Skip to main content
SpringerLink
Log in

Complementary screening, identification and application of a novel class II 5-enopyruvylshikimate-3-phosphate synthase from Bacillus cereus

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Bacillus cereus was identified and overexpressed by genomic library construction and complementary screening. The enzyme was then purified to homogeneity. We also transformed the aroA B. cereus gene into Arabidopsis thaliana by a floral dip method, and demonstrated that transgenic A. thaliana plants exhibited significant glyphosate resistance compared with the wild type. These results strongly suggested that the strategy was highly efficient and advantageous for rapidly cloning aroA genes from microorganisms in natural environments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ausubel FM, Brent RE, Kingston DD, Moore JG, Seidman JAS, Struhl K (1997) Short protocols in molecular biology, 3rd edn. Wiley, New York

    Google Scholar 

  • Baerson SR, Rodriguez DJ, Tran M, Feng Y, Biest NA, Dill GM (2002) Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol 129(3):1265–1275. doi:10.1104/pp.001560

    Article  CAS  Google Scholar 

  • Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lu W, Wang J, Lin M, Wang GA (2012) Novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE 7(6):e38718. doi:10.1371/journal.pone.0038718

    Article  CAS  Google Scholar 

  • Chen L, Pradhan S, Evans TC Jr (2001) Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain. Gene 263(1–2):39–48

    Article  CAS  Google Scholar 

  • Della-Cioppa G, Bauer SC, Klein BK, Shah DM, Fraley RT, Kishore GM (1986) Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proc Natl Acad Sci U S A 83(18):6873–6877

    Article  CAS  Google Scholar 

  • Dill GM, Cajacob CA, Padgette SR (2008) Glyphosate-resistant crops: adoption, use and future considerations. Pest Manag Sci 64(4):326–331. doi:10.1002/ps.1501

    Article  CAS  Google Scholar 

  • Dun BQ, Wang XJ, Lu W, Zhao ZL, Hou SN, Zhang BM, Li GY, Evans TC Jr, Xu MQ, Lin M (2007) Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco. Appl Environ Microbiol 73(24):7997–8000. doi:10.1128/AEM.00956-07

    Article  CAS  Google Scholar 

  • Eschenburg S, Healy ML, Priestman MA, Lushington GH, Schonbrunn E (2002) How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli. Planta 216(1):129–135. doi:10.1007/s00425-002-0908-0

    Article  CAS  Google Scholar 

  • Franz JE, Mao MK, Sikorski JA (1997) Glyphosate: a unique global herbicide. American chemical society monograph 189 Washington DC

  • Funke T, Han H, Healy-Fried ML, Fischer M, Schonbrunn E (2006) Molecular basis for the herbicide resistance of Roundup Ready crops. Proc Natl Acad Sci U S A 103(35):13010–13015. doi:10.1073/pnas.0603638103

    Article  CAS  Google Scholar 

  • Funke T, Yang Y, Han H, Healy-Fried M, Olesen S, Becker A, Schonbrunn E (2009) Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli. J Biol Chem 284(15):9854–9860. doi:10.1074/jbc.M809771200

    Article  CAS  Google Scholar 

  • He M, Yang ZY, Nie YF, Wang J, Xu P (2001) A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate. Biochim Biophys Acta 1568(1):1–6

    Article  CAS  Google Scholar 

  • He M, Nie YF, Xu P (2003) A T42 M substitution in bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) generates enzymes with increased resistance to glyphosate. Biosci Biotechnol Biochem 67(6):1405–1409

    Article  CAS  Google Scholar 

  • Klee HJ, Muskopf YM, Gasser CS (1987) Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol Gen Genet 210(3):437–442

    Article  CAS  Google Scholar 

  • Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100(1):95–97

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiol 15(3):473–497

    Article  CAS  Google Scholar 

  • Peng RH, Tian YS, Xiong AS, Zhao W, Fu XY, Han HJ, Chen C, Jin XF, Yao QH (2012) A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity. PLoS ONE 7(8):e39579. doi:10.1371/journal.pone.0039579

    Article  CAS  Google Scholar 

  • Schönbrunn E, Eschenburg S, Shuttleworth WA, Schloss JV, Amrhein N, Evans JN, Kabsch W (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc Natl Acad Sci U S A 98(4):1376–1380. doi:10.1073/pnas.98.4.1376

    Article  Google Scholar 

  • Steinrucken HC, Amrhein N (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem Biophys Res Commun 94(4):1207–1212

    Article  CAS  Google Scholar 

  • Sun YC, Chen YC, Tian ZX, Li FM, Wang XY, Zhang J, Xiao ZL, Lin M, Gilmartin N, Dowling DN, Wang YP (2005) Novel AroA with high tolerance to glyphosate, encoded by a gene of Pseudomonas putida 4G–1 isolated from an extremely polluted environment in China. Appl Environ Microbiol 71(8):4771–4776. doi:10.1128/AEM.71.8.4771-4776.2005

    Article  CAS  Google Scholar 

  • Tian YS, Peng RH, Xu J, Zhao W, Gao F, Fu XY, Xiong AS, Yao QH (2010a) Mutations in two amino acids in phyI1 s from Aspergillus niger 113 improve its phytase activity. World J Microbiol Biotechnol 26:903–907. doi:10.1007/s11274-009-0251-8

    Article  Google Scholar 

  • Tian YS, Xiong AS, Xu J, Zhao W, Gao F, Fu XY, Xu H, Zheng JL, Peng RH, Yao QH (2010b) Isolation from Ochrobactrum anthropi of a novel class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate. Appl Environ Microbiol 76(17):6001–6005. doi:10.1128/AEM.00770-10

    Article  CAS  Google Scholar 

  • Tian YS, Xu J, Xiong AS, Zhao W, Fu XY, Peng RH, Yao QH (2011) Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase. Appl Environ Microbiol 77(23):8409–8414. doi:10.1128/AEM.05271-11

    Article  CAS  Google Scholar 

  • Tian YS, Xu J, Xiong AS, Zhao W, Gao F, Fu XY, Peng RH, Yao QH (2012) Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from Halothermothrix orenii H168 in Escherichia coli and transgenic Arabidopsis. Appl Microbiol Biotechnol 93(1):241–250. doi:10.1007/s00253-011-3443-8

    Article  Google Scholar 

  • Wang HY, Li YF, Xie LX, Xu P (2003) Expression of a bacterial aroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants. J Plant Res 116(6):455–460. doi:10.1007/s10265-003-0120-8

    Article  CAS  Google Scholar 

  • Xu J, Tian YS, Peng RH, Xiong AS, Zhu B, Jin XF, Gao F, Fu XY, Hou XL, Yao QH (2010) AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. Planta 231(6):1251–1260. doi:10.1007/s00425-010-1122-0

    Article  CAS  Google Scholar 

  • Yan HQ, Chang SH, Tian ZX, Zhang L, Sun YC, Li Y, Wang J, Wang YP (2011) Novel AroA from Pseudomonas putida confers tobacco plant with high tolerance to glyphosate. PLoS ONE 6(5):e19732. doi:10.1371/journal.pone.0019732

    Article  CAS  Google Scholar 

  • Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2):641–646. doi:10.1038/nprot.2006.97

    Article  CAS  Google Scholar 

  • Zhou M, Xu H, Wei X, Ye Z, Wei L, Gong W, Wang Y, Zhu Z (2006) Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy. Plant Physiol 140(1):184–195. doi:10.1104/pp.105.068577

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The research was supported financially by the Shanghai National Science Foundation (11ZR14324000), the Development Fund of Shanghai Academy of Agricultural Sciences (No. 2011-10)], the Key Project Fund of the Shanghai Municipal Committee of Agriculture (No. 2009-6-4), the Key Project Fund of the Shanghai Municipal Committee of Agriculture (No. 2011-1-8), International Scientific and Technological Cooperation (2010DFA62320, 11230705900) and National Natural Science Foundation (