Cell Biology and Toxicology

, Volume 30, Issue 4, pp 207–232 | Cite as

cDNA microarray assessment of early gene expression profiles in Escherichia coli cells exposed to a mixture of heavy metals

  • María T. Gómez-Sagasti
  • José M. Becerril
  • Iker Martín
  • Lur Epelde
  • Carlos Garbisu
Original Research


Many contaminated sites are characterized by the presence of different metals, thus increasing the complexity of toxic responses in exposed organisms. Within toxicogenomics, transcriptomics can be approached through the use of microarrays aimed at producing a genetic fingerprint for the response of model organisms to the presence of chemicals. We studied temporal changes in the early gene expression profiles of Escherichia coli cells exposed to three metal doses of a polymetallic solution over three exposure times, through the application of cDNA microarray technology. In the absence of metals, many genes belonging to a variety of cellular functions were up- and down-regulated over time. At the lowest metal dose, an activation of metal-specific transporters (Cus and ZraP proteins) and a mobilization of glutathione transporters involved in metal sequestration and trafficking was observed over time; this metal dose resulted in the generation of ROS capable of stimulating the transcription of Mn-superoxide dismutase, the assembly of Fe-S clusters and the synthesis of cysteine. At the intermediate dose, an overexpression of ROS scavengers (AhpF, KatG, and YaaA) and heat shock proteins (ClpP, HslV, DnaK, and IbpAB) was observed. Finally, at the highest dose, E. coli cells showed a repression of genes related with DNA mutation correctors (MutY glycopeptidases).


Exposure time Metal toxicity Toxicogenomics Trace elements Transcriptomics 



This work has been financially supported by the following projects: 7/12/TK/2009/3 LURCHIP (Biscay County Council), BERRILUR3-Etortek (Basque Government), and MINECO AGL2012-39715-CO3-01/02. M.T. Gómez-Sagasti is the recipient of a predoctoral fellowship from the Department of Education, Universities and Research, Basque Government. Technical support by Javier Etxebarria and Amaia García from GAIKER is gratefully acknowledged.


  1. Addis P, Shecterle LM, St. Cyr JA. Cellular protection during oxidative stress: a potential role for D-ribose and antioxidants. J Diet Suppl. 2012;9:178–82.PubMedCrossRefGoogle Scholar
  2. Agrawal SB, Agrawal M, Lee EH, Kramer GF, Pillai P. Changes in polyamine and glutathione contents of a green alga, Chlorogonium elongatum (Dang) France exposed to mercury. Environ Exp Bot. 1992;32:145–51.CrossRefGoogle Scholar
  3. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245–50.PubMedCrossRefGoogle Scholar
  4. Aryee M, Gutierrez-Pabello J, Kramnik I, Maiti T, Quackenbush J. An improved empirical Bayes approach to estimating differential gene expression in microarray time-course data: BETR (Bayesian Estimation of Temporal Regulation). BMC Bioinforma. 2009;10:409.CrossRefGoogle Scholar
  5. Asad NR, Asad L, de Almeida CEB, Felzenszwalb I, Cabral-Neto JB, Leitão AC. Several pathways of hydrogen peroxide action that damage the Escherichia coli genome. Genet Mol Biol. 2004;27:291–303.CrossRefGoogle Scholar
  6. Baek YW, An YJ. Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus. Sci Total Environ. 2011;409:1603–8.PubMedCrossRefGoogle Scholar
  7. Bagai I, Liu W, Rensing C, Blackburn NJ, McEvoy MM. Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system. J Biol Chem. 2007;282:35695–702.PubMedCrossRefGoogle Scholar
  8. Barras F, Fontecave M. Cobalt stress in Escherichia coli and Salmonella enterica: Molecular bases for toxicity and resistance. Metallomics. 2011;3:1130–4.PubMedCrossRefGoogle Scholar
