1887

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28593-0
2006-06-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/6/1765.html?itemId=/content/journal/micro/10.1099/mic.0.28593-0&mimeType=html&fmt=ahah

References

  1. Ames, G. F., Prody, C. & Kustu, S. ( 1984; ). Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol 160, 1181–1183.
    [Google Scholar]
  2. Arnesano, F., Banci, L., Bertini, I., Mangani, S. & Thompsett, A. R. ( 2003; ). A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites. Proc Natl Acad Sci U S A 100, 3814–3819.[CrossRef]
    [Google Scholar]
  3. Bender, C. L. & Cooksey, D. A. ( 1987; ). Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato. J Bacteriol 169, 470–474.
    [Google Scholar]
  4. Borremans, B., Hobman, J. L., Provoost, A., Brown, N. L. & van Der Lelie, D. ( 2001; ). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol 183, 5651–5658.[CrossRef]
    [Google Scholar]
  5. Brim, H., Heyndrickx, M., de Vos, P., Wilmotte, A., Springael, D., Schlegel, H. & Mergeay, M. ( 1999; ). Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs. Syst Appl Microbiol 22, 258–268.[CrossRef]
    [Google Scholar]
  6. Brown, N. L., Rouch, D. A. & Lee, B. T. ( 1992; ). Copper resistance determinants in bacteria. Plasmid 27, 41–51.[CrossRef]
    [Google Scholar]
  7. Cha, J. S. & Cooksey, D. A. ( 1991; ). Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proc Natl Acad Sci U S A 88, 8915–8919.[CrossRef]
    [Google Scholar]
  8. Chen, Y. T., Chang, H. Y., Lai, Y. C., Pan, C. C., Tsai, S. F. & Peng, H. L. ( 2004; ). Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene 337, 189–198.[CrossRef]
    [Google Scholar]
  9. Corbisier, P. ( 1997; ). Bacterial metal-lux biosensors for a rapid determination of the heavy metal bioavailability and toxicity in solid samples. Res Microbiol 148, 534–536.[CrossRef]
    [Google Scholar]
  10. Diels, L. & Mergeay, M. ( 1990; ). DNA probe-mediated detection of resistant bacteria from soils highly polluted by heavy metals. Appl Environ Microbiol 56, 1485–1491.
    [Google Scholar]
  11. Diels, L., Sadouk, A. & Mergeay, M. ( 1989; ). Large plasmids governing multiple resistance to heavy metals: a genetic approach. Toxicol Environ Chem 23, 79–89.[CrossRef]
    [Google Scholar]
  12. Ettema, T. J., Huynen, M. A., de Vos, W. M. & van der Oost, J. ( 2003; ). trash: a novel metal-binding domain predicted to be involved in heavy-metal sensing, trafficking and resistance. Trends Biochem Sci 28, 170–173.[CrossRef]
    [Google Scholar]
  13. Figurski, D. H. & Helinski, D. R. ( 1979; ). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[CrossRef]
    [Google Scholar]
  14. Francki, K. T., Chang, B. J., Mee, B. J., Collignon, P. J., Susai, V. & Keese, P. K. ( 2000; ). Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria. FEMS Immunol Med Microbiol 29, 115–121.[CrossRef]
    [Google Scholar]
  15. Gilmour, M. W., Thomson, N. R., Sanders, M., Parkhill, J. & Taylor, D. E. ( 2004; ). The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics. Plasmid 52, 182–202.[CrossRef]
    [Google Scholar]
  16. Goris, J., De Vos, P., Coenye, T. & 7 other authors ( 2001; ). Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend. Int J Syst Evol Microbiol 51, 1773–1782.[CrossRef]
    [Google Scholar]
  17. Grass, G., Grosse, C. & Nies, D. H. ( 2000; ). Regulation of the cnr cobalt and nickel resistance determinant from Ralstonia sp. strain CH34. J Bacteriol 182, 1390–1398.[CrossRef]
    [Google Scholar]
  18. Grosse, C., Anton, A., Hoffmann, T., Franke, S., Schleuder, G. & Nies, D. H. ( 2004; ). Identification of a regulatory pathway that controls the heavy-metal resistance system Czc via promoter czcNp in Ralstonia metallidurans. Arch Microbiol 182, 109–118.
    [Google Scholar]
  19. Juhnke, S., Peitzsch, N., Hübener, N., Grosse, C. & Nies, D. ( 2002; ). New genes involved in chromate resistance in Ralstonia metallidurans strain CH34. Arch Microbiol 179, 15–25.[CrossRef]
    [Google Scholar]
  20. Lee, S. M., Grass, G., Rensing, C., Barrett, S. R., Yates, C. J., Stoyanov, J. V. & Brown, N. L. ( 2002; ). The Pco proteins are involved in periplasmic copper handling in Escherichia coli. Biochem Biophys Res Commun 295, 616–620.[CrossRef]
    [Google Scholar]
  21. Legatzki, A., Grass, G., Anton, A., Rensing, C. & Nies, D. H. ( 2003; ). Interplay of the Czc system and two P-type ATPases in conferring metal resistance to Ralstonia metallidurans. J Bacteriol 185, 4354–4361.[CrossRef]
