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Abstract

The four replicons of 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 locus on the 2.1 Mb megaplasmid (MPL) of , a closely related plant pathogen. One of the 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 () 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 CH34, the 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 cluster. Megaplasmidic genes were also induced, but at a much lower level, with the exception of the highly expressed MPL . 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 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 and in an initial phase, while other genes, such as , 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 genes.

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2006-06-01
2024-03-19
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