  9. Bolstad BM, Irizarry RA, Åstrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–93.PubMedCrossRefGoogle Scholar
  10. Booth IR, Blount P. The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves. J Bacteriol. 2012;194:4802–9.PubMedCrossRefPubMedCentralGoogle Scholar
  11. Bruins MR, Kapil S, Oehme FW. Microbial resistance to metals in the environment. Ecotoxicol Environ Saf. 2000;45:198–207.PubMedCrossRefGoogle Scholar
  12. Bruno-Barcena JM, Azcarate-Peril MA, Hassan HM. Role of antioxidant enzymes in bacterial resistance to organic acids. Appl Environ Microbiol. 2010;76:2747–53.PubMedCrossRefPubMedCentralGoogle Scholar
  13. Chassagnole C, Quentin E, Fell DA, de Atauri P, Mazat JP. Dynamic simulation of pollutant effects on the threonine pathway in Escherichia coli. C R Biol. 2003;326:501–8.PubMedCrossRefGoogle Scholar
  14. Cizewski Culotta V, Joh H-D, Lin S-J, Hudak Slekar K, Strain J. A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J Biol Chem. 1995;270:29991–7.CrossRefGoogle Scholar
  15. Cui J, Kaandorp JA, Lloyd CM. Simulating in vitro transcriptional response of zinc homeostasis system in Escherichia coli. BMC Syst Biol. 2008;2:1752–0509.CrossRefGoogle Scholar
  16. Decker K, Gerhardt F, Boos W. The role of the trehalose system in regulating the maltose regulon of Escherichia coli. Mol Microbiol. 1999;32:777–88.PubMedCrossRefGoogle Scholar
  17. Eggen RIL, Behra R, Burkhardt-Holm P, Escher BI, Schweigert N. Peer reviewed: Challenges in ecotoxicology. Environ Sci Technol. 2004;38:58A–64.PubMedCrossRefGoogle Scholar
  18. Ercal N, Gurer-Orhan H, Aykin-Burns N. Toxic metals and oxidative stress part I: Mechanisms involved in metal-induced oxidative damage. Curr Top Med Chem. 2001;1:529–39.PubMedCrossRefGoogle Scholar
  19. Fantino JR, Py B, Fontecave M, Barras F. A genetic analysis of the response of Escherichia coli to cobalt stress. Environ Microbiol. 2010;12:2846–57.PubMedGoogle Scholar
  20. Fluman N, Bibi E. Bacterial multidrug transport through the lens of the major facilitator superfamily. Biochim Biophys Acta. 2009;5:738–47.CrossRefGoogle Scholar
  21. Gama-Castro S, Salgado H, Peralta-Gil M, Santos-Zavaleta A, Muniz-Rascado L, Solano-Lira H, et al. RegulonDB version 7.0: Transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory response units (Gensor Units). Nucleic Acids Res. 2011;39:D98–105.PubMedCrossRefPubMedCentralGoogle Scholar
  22. Geslin C, Llanos J, Prieur D, Jeanthon C. The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity. Res Microbiol. 2001;152:901–5.PubMedCrossRefGoogle Scholar
  23. Giller KE, Witter E, McGrath SP. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review. Soil Biol Biochem. 1998;30:1389–414.CrossRefGoogle Scholar
  24. Goetz AK, Singh BP, Battalora M, Breier JM, Bailey JP, Chukwudebe AC, et al. Current and future use of genomics data in toxicology: Opportunities and challenges for regulatory applications. Regul Toxicol Pharmacol. 2011;61:141–53.PubMedCrossRefGoogle Scholar
  25. Graham AI, Hunt S, Stokes SL, Bramall N, Bunch J, Cox AG, et al. Severe zinc depletion of Escherichia coli. J Biol Chem. 2009;284:18377–89.PubMedCrossRefPubMedCentralGoogle Scholar
  26. Hantke K. Members of the Fur protein family regulate iron and zinc transport in E. coli and characteristics of the Fur-regulated FhuF protein. J Mol Microbiol Biotechnol. 2002;4:217–22.PubMedGoogle Scholar
  27. Helbig K, Grosse C, Nies DH. Cadmium toxicity in glutathione mutants of Escherichia coli. J Bacteriol. 2008;190:5439–54.PubMedCrossRefPubMedCentralGoogle Scholar
  28. Hu P, Brodie EL, Suzuki Y, McAdams HH, Andersen GL. Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. J Bacteriol. 2005;187:8437–49.PubMedCrossRefPubMedCentralGoogle Scholar