    [Google Scholar]
  22. Lim, C. K. & Cooksey, D. A. ( 1993; ). Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae. J Bacteriol 175, 4492–4498.
    [Google Scholar]
  23. Mellano, M. A. & Cooksey, D. A. ( 1988; ). Induction of the copper resistance operon from Pseudomonas syringae. J Bacteriol 170, 4399–4401.
    [Google Scholar]
  24. Mergeay, M. ( 1995; ). Heavy metal resistances in microbial ecosystems. In Molecular Microbial Ecology Manual, pp. 6.1.7.1–6.1.7.17. Dordrecht: Kluwer.
  25. Mergeay, M. ( 2000; ). Bacteria adapted to industrial biotopes: metal-resistant Ralstonia. In Bacterial Stress Responses, pp. 403–414. Edited by R. Hengge-Aronis & G. Storz. Washington, DC: American Society for Microbiology.
  26. Mergeay, M., Houba, C. & Gerits, J. ( 1978; ). Extrachromosomal inheritance controlling resistance to cadmium, cobalt, copper and zinc ions: evidence from curing in a Pseudomonas [proceedings]. Arch Int Physiol Biochim 86, 440–442.
    [Google Scholar]
  27. Mergeay, M., Nies, D., Schlegel, H. G., Gerits, J., Charles, P. & Van Gijsegem, F. ( 1985; ). Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162, 328–334.
    [Google Scholar]
  28. Mergeay, M., Monchy, S., Vallaeys, T. & 7 other authors ( 2003; ). Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS Microbiol Rev 27, 385–410.[CrossRef]
    [Google Scholar]
  29. Mills, S. D., Lim, C. K. & Cooksey, D. A. ( 1994; ). Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae. Mol Gen Genet 244, 341–351.
    [Google Scholar]
  30. Monchy, S., Vallaeys, T., Bossus, A. & Mergeay, M. ( 2006; ). Metal efflux P1-ATPase genes of Cupriavidus metallidurans CH34: a transcriptomic approach. Int J Environ Anal Chem (in press).
    [Google Scholar]
  31. Nies, D. H. ( 2000; ). Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34. Extremophiles: Life Under Extreme Conditions 4, 77–82.[CrossRef]
    [Google Scholar]
  32. Nies, D. H. ( 2003; ). Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27, 313–339.[CrossRef]
    [Google Scholar]
  33. Noel-Georis, I., Vallaeys, T., Chauvaux, R., Monchy, S., Falmagne, P., Mergeay, M. & Wattiez, R. ( 2004; ). Global analysis of the Ralstonia metallidurans proteome: prelude for the large-scale study of heavy metal response. Proteomics 4, 151–179.[CrossRef]
    [Google Scholar]
  34. Pearson, W. R. & Lipman, D. J. ( 1988; ). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.[CrossRef]
    [Google Scholar]
  35. Rensing, C. & Grass, G. ( 2003; ). Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiol Rev 27, 197–213.[CrossRef]
    [Google Scholar]
  36. Rensing, C., Fan, B., Sharma, R., Mitra, B. & Rosen, B. P. ( 2000; ). CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci U S A 97, 652–656.[CrossRef]
    [Google Scholar]
  37. Salanoubat, M., Genin, S., Artiguenave, F. & 25 other authors ( 2002; ). Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415, 497–502.[CrossRef]
    [Google Scholar]
  38. Schlegel, H. G., Kaltwasser, H. & Gottschalk, G. ( 1961; ). A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies. Arch Mikrobiol 38, 209–222.[CrossRef]
    [Google Scholar]
  39. Schmidt, T. & Schlegel, H. G. ( 1994; ). Combined nickel–cobalt–cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. J Bacteriol 176, 7045–7054.
    [Google Scholar]
  40. Staskawicz, B., Dahlbeck, D., Keen, N. & Napoli, C. ( 1987; ). Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 5789–5794.
    [Google Scholar]
  41. Taghavi, S., Mergeay, M. & van der Lelie, D. ( 1997; ). Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality. Plasmid 37, 22–34.[CrossRef]
    [Google Scholar]
  42. Tetaz, T. J. & Luke, R. K. ( 1983; ). Plasmid-controlled resistance to copper in Escherichia coli. J Bacteriol 154, 1263–1268.
    [Google Scholar]
  43. Tricot, C., van Aelst, S., Wattiez, R., Mergeay, M., Stalon, V. & Wouters, J. ( 2005; ). Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans. Acta Crystallograph Sect F Struct Biol Cryst Commun 61, 825–827.[CrossRef]
    [Google Scholar]
  44. Vandamme, P. & Coenye, T. ( 2004; ). Taxonomy of the genus Cupriavidus: a tale of lost and found. Int J Syst Evol Microbiol 54, 2285–2289.[CrossRef]
    [Google Scholar]
  45. Vaneechoutte, M., Kampfer, P., De Baere, T., Falsen, E. & Verschraegen, G. ( 2004; ). Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol 54, 317–327.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28593-0
Loading
/content/journal/micro/10.1099/mic.0.28593-0
Loading

