Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34.
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.
Monchy
Sébastien
S
Service de Confirmation des Macromolécules Biologiques et de Bioinformatique, Université Libre de Bruxelles, Belgium.|Laboratories for Microbiology and Radiobiology, SCK.CEN, Boeretang 200, B-2400 Mol, Belgium.
Benotmane
Mohammed A
MA
Laboratories for Microbiology and Radiobiology, SCK.CEN, Boeretang 200, B-2400 Mol, Belgium.
Wattiez
Ruddy
R
Département de Protéomique et de Biochimie des Protéines, University of Mons-Hainaut, B-7000 Mons, Belgium.
van Aelst
Sébastien
S
Laboratoire de Microbiologie de l'Université Libre de Bruxelles, campus CERIA, 1 av. E. Gryzon, 1070 Brussels, Belgium.|Laboratories for Microbiology and Radiobiology, SCK.CEN, Boeretang 200, B-2400 Mol, Belgium.
Auquier
Vanessa
V
Centre de Biologie Structurale et de Bioinformatique: Structure et Fonction des Membranes Biologiques, Université Libre de Bruxelles, Belgium.
Borremans
Brigitte
B
Environmental Technology, Vito, B-2400 Mol, Belgium.
Mergeay
Max
M
Laboratories for Microbiology and Radiobiology, SCK.CEN, Boeretang 200, B-2400 Mol, Belgium.
Taghavi
Safiyh
S
Brookhaven National Laboratory, Biology Department, Upton, NY 11973-5000, USA.|Environmental Technology, Vito, B-2400 Mol, Belgium.
van der Lelie
Daniel
D
Brookhaven National Laboratory, Biology Department, Upton, NY 11973-5000, USA.
Vallaeys
Tatiana
T
INRA, Département de Microbiologie, F-78850 Thiverval Grignon, France.|Laboratories for Microbiology and Radiobiology, SCK.CEN, Boeretang 200, B-2400 Mol, Belgium.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042521:Burkholderiaceae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D003300:Copper / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D020543:Proteome; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D020543:Proteome; D003300:Copper