  29. Ivask A, Bondarenko O, Jepihhina N, Kahru A. Profiling of the reactive oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene nanoparticles using a set of recombinant luminescent Escherichia coli strains: Differentiating the impact of particles and solubilised metals. Anal Bioanal Chem. 2010;398:701–16.PubMedCrossRefGoogle Scholar
  30. Kalantari N, Ghaffari S. Evaluation of toxicity of heavy metals for Escherichia coli growth. Iran J Environ Health. 2008;5:173–8.Google Scholar
  31. Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000;28:27–30.PubMedCrossRefPubMedCentralGoogle Scholar
  32. Karp PD, Riley M, Saier M, Paulsen IT, Collado-Vides J, Paley SM, et al. The EcoCyc Database. Nucleic Acids Res. 2002;30:56–8.PubMedCrossRefPubMedCentralGoogle Scholar
  33. Kershaw CJ, Brown NL, Constantinidou C, Patel MD, Hobman JL. The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations. Microbiology. 2005;151:1187–98.PubMedCrossRefGoogle Scholar
  34. Kimura T, Nishioka H. Intracellular generation of superoxide by copper sulphate in Escherichia coli. Mutat Res. 1997;389:237–42.PubMedCrossRefGoogle Scholar
  35. Kitagawa M, Matsumura Y, Tsuchido T. Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli. FEMS Microbiol Lett. 2000;184:165–71.PubMedCrossRefGoogle Scholar
  36. Krämer U, Talke IN, Hanikenne M. Transition metal transport. FEBS Lett. 2007;581:2263–72.PubMedCrossRefGoogle Scholar
  37. Kusano T, Berberich T, Tateda C, Takahashi Y. Polyamines: Essential factors for growth and survival. Planta. 2008;228:367–81.PubMedCrossRefGoogle Scholar
  38. Lee LJ, Barrett JA, Poole RK. Genome-wide transcriptional response of chemostat-cultured Escherichia coli to zinc. J Bacteriol. 2005;187:1124–34.PubMedCrossRefPubMedCentralGoogle Scholar
  39. Lee C, Kim J, Shin SG, Hwang S. Absolute and relative qPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol. 2006;123:273–80.PubMedCrossRefGoogle Scholar
  40. Lee C, Lee S, Shin SG, Hwang S. Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotechnol. 2008;78:371–6.PubMedCrossRefGoogle Scholar
  41. Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, Schroeder JI. Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell. 2004;16:596–615.PubMedCrossRefPubMedCentralGoogle Scholar
  42. Liu Y, Bauer SC, Imlay JA. The YaaA protein of the Escherichia coli OxyR Regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron. J Bacteriol. 2011;193:2186–96.PubMedCrossRefPubMedCentralGoogle Scholar
  43. Long AD, Mangalam HJ, Chan BYP, Tolleri L, Hatfield GW, Baldi P. Improved statistical inference from DNA microarray data using analysis of variance and a bayesian statistical framework. J Biol Chem. 2001;276:19937–44.PubMedCrossRefGoogle Scholar
  44. Mellies J, Thomas K, Turvey M, Evans N, Crane J, Boedeker E, et al. Zinc-induced envelope stress diminishes type III secretion in enteropathogenic Escherichia coli. BMC Microbiol. 2012;12:123.PubMedCrossRefPubMedCentralGoogle Scholar
  45. Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet. 2010;11:31–46.PubMedCrossRefGoogle Scholar
  46. Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD. Genetic and physiological responses of Bacillus subtilis to metal ion stress. Mol Microbiol. 2005;57:27–40.PubMedCrossRefGoogle Scholar
  47. Morey JS, Ryan JC, Van Dolah FM. Microarray validation: Factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online. 2006;8:175–93.PubMedCrossRefPubMedCentralGoogle Scholar
  48. Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, et al. The transcriptional landscape of the yeast whole genome defined by RNA sequencing. Science. 2008;320:1344–9.PubMedCrossRefPubMedCentralGoogle Scholar
  49. Nagalakshmi U, Waern K, Snyder M. RNA‐Seq: a method for comprehensive transcriptome analysis. Curr Protocols Mol Biol. 2010;Chapter 4:Unit 4.11.1-13. doi:  10.1002/0471142727.mb0411s89.