Data & Media loading...

Oligonucleotides spotted on the microarrays for both pMOL30 and MPL gene clusters. [PDF](13 kb) Primer sequences for all genes investigated for expression analysis using quantitative PCR. [PDF](14 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 5 min of copper induction. [PDF](17 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 60 min of copper induction. [PDF](17 kb)

PDF

Oligonucleotides spotted on the microarrays for both pMOL30 and MPL gene clusters. [PDF](13 kb) Primer sequences for all genes investigated for expression analysis using quantitative PCR. [PDF](14 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 5 min of copper induction. [PDF](17 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 60 min of copper induction. [PDF](17 kb)

PDF

Oligonucleotides spotted on the microarrays for both pMOL30 and MPL gene clusters. [PDF](13 kb) Primer sequences for all genes investigated for expression analysis using quantitative PCR. [PDF](14 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 5 min of copper induction. [PDF](17 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 60 min of copper induction. [PDF](17 kb)

PDF

Oligonucleotides spotted on the microarrays for both pMOL30 and MPL gene clusters. [PDF](13 kb) Primer sequences for all genes investigated for expression analysis using quantitative PCR. [PDF](14 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 5 min of copper induction. [PDF](17 kb) Transcriptomic analysis of pMOL30 genes by quantitative PCR after 60 min of copper induction. [PDF](17 kb)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error