the preliminary annotation by the Joint Genome Institute, and by using transcriptomic and proteomic data. When introduced into plasmid-cured derivatives of C. metallidurans CH34, the cop locus was able to restore the wild-type MIC, albeit with a biphasic survival curve, with respect to applied Cu(II) concentration. Quantitative-PCR data showed that the 19 ORFs were induced from 2- to 1159-fold when cells were challenged with elevated Cu(II) concentrations. Microarray data showed that the genes that were most induced after a Cu(II) challenge of 0.1 mM belonged to the pMOL30 cop cluster. Megaplasmidic cop genes were also induced, but at a much lower level, with the exception of the highly expressed MPL copD. Proteomic data allowed direct observation on two-dimensional gel electrophoresis, and via mass spectrometry, of pMOL30 CopK, CopR, CopS, CopA, CopB and CopC proteins. Individual cop gene expression depended on both the Cu(II) concentration and the exposure time, suggesting a sequential scheme in the resistance process, involving genes such as copK and copT in an initial phase, while other genes, such as copH, seem to be involved in a late response phase. A concentration of 0.4 mM Cu(II) was the highest to induce maximal expression of most cop genes.

Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34

“…Moreover, the deletion of the two megaplasmids with one mer determinant on either plasmid decreased mercury resistance (7,52,53) but not gold resistance. This leaves the pMOL30-harbored cop determinant with more than 20 genes involved in copper transformation (28,54), which might "mishandle" gold ions.…”

“…Unfortunately, there is some confusion with names since "CopA" designates a copper-exporting P IB1 -type ATPase from E. coli but also a periplasmic copper oxidase in other bacteria, including C. metallidurans. This bacterium contains a chromosomal cupC/AR determinant and a huge copper resistance cluster on plasmid pMOL30 (5,28), which both might be involved in periplasmic modification of gold complexes.…”

Insights learned from pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1 and prevalence of accessory plasmids in other Bradyrhizobium sp. strains

“…tomato (Mills et al, 1994). Moreover, genomic analysis has revealed a large number of similar genes on bacterial plasmids and chromosomes for a variety of other microorganisms (Mergeay et al, 1995;Francki et al, 2000;Monchy et al, 2006). The pco/copABCD operon, transcribed from a copper-inducible promoter, is controlled by the two-component regulatory module pco/copRS (Mills et al, 1994).…”

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans

“…4 B and C). The cup/cop clusters contain genes encoding for sensor-, chaperone-, efflux-, cytochrome C-, and oxido-reductase proteins regulating the cyto-and periplasmic detoxification of Cu(I/II) (25). The DNAbinding transcriptional activator CupR (Rmet3525), an ortholog of CueR in Escherichia coli and GolS in S. enterica, the Cutransporting P-type ATPase CopA, as well as CopI, and the putative electron transferring cytochrome C-type copJ were strongly upregulated, suggesting their involvement in efflux and reduction of Au(III/I) to Au(0) (12,26,27).…”