  50. Nandakumar R, Espirito SC, Madayiputhiya N, Grass G. Quantitative proteomic profiling of the Escherichia coli response to metallic copper surfaces. Biometals. 2011;24:429–44.PubMedCrossRefGoogle Scholar
  51. Neumann M, Leimkuhler S. Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli. FEBS J. 2008;275:5678–89.PubMedCrossRefGoogle Scholar
  52. Nies DH. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev. 2003;27:313–39.PubMedCrossRefGoogle Scholar
  53. Noinaj N, Guillier M, Barnard TJ, Buchanan SK. TonB-dependent transporters: Regulation, structure, and function. Annu Rev Microbiol. 2010;64:43–60.PubMedCrossRefPubMedCentralGoogle Scholar
  54. Panek HR, O'Brian MR. KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum. J Bacteriol. 2004;186:7874–80.PubMedCrossRefPubMedCentralGoogle Scholar
  55. Park S, Ely RL. Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals. Appl Environ Microbiol. 2008;74:5475–82.PubMedCrossRefPubMedCentralGoogle Scholar
  56. Peng L, Lifang R, Hongyu X, Xi L, Chaocan Z. Study on the toxic effect of lead(II) ion on Escherichia coli. Biol Trace Elem Res. 2007;115:195–202.PubMedCrossRefGoogle Scholar
  57. Pfaffl MW. Relative quantification. Real Time qPCR. Taylor & Francis Group; 2006, pp. 63–82.Google Scholar
  58. Pope MA, Porello SL, David SS. Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G:A substrates. J Biol Chem. 2002;277:22605–15.PubMedCrossRefGoogle Scholar
  59. Py B, Barras F. Building Fe-S proteins: Bacterial strategies. Nat Rev Microbiol. 2010;8:436–46.PubMedCrossRefGoogle Scholar
  60. Ranquet C, Ollagnier-de-Choudens S, Loiseau L, Barras F, Fontecave M. Cobalt stress in Escherichia coli. The effect on the iron-sulfur proteins. J Biol Chem. 2007;282:30442–51.PubMedCrossRefGoogle Scholar
  61. Rispoli F, Angelov A, Badia D, Kumar A, Seal S, Shah V. Understanding the toxicity of aggregated zero valent copper nanoparticles against Escherichia coli. J Hazard Mater. 2010;180:212–6.PubMedCrossRefGoogle Scholar
  62. Robbens J, van der Ven K, Maras M, Blust R, De Coen W. Ecotoxicological risk assessment using DNA chips and cellular reporters. Trends Biotechnol. 2007;25:460–6.PubMedCrossRefGoogle Scholar
  63. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.PubMedGoogle Scholar
  64. Sawers G. A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon. Mol Microbiol. 2001;39:1285–98.PubMedCrossRefGoogle Scholar
  65. Schmidt R, Zahn R, Bukau B, Mogk A. ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway. Mol Microbiol. 2009;72:506–17.PubMedCrossRefGoogle Scholar
  66. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101–8.PubMedCrossRefGoogle Scholar
  67. Seaver LC, Imlay JA. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J Bacteriol. 2001;183:7182–9.PubMedCrossRefPubMedCentralGoogle Scholar
  68. Sharma SS, Dietz K-J. The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J Exp Bot. 2006;57:711–26.PubMedCrossRefGoogle Scholar
  69. Sharma SK, Goloubinoff P, Christen P. Heavy metal ions are potent inhibitors of protein folding. Biochem Biophys Res Commun. 2008;372:341–5.PubMedCrossRefGoogle Scholar
  70. Sipos K, Lange H, Fekete Z, Ullmann P, Lill R, Kispal G. Maturation of cytosolic iron-sulfur proteins requires glutathione. J Biol Chem. 2002;277:26944–9.PubMedCrossRefGoogle Scholar
  71. Soukas A, Cohen P, Socci ND, Friedman JM. Leptin-specific patterns of gene expression in white adipose tissue. Gene Dev. 2000;14:963–80.PubMedPubMedCentralGoogle Scholar
  72. Teitzel GM, Geddie A, De Long SK, Kirisits MJ, Whiteley M, Parsek MR. Survival and growth in the presence of elevated copper: Transcriptional profiling of copper-stressed Pseudomonas aeruginosa. J Bacteriol. 2006;188:7242–56.PubMedCrossRefPubMedCentralGoogle Scholar
  73. Tkachenko A, Nesterova L, Pshenichnov M. The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli. Arch Microbiol. 2001;176:155–7.PubMedCrossRefGoogle Scholar
  74. Valls M, Lorenzo V. Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol Rev. 2002;26:327–38.PubMedCrossRefGoogle Scholar
  75. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paene A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:research0034.0031–11.CrossRefGoogle Scholar
  76. Veinger L, Diamant S, Buchner J, Goloubinoff P. The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network. J Biol Chem. 1998;273:11032–7.PubMedCrossRefGoogle Scholar
  77. Wang A, Crowley DE. Global gene expression responses to cadmium toxicity in Escherichia coli. J Bacteriol. 2005;187:3259–66.PubMedCrossRefPubMedCentralGoogle Scholar
  78. Wang S, Deng K, Zaremba S, Deng X, Lin C, Wang Q, et al. Transcriptomic response of Escherichia coli O157:H7 to oxidative stress. Appl Environ Microbiol. 2009;75:6110–23.PubMedCrossRefPubMedCentralGoogle Scholar
  79. Waters MD, Fostel JM. Toxicogenomics and systems toxicology: Aims and prospects. Nat Rev Genet. 2004;5:936–48.PubMedCrossRefGoogle Scholar
  80. Worden CR, Kovac WK, Dorn LA, Sandrin TR. Environmental pH affects transcriptional responses to cadmium toxicity in Escherichia coli K-12 (MG1655). FEMS Microbiol Lett. 2009;293:58–64.PubMedCrossRefGoogle Scholar
  81. Wortham B, Oliveira M, Patel C. Polyamines in bacteria: Pleiotropic effects yet specific mechanisms. In: Perry R, Fetherston J, editors. The Genus Yersinia. New York: Springer; 2007. p. 106–15.CrossRefGoogle Scholar
  82. Xu FF, Imlay JA. Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron-sulfur clusters when they toxify Escherichia coli. Appl Environ Microbiol. 2012;78:3614–21.PubMedCrossRefPubMedCentralGoogle Scholar
  83. Yamamoto K, Ishihama A. Transcriptional response of Escherichia coli to external copper. Mol Microbiol. 2005a;56:215–27.PubMedCrossRefGoogle Scholar
  84. Yamamoto K, Ishihama A. Transcriptional response of Escherichia coli to external zinc. J Bacteriol. 2005b;187:6333–40.PubMedCrossRefPubMedCentralGoogle Scholar
  85. Yeung KY, Haynor DR, Ruzzo WL. Validating clustering for gene expression data. Bioinformatics. 2001;17:309–18.PubMedCrossRefGoogle Scholar
  86. Zhou J, Rudd KE. EcoGene 3.0. Nucleic Acids Res. 2013;41:D613–24.PubMedCrossRefPubMedCentralGoogle Scholar
  87. Zimmermann M, Udagedara SR, Sze CM, Ryan TM, Howlett GJ, Xiao Z, et al. PcoE–a metal sponge expressed to the periplasm of copper resistance Escherichia coli. Implication of its function role in copper resistance. J Inorg Biochem. 2012;115:186–97.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • María T. Gómez-Sagasti
    • 1
  • José M. Becerril
    • 2
  • Iker Martín
    • 1
  • Lur Epelde
    • 1
  • Carlos Garbisu
    • 1
  1. 1.NEIKER-Tecnalia, Department of Ecology and Natural ResourcesSoil Microbial Ecology GroupDerioSpain
  2. 2.Department of Plant Biology and EcologyUniversity of the Basque CountryBilbaoSpain

Personalised recommendations