Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans Are Specialized in the Maximal Viable Response to Heavy Metals

Abstract

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...

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans

“…Another study showed that multiple-metal responses by genes belonging to the cop, cnr, mer, and pbr loci occurred after challenging C. metallidurans CH34 with Cd 2ϩ , Pb 2ϩ , Zn 2ϩ , and Cu 2ϩ (28). This suggests that C. metallidurans uses several strategies to reduce the intracellular concentration of toxic metal ions.…”

“…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). Experiments with C. metallidurans had demonstrated the induction of cupR with Au(I) (28). This suggests that the cup/cop system is a general metal response system that is readily inducible at low levels of metal stress, rather than a Cu-specific resistance system.…”

Structure of the Periplasmic Stress Response Protein CpxP

“…ZraP is a metal-binding protein that is induced by a two-component system that responds to zinc or lead (33,42), while CnrX is thought to act as a metal sensor that binds cobalt, nickel, and potentially copper in an extracytoplasmic function (ECF)-sigma factor signaling pathway (18,19,34,38,61). Interestingly, copper ions have been implicated as an inducer of the Cpx pathway (26,(67)(68)(69).…”

Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation

“…Cupriavidus metallidurans CH34 [28,29], Escherichia coli JM109 (pTP6) [26] and Cupriavidus necator JMP134 (pJP4) [30] were grown in Luria Bertani (LB) medium (contained per l: 10 g tryptone, 5 g yeast extract and 10 g NaCl) [31] [28]. Succinate (0.3%) was used as sole carbon source.…”