Antonie van Leeuwenhoek

, Volume 112, Issue 1, pp 127–139 | Cite as

Detoxification and reduction of selenite to elemental red selenium by Frankia

  • Medhat RehanEmail author
  • Abdullah S. Alsohim
  • Gomaah El-Fadly
  • Louis S. Tisa
Original Paper


Four Frankia strains (EuI1c, CN3, ACN14a and CcI3) were tested for selenite tolerance. Frankia inefficax strain EuI1c was resistant to selenite with a MIC value of 518.8 µg ml−1. After 48 h incubation with selenite, a reddish precipitate began to appear in these cultures. The red color suggests the reduction of the toxic, soluble, and colorless sodium selenite (Na2SeO32−) to the nontoxic, insoluble, and red colored elemental selenium (Seº). Analysis showed F. inefficax strain EuI1c cultures exposed to 17.3 and 86.5 µg ml−1selenite completely reduced all of the selenite after 5 and 8 days, respectively. When observed under Scanning Electron Microscopy, selenite-resistant F. inefficax strain EuI1c grown with selenite formed nanosphere particles on the hyphal surface as free deposits or in aggregates and inside the hyphae. EDAX analysis of the nanosphere particles determined that they are composed of selenium with up to 27.3-fold increase in intensity as compared to control cells. FTIR Spectroscopy of selenite-stressed cells showed cell surface changes in fatty acids, polysaccharides, carbohydrates and phosphate groups. This result suggests a mechanism for selenite reduction and nanosphere transport through cell membrane in this strain. Native gel electrophoresis of extracted cell-free protein revealed one band showing activity after staining with selenite and NADH. SDS-PAGE analysis revealed the presence of several bands with one dominant band of 37.8 kDa. Mass spectrometry analysis of the bands determined that the main proteins were a periplasmic-binding protein, sulfate ABC transporter and extracellular ligand-binding receptor.


Frankia Metal resistance Elemental selenium Nanospheres 



Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2787. This investigation was supported in part by USDA National Institute of Food and Agriculture Hatch Project 022821 (LST), and by the College of Life Science and Agriculture, The University of New Hampshire-Durham. Funding support was provided by King Abdulaziz City for Science and Technology (KACST) as a part of project No. AT 33-15. MR was supported by an Egyptian Channel Fellowship from the Egyptian Bureau of Education and Culture. We thank Robert Mooney for his help with the photography and Rebecca Wagers, Joel Richards, and Glenn Krumholz for their contributions to the early phases of this manuscript.

Author contributions

MR, GE and LT contributed to experimental design. MR performed the experiments. MR and LT analysed data and wrote the manuscript. ASA support part of the project by fund from King Abdulaziz City.

Conflict of interest

Authors declare that they have no conflict of interest.

Supplementary material

10482_2018_1196_MOESM1_ESM.docx (1.1 mb)
Supplementary material 1 (DOCX 1147 kb)


