We fully annotated two large plasmids, pMOL28 (164 open reading frames [ORFs]; 171,459 bp) and pMOL30 (247 ORFs; 233,720 bp), in the genome of Cupriavidus metallidurans CH34. pMOL28 contains a backbone of maintenance and transfer genes resembling those found in plasmid pSym of C. taiwanensis and plasmid pHG1 of C. eutrophus, suggesting that they belong to a new class of plasmids. Genes involved in resistance to the heavy metals Co(II), Cr(VI), Hg(II), and Ni(II) are concentrated in a 34-kb region on pMOL28, and genes involved in resistance to Ag(I), Cd(II), Co(II), Cu(II), Hg(II), Pb(II), and Zn(II) occur in a 132-kb region on pMOL30. We identified three putative genomic islands containing metal resistance operons flanked by mobile genetic elements, one on pMOL28 and two on pMOL30. Transcriptomic analysis using quantitative PCR and microarrays revealed metal-mediated up-regulation of 83 genes on pMOL28 and 143 genes on pMOL30 that coded for all known heavy metal resistance proteins, some new heavy metal resistance proteins (czcJ, mmrQ, and pbrU), membrane proteins, truncated transposases, conjugative transfer proteins, and many unknown proteins. Five genes on each plasmid were down-regulated; for one of them, chrI localized on pMOL28, the down-regulation occurred in the presence of five cations. We observed multiple crossresponses (induction of specific metal resistance by other metals), suggesting that the cellular defense of C. metallidurans against heavy metal stress involves various regulons and probably has multiple stages, including a more general response and a more metal-specific response.Cupriavidus (formerly Ralstonia) metallidurans CH34 is a facultatively hydrogenotrophic, metal-resistant bacterium isolated from the sludge of a zinc decantation tank in Belgium that was contaminated with high concentrations of several heavy metals (2,15,23,25). The Joint Genome Institute (http://genome.jgi-psf.org /finished_microbes/ralme/ralme.home.html) sequenced the entire genome of this strain, which revealed four replicons: the chromosome (3.9 Mb), a megaplasmid (2.6 Mb), and two large plasmids, pMOL28 (171 kb) and pMOL30 (234 kb). C. metallidurans CH34 displays a variety of responses or resistance to several heavy metals; plasmid curing and transfer experiments demonstrated that the two plasmids are involved in metal resistance.Plasmid pMOL28 was associated with resistance to Ni(II), Co(II), CrO 4 2Ϫ , and Hg(II), and pMOL30 was associated with resistance to Ag(I), Cd(II), Co(II), Cu(II), Hg(II), Pb(II), and Zn(II). Cloning and sequencing of several fragments from both plasmids revealed many determinants of resistance (cnr, chr, and mer for pMOL28; czc, pbr, mer, sil, and cop for pMOL30) (1,9,18,22,27,30). The mechanisms of resistance included chemoosmotic efflux of cations with proton antiporters (HME-RND family) encoded by czcCBA, cnrCBA, and silCBA, cation diffusion facilitators such as CzcD and CnrT, and P-type ATPases for cytoplasmic detoxification (pbrA, copF, and czcP). The large copVTMKNS1R1A1B1C1D1IJGFLQ...
Classification of metal-resistant bacteria from industrial biotopes as), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain ; an emendation of the latter species description is therefore proposed.
The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni 2؉ and 2.0 mM Co 2؉ in a noncomplexing medium for metals. The two promoter regions P Y (upstream cnrY) and P H (upstream cnrH) were probed and characterized using this vector and were found to control the nickelinducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.The resistance of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) against multiple heavy metals can be regarded as a phenomenon in its own right (15) and has found increasing applications within the field of environmental technology (29). The metal resistance determinants, which are localized predominantly on either of the two indigenous megaplasmids pMOL28 (15, 28) and pMOL30 (15), have been recently reviewed by Taghavi et al. (27). One important application has been the development of metal-specific biosensors based on in vivo and in vitro gene fusions of CH34 heavy metal resistance determinants. This was possible only through a detailed knowledge of the regulation of the metal resistances at the molecular level (2).The cnr operon, located on pMOL28 (13, 28), encodes a phenotype of inducible resistance to 5 mM Co 2ϩ and 3 mM Ni 2ϩ in minimal medium. R...
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