Biological Trace Element Research

, Volume 189, Issue 1, pp 291–300 | Cite as

Cloning, Expression, and Bioinformatics Analysis of Heavy Metal Resistance Gene afe_1862 from Acidithiobacillus ferrooxidans L1 in Escherichia coli

  • Feifan LengEmail author
  • Yuanli Li
  • Wen Luo
  • Qingwei Wei
  • Yanjun Jing
  • Xiaoli Wang
  • Mingjun Yang
  • Yonggang WangEmail author


Molecular studies of copper and cadmium resistances in acidophilic bacteria are significant in biomining. In this study, afe_1862, which encodes a heavy metal-binding protein in Acidithiobacillus ferrooxidans L1, was amplified using PCR, cloned into the pET32a plasmid, and sequenced. Following SDS-PAGE analysis, optimization of the expression conditions and heterologous overexpression of afe_1862 in Escherichia coli BL21 in the presence of Cu2+ and Cd2+ were studied as well. The results indicated that AFE_1862 has higher resistance to Cu2+ than Cd2+. Bioinformatics analysis illustrated that AFE_1862 has a conserved HMA domain containing heavy metal-binding sites, which may play a role in transporting or detoxifying heavy metals.


Acidithiobacillus ferrooxidans Metal resistance Expression Bioinformatics afe_1862 



The authors would like to give thanks to the participants, coordinators, and administrators for their supports during the study. This work was supported by Chinese National Natural Science Foundation (No. 31460032, 31760028 and 81660581).

Compliance with Ethical Standards

Conflict of Interest

All authors declare that they have no conflicts of interest.

Supplementary material

12011_2018_1462_MOESM1_ESM.docx (2.6 mb)
ESM 1 (DOCX 2620 kb)