  1. Baker E, Tang Y, Chu F, Tisa LS (2015) Molecular responses of Frankia sp. strain QA3 to naphthalene. Can J Microbiol 61:281–292. CrossRefGoogle Scholar
  2. Bebien M, Kirsch J, Mejean V, Vermeglio A (2002) Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli. Microbiology 148:3865–3872. CrossRefGoogle Scholar
  3. Berks BC, Richardson DJ, Robinson C, Reilly A, Aplin RT, Ferguson SJ (1994) Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. Eur J Biochem 220:117–124CrossRefGoogle Scholar
  4. Biswas KC, Barton LL, Tsui WL, Shuman K, Gillespie J, Eze CS (2011) A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite. J Microbiol Methods 86:140–144. CrossRefGoogle Scholar
  5. Debieux CM et al (2011) A bacterial process for selenium nanosphere assembly. Proc Natl Acad Sci 108:13480CrossRefGoogle Scholar
  6. Dhanjal S, Cameotra SS (2010) Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil. Microb Cell Fact 9:52. CrossRefGoogle Scholar
  7. Diem HG, Dommergues YR (1990) Current and potential uses and management of casuarinaceae in the tropics and subtropics. In: Schwintzer CR, Tjepkema JD (eds) The biology of frankia and actinorhizal plants. Academic Press, New York, pp 317–342.
  8. Etezad SM, Khajeh K, Soudi M, Ghazvini PTM, Dabirmanesh B (2009) Evidence on the presence of two distinct enzymes responsible for the reduction of selenate and tellurite in Bacillus sp. STG-83. Enzyme Microb Technol 45:1–6. CrossRefGoogle Scholar
  9. Furnholm TR, Tisa LS (2014) The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia. BMC Genom 15:1092. CrossRefGoogle Scholar
  10. Furnholm T, Beauchemin N, Tisa LS (2012) Development of a semi-high-throughput growth assay for the filamentous actinobacteria Frankia. Arch Microbiol 194:13–20. CrossRefGoogle Scholar
  11. Furnholm T, Rehan M, Wishart J, Tisa LS (2017) Pb2 + tolerance by Frankia sp. strain EAN1pec involves surface-binding. Microbiology 163:472–487. CrossRefGoogle Scholar
  12. Ghosh A, Mohod AM, Paknikar KM, Jain RK (2008) Isolation and characterization of selenite- and selenate-tolerant microorganisms from selenium-contaminated sites. J Microbiol Biotechnol 24:1607–1611. CrossRefGoogle Scholar
  13. Gonzalez-Gil G, Lens PNL, Saikaly PE (2016) Selenite reduction by anaerobic microbial aggregates: microbial community structure and proteins associated to the produced selenium spheres. Front Microbiol. Google Scholar
  14. Hladun KR, Smith BH, Mustard JA, Morton RR, Trumble JT (2012) Selenium toxicity to honey bee (Apis mellifera L.) pollinators: effects on behaviors and survival. PLoS ONE 7:e34137. CrossRefGoogle Scholar
  15. Hunter WJ (2007) An Azospira oryzae (syn Dechlorosoma suillum) strain that reduces selenate and selenite to elemental red selenium. Curr Microbiol 54:376–381. CrossRefGoogle Scholar
  16. Hunter WJ, Manter DK (2009) Reduction of selenite to elemental red selenium by Pseudomonas sp. Strain CA5. Curr Microbiol 58:493–498. CrossRefGoogle Scholar
  17. Jain R, Matassa S, Singh S, van Hullebusch ED, Esposito G, Lens PNL (2016) Reduction of selenite to elemental selenium nanoparticles by activated sludge. Environ Sci Pollut Res 23:1193–1202. CrossRefGoogle Scholar
  18. Kessi J, Ramuz M, Wehrli E, Spycher M, Bachofen R (1999) Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum. Appl Environ Microbiol 65:4734–4740Google Scholar
  19. Krafft T, Bowen A, Theis F, Macy JM (2000) Cloning and sequencing of the genes encoding the periplasmic-cytochrome B-containing selenate reductase of Thauera selenatis. DNA Seq 10:365–377CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  21. Lampis S et al (2017) Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: novel clues on the route to bacterial biogenesis of selenium nanoparticles. J Hazard Mater 324:3–14. CrossRefGoogle Scholar
  22. Li B et al (2014) Reduction of selenite to red elemental selenium by Rhodopseudomonas palustris Strain N. PLoS ONE 9:e95955. CrossRefGoogle Scholar
  23. Lortie L, Gould WD, Rajan S, McCready RGL, Cheng KJ (1992) Reduction of selenate and selenite to elemental selenium by a Pseudomonas stutzeri isolate. Appl Environ Microbiol 58:4042–4044Google Scholar
  24. Losi ME, Frankenberger WT (1997) Reduction of selenium oxyanions by Enterobacter cloacae SLD1a- 1: isolation and growth of the bacterium and its expulsion of selenium particles. Appl Environ Microbiol 63:3079–3084Google Scholar
  25. Ma J, Kobayashi DY, Yee N (2007) Chemical kinetic and molecular genetic study of selenium oxyanion reduction by Enterobacter cloacae SLD1a-1. Environ Sci Technol 41:7795–7801. CrossRefGoogle Scholar
  26. MacFarquhar JK et al (2010) Acute selenium toxicity associated with a dietary supplement. Arch Intern Med 170:256–261. CrossRefGoogle Scholar
  27. Mishra RR, Prajapati S, Das J, Dangar TK, Das N, Thatoi H (2011) Reduction of selenite to red elemental selenium by moderately halotolerant Bacillus megaterium strains isolated from Bhitarkanika mangrove soil and characterization of reduced product. Chemosphere 84:1231–1237CrossRefGoogle Scholar
  28. Myers AK, Tisa LS (2004) Isolation of antibiotic-resistant and antimetabolite-resistant mutants of Frankia strains EuI1c and Cc1.17. Can J Microbiol 50:261–267. CrossRefGoogle Scholar
  29. Oremland RS et al (2004) Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria. Appl Environ Microbiol 70:52–60CrossRefGoogle Scholar
  30. Pieniz S, Okeke BC, Andreazza R, Brandelli A (2011) Evaluation of selenite bioremoval from liquid culture by Enterococcus species. Microbiol Res 166:176–185. CrossRefGoogle Scholar
  31. Raines AM, Sunde RA (2011) Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents. BMC Genom 12:26. CrossRefGoogle Scholar
  32. Rehan M (2017) Microbial bioremediation. Rev J Agric Vet Sci 10:147–162Google Scholar
  33. Rehan M, Furnholm T, Finethy RH, Chu F, El-Fadly G, Tisa LS (2014a) Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport. Appl Microbiol Biotechnol 98:8005–8015CrossRefGoogle Scholar
  34. Rehan M, Kluge M, Fränzle S, Kellner H, Ullrich R, Hofrichter M (2014b) Degradation of atrazine by Frankia alni ACN14a: gene regulation, dealkylation, and dechlorination. Appl Microbiol Biotechnol 98:6125–6135CrossRefGoogle Scholar
  35. Rehan M, Swanson E, Tisa LS (2016) Frankia as a biodegrading agent. Actinobacteria-basics and biotechnological applications. InTech, RijekaGoogle Scholar
  36. Rehan M et al (2017) Opening the s-triazine ring and biuret hydrolysis during conversion of atrazine by Frankia sp. strain EuI1c. Int Biodeterior Biodegrad 117:14–21. CrossRefGoogle Scholar
  37. Richards JW, Krumholz GD, Chval MS, Tisa LS (2002) Heavy metal resistance patterns of Frankia strains. Appl Environ Microbiol 68:923–927. CrossRefGoogle Scholar
  38. Ridgway KP, Marland LA, Harrison AF, Wright J, Young JP, Fitter AH (2004) Molecular diversity of Frankia in root nodules of Alnus incana grown with inoculum from polluted urban soils. FEMS Microbiol Ecol 50:255–263. CrossRefGoogle Scholar
  39. Ridley H, Watts CA, Richardson DJ, Butler CS (2006) Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions. Appl Environ Microbiol 72:5173–5180. CrossRefGoogle Scholar
  40. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179CrossRefGoogle Scholar
  41. Sabaty M, Avazeri C, Pignol D, Vermeglio A (2001) Characterization of the reduction of selenate and tellurite by nitrate reductases. Appl Environ Microbiol 67:5122–5126. CrossRefGoogle Scholar
  42. Schroder I, Rech S, Krafft T, Macy JM (1997) Purification and characterization of the selenate reductase from Thauera selenatis. J Biol Chem 272:23765–23768CrossRefGoogle Scholar
  43. Smith PK et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefGoogle Scholar
  44. Song D, Li X, Cheng Y, Xiao X, Lu Z, Wang Y, Wang F (2017) Aerobic biogenesis of selenium nanoparticles by Enterobacter cloacae Z0206 as a consequence of fumarate reductase mediated selenite reduction. Sci Rep 7:3239. CrossRefGoogle Scholar
  45. Tan Y, Yao R, Wang R, Wang D, Wang G, Zheng S (2016) Reduction of selenite to Se(0) nanoparticles by filamentous bacterium Streptomyces sp. ES2-5 isolated from a selenium mining soil. Microb Cell Fact 15:157. CrossRefGoogle Scholar
  46. Tisa LS, Chval MS, Krumholz GD, Richards J (1999) Antibiotic resistance patterns of Frankia strains. Can J Bot 77:1257–1260. Google Scholar
  47. Turick CE, Caccavo F Jr, Tisa LS (2003) Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin. FEMS Microbiol Lett 220:99–104CrossRefGoogle Scholar
  48. Turner RJ, Weiner JH, Taylor DE (1998) Selenium metabolism in Escherichia coli. Biometals 11:223–227. CrossRefGoogle Scholar
  49. van der Heide T, Poolman B (2002) ABC transporters: one, two or four extracytoplasmic substrate-binding sites? EMBO Rep 3:938–943. CrossRefGoogle Scholar
  50. Vinceti M, Mandrioli J, Borella P, Michalke B, Tsatsakis A, Finkelstein Y (2014) Selenium neurotoxicity in humans: bridging laboratory and epidemiologic studies. Toxicol Lett 230:295–303. CrossRefGoogle Scholar
  51. Watts CA, Ridley H, Condie KL, Leaver JT, Richardson DJ, Butler CS (2003) Selenate reduction by Enterobacter cloacae SLD1a-1 is catalysed by a molybdenum-dependent membrane-bound enzyme that is distinct from the membrane-bound nitrate reductase. FEMS Microbiol Lett 228:273–279CrossRefGoogle Scholar
  52. Wheeler CT, Miller IM (1990) Current and potential uses of actinorhizal plants in Europe. In: Schwintzer CR, Tjepkema JD (eds) The biology of Frankia and actinorhizal plants. Academic Press, New York, pp 365–389. CrossRefGoogle Scholar
  53. Yanke LJ, Bryant RD, Laishley EJ (1995) Hydrogenase I of Clostridium pasteurianum functions as a novel selenite reductase. Anaerobe 1:61–67CrossRefGoogle Scholar
  54. Yee N, Ma J, Dalia A, Boonfueng T, Kobayashi DY (2007) Se(VI) reduction and the precipitation of Se(0) by the facultative bacterium Enterobacter cloacae SLD1a-1 are regulated by FNR. Appl Environ Microbiol 73:1914–1920. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Medhat Rehan
    • 1
    • 3
    Email author
  • Abdullah S. Alsohim
    • 3
  • Gomaah El-Fadly
    • 1
  • Louis S. Tisa
    • 2
  1. 1.Department of GeneticsKafrelsheikh UniversityKafr ElsheikhEgypt
  2. 2.Department of Molecular, Cellular, and Biomedical SciencesUniversity of New HampshireDurhamUSA
  3. 3.Department of Plant Production and Protection, College of Agriculture and Veterinary MedicineQassim UniversityBuraydahSaudi Arabia

Personalised recommendations