Journal of Molecular Evolution

, Volume 86, Issue 2, pp 103–110 | Cite as

Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity

  • Kenric J. Hoegler
  • Michael H. Hecht
Original Article


When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells—with their large populations and short generation times—frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.


Evolution of resistance Antimicrobial resistance Acquired resistance by gene duplication Resistance to metal toxicity 



We thank Wayne Patrick for generously gifting us an aliquot of his lab’s pooled ASKA collection. We also thank Shlomo Zarzhitsky for help with Fig. 2. This work was supported by the National Science Foundation, Grant Number: MCB-1409402.

Supplementary material

239_2018_9830_MOESM1_ESM.docx (481 kb)
Supplementary material 1 (DOCX 480 KB)


  1. Alam M, Imran M (2014) Multiple antibiotic resistances in metal tolerant E. coli from hospital waste water. Bioinformation 10(5):267–272CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aminov RI (2010) A brief history of the antibiotic era: lessons learned and challenges for the future. Front Microbiol 1:134CrossRefPubMedPubMedCentralGoogle Scholar
  3. Anes J, McCusker MP, Fanning S, Martins M. 2015. The ins and outs of RND efflux pumps in Escherichia coli. Front Microbiol 6:587CrossRefPubMedPubMedCentralGoogle Scholar
  4. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1CrossRefGoogle Scholar
  5. Brocklehurst KR, Morby AP (2000) Metal-ion tolerance in Escherichia coli: analysis of transcriptional profiles by gene-array technology. Microbiology 146:2277–2282CrossRefPubMedGoogle Scholar
  6. Ciampi MS (2006) Rho-dependent terminators and transcription termination. Microbiology 152(9):2515–2528CrossRefPubMedGoogle Scholar
  7. Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74(3):417–433CrossRefPubMedPubMedCentralGoogle Scholar
  8. Digianantonio KM, Hecht MH (2016) A protein constructed de novo enables cell growth by altering gene regulation. Proc Natl Acad Sci USA 113:2400–2405CrossRefPubMedPubMedCentralGoogle Scholar
  9. Digianantonio KM, Korolev M, Hecht MH (2017) A non-natural protein rescues cells deleted for a key enzyme in central metabolism. ACS Synth Biol 6:694–700CrossRefPubMedGoogle Scholar
  10. Fisher MA, McKinley KL, Bradley LH, Viola SR, Hecht MH (2011) De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth. PLoS ONE 6(1):e15364CrossRefPubMedPubMedCentralGoogle Scholar
  11. Freeman JL, Persans MW, Nieman K, Salt DE (2005) Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense. Appl Environ Microbiol 71(12):8627–8633CrossRefPubMedPubMedCentralGoogle Scholar
  12. Grass G, Fan B, Rosen BP, Franke S, Nies DH, Rensing C (2001) ZitB (YbgR), a Member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli. J Bacteriol 183(15):4664–4667CrossRefPubMedPubMedCentralGoogle Scholar
  13. Grass G, Rensing C, Solioz M (2011) Metallic copper as an antimicrobial surface. Appl Environ Microbiol 77(5):1541–1547CrossRefPubMedGoogle Scholar
  14. Graves JL Jr, Tajkarimi M, Cunningham Q, Campbell A, Nonga H, Harrison SH, Barrick JE (2015) Rapid evolution of silver nanoparticle resistance in Escherichia coli. Front Genet 6:42CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2(2):95–108CrossRefPubMedGoogle Scholar
  16. Herron MD, Doebeli M. 2013. Parallel evolutionary dynamics of adaptive diversification in Escherichia coli. PLoS Biol 11(2):e1001490. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hobman JL, Crossman LC. 2015. Bacterial antimicrobial metal ion resistance. J Med Microbiol 64(5):471–497CrossRefPubMedGoogle Scholar
  18. Hoegler KJ, Hecht MH (2016) A de novo protein confers copper resistance in Escherichia coli. Protein Sci 25:1249–1259CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hughes D, Andersson DI (2015) Evolutionary consequences of drug resistance: shared principles across diverse targets and organisms. Nat Rev Genet 16:459–471CrossRefPubMedGoogle Scholar
  20. Jensen RA (1976) Enzyme recruitment in evolution of new function. Ann Rev Microbiol 30(1):409–425CrossRefGoogle Scholar
  21. Jerez CA. (2013) Molecular characterization of copper and cadmium resistance determinants in the biomining thermoacidophilic archaeon Sulfolobus metallicus. Archaea 2013Google Scholar
  22. Jozefczak M, Remans T, Vangronsveld J, Cuypers A (2012) Glutathione is a key player in metal-induced oxidative stress defenses. Int J Mol Sci 13(3):3145–3175CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kacar B, Gaucher EA (2012) Towards the recapitulation of ancient history in the laboratory: combining synthetic biology with experimental evolution. Artif Life 13:11–18Google Scholar
  24. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2006) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12(5):291–299CrossRefGoogle Scholar
  25. Lemire JA, Harrison JJ, Turner RJ (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Microbiol 11(6):371–384CrossRefPubMedGoogle Scholar
  26. Levin BR, Baquero F, Johnsen PJ (2014) A model-guided analysis and perspective on the evolution and epidemiology of antibiotic resistance and its future. Curr Opin Microbiol 19:83–89CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lok CN, Ho CM, Chen R, Tam PK, Chiu JF, Che CM (2008) Proteomic identification of the Cus system as a major determinant of constitutive Escherichia coli silver resistance of chromosomal origin. J Proteome Res 7(6):2351–2356CrossRefPubMedGoogle Scholar
  28. Montealegre MC, Roh JH, Rae M, Davlieva MG, Singh KV, Shamoo Y, Murray BE (2017) Differential penicillin-binding protein 5 (PBP5) levels in the Enterococcus faecium clades with different levels of ampicillin resistance. Antimicrob Agents Chemother 61:e02034-16CrossRefPubMedGoogle Scholar
  29. Näsvall J, Sun L, Roth JR, Andersson DI (2012) Real-time evolution of new genes by innovation, amplification, and divergence. Science 338(6105):384–387CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nies DH, Silver S (2007) Molecular microbiology of heavy metals, Vol 6, Springer, Berlin, pp 118–142CrossRefGoogle Scholar
  31. Notebaart RA, Kintses B, Feist AM, Papp B (2018) Underground metabolism: network-level perspective and biotechnological potential. Curr Opin Biotechnol 49:108–114CrossRefPubMedGoogle Scholar
  32. Ohno S (2013) Evolution by gene duplication. Springer, New YorkGoogle Scholar
  33. Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007) Multicopy suppression underpins metabolic evolvability. Mol Biol Evol 24(12):2716–2722CrossRefPubMedPubMedCentralGoogle Scholar
  34. Randall CP, Gupta A, Jackson N, Busse D, O’Neill AJ (2015) Silver resistance in Gram-negative bacteria: a dissection of endogenous and exogenous mechanisms. J Antimicrob Chemother 70:1037–1046PubMedPubMedCentralGoogle Scholar
  35. Soo VW, Hanson-Manful P, Patrick WM (2011) Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli. Proc Natl Acad Sci USA 108(4):1484–1489CrossRefGoogle Scholar
  36. Tajkarimi M, Rhinehardt K, Thomas M, Ewunkem JA, Campbell A, Boyd S, Turner D, Harrison SH, Graves JL (2017) Selection for ionic- confers silver nanoparticle resistance in Escherichia coli. JSM Nanotechnol Nanomed 5(1):1047Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departments of Chemistry and Molecular BiologyPrinceton UniversityPrincetonUSA

Personalised recommendations