  1. 1.
    Xu L, Song Q, Mao C (2004) Metal-binding proteins or peptides in the bioremediation of environmental heavy metals. Prog Biotechnol 24:39–43 (in Chinese)Google Scholar
  2. 2.
    Mao C, Xue Y, Wang H (2001) Progress of microbial display technology in the bioremediation of heavy metals. Prog Biotechnol 21:49–51 (in Chinese)Google Scholar
  3. 3.
    Castagnetto JM, Hennessy SW, Roberts VA, Getzoff ED, Tainer JA, Pique ME (2002) MDB: the Metalloprotein Database and Browser at The Scripps Research Institute. Nucleic Acids Res 30:379–382CrossRefGoogle Scholar
  4. 4.
    Dhal NK, Pattanayak B, Padhi S (2014) Genetic engineering to express metal binding proteins and peptides: implications for bioremediation. Biolife 2:442–451Google Scholar
  5. 5.
    Mejáre M, Bülow L (2001) Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends Biotechnol 19:67–73CrossRefGoogle Scholar
  6. 6.
    Mejàre M, Ljung S, Bülow L (1998) Selection of cadmium specific hexapeptides and their expression as OmpA fusion proteins in Escherichia coli. Protein Eng 11:489–494CrossRefGoogle Scholar
  7. 7.
    Harvey PI, Crundwell FK (1996) The effect of As (III) on the growth of Thiobacillus ferrooxidans in an electrolytic cell under controlled redox potentials. Miner Eng 9:1059–1068CrossRefGoogle Scholar
  8. 8.
    Zhang Y, Wu X, Liu D, Duan H, Fan H (2013) Sequencing and bioinformatics analysis of the metal-related genes in Acidithiobacillus ferrooxidans strain DC. Folia Microbiol 58:551–560CrossRefGoogle Scholar
  9. 9.
    Chan J, Huang Z, Merrifield ME, Salgado MT, Stillman MJ (2002) Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques. Coord Chem Rev 233–234:319–339CrossRefGoogle Scholar
  10. 10.
    Liu T, Nakashima S, Hirose K, Uemura Y, Shibasaka M, Katsuhara M, Kasamo K (2003) A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous Cyanobacterium oscillatoria brevis 1. FEBS Lett 542:159–163CrossRefGoogle Scholar
  11. 11.
    Chaturvedi AK, Mishra A, Tiwari V, Jha B (2012) Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli. Gene 499:280–287CrossRefGoogle Scholar
  12. 12.
    Navarro CA, Bernath DV, Bussenius CM, Castillo RA, Jerez CA (2016) Cytoplasmic CopZ-like protein and periplasmic rusticyanin and AcoP proteins as possible copper resistance determinants in Acidithiobacillus ferrooxidans ATCC23270. Appl Environ Microbiol 82:1015–1022CrossRefGoogle Scholar
  13. 13.
    Xueling W, Yuan P, Qi H, Hou D, Miao B, Qiu G (2011) Bioleaching of chalcopyrite by Acidithiobacillus ferrooxidans DY15, DY26 and DC and difference expressions of gene Afe0022. Trans Nonferrous Metals Soc China 21:932–938Google Scholar
  14. 14.
    Kumar MS, Kaur G, Sandhu AK (2014) Genomic DNA isolation from fungi, algae, plant, bacteria and human blood using CTAB. IJSR 3:617–617Google Scholar
  15. 15.
    Gasteiger E, Hoogland C, Gattiker A, Duvaud SE, Wilkins MR, Appel RD, Bairoch A (1999) Protein identification and analysis tools on the ExPASy server. Methods Mol Biol 112:531–532Google Scholar
  16. 16.
    Petersen TN, Brunak S, Von HG, Nielsen H (2011) SIGNALP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefGoogle Scholar
  17. 17.
    Andreozzi S, Chakrabarti A, Soh KC, Burgard A, Yang TH, van Dien S, Miskovic L, Hatzimanikatis V (2016) Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models. Metab Eng 35:148–159CrossRefGoogle Scholar
  18. 18.
    Geourjon C, Deléage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684Google Scholar
  19. 19.
    Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36CrossRefGoogle Scholar
  20. 20.
    Gupta R, Brunak S (2002) Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput 7:310–322Google Scholar
  21. 21.
    Jensen LJ et al (2002) Prediction of human protein function from post-translational modifications and localization features. J Mol Biol 319:1257–1265CrossRefGoogle Scholar
  22. 22.
    Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452CrossRefGoogle Scholar
  23. 23.
    Azam M, Jan AT, Kumar A, Siddiqui K, Mondal AH, Haq QMR (2018) Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna. Braz J Microbiol 49:471-480Google Scholar
  24. 24.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132CrossRefGoogle Scholar
  25. 25.
    Chen GQ, Meng P, Liu LL, Chen G, Wang P (2011) In silico cloning and characterization of Sorghum APX gene. J Bioinformatics 16:557–562 (in Chinese)Google Scholar
  26. 26.
    Lin YZ, Guo H, Huang SW, Liu CX, Liu TY, Chen XY (2010) In silico cloning and bioinformatics analysis of DREB1 gene from Pinus teada. J Bioinformatics 8:43–46 (in Chinese)Google Scholar
  27. 27.
    Lamb AL, Torres AS, O'Halloran TV, Rosenzweig AC (2001) Heterodimeric structure of superoxide dismutase in complex with its metallochaperone. Nat Struct Biol 8:751–755CrossRefGoogle Scholar
  28. 28.
    Wernimont AK, Huffman DL, Lamb AL, O'Halloran TV, Rosenzweig AC (2000) Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins. Nat Struct Biol 7:766–771CrossRefGoogle Scholar
  29. 29.
    Wu X, Zhang Z, Liu L, Deng F, Liu X, Qiu G (2014) Metal resistance-related genes are differently expressed in response to copper and zinc ion in six Acidithiobacillus ferrooxidans strains. Curr Microbiol 69:775–784CrossRefGoogle Scholar
  30. 30.
    Mattie MD, Freedman JH (2004) Copper-inducible transcription: regulation by metal- and oxidative stress-responsive pathways. Am J Physiol Cell Physiol 286:C293–C301CrossRefGoogle Scholar
  31. 31.
    Hartwig A, Asmuss M, Ehleben I, Herzer U, Kostelac D, Pelzer A, Schwerdtle T, Bürkle A (2002) Interference by toxic metal ions with DNA repair processes and cell cycle control: molecular mechanisms. Environ Health Perspect 110:797–799CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.School of Life Science and EngineeringLanzhou University of TechnologyLanzhouChina
  2. 2.Lanzhou Institute of Husbandry and Pharmaceutical Science of CAASLanzhouChina

Personalised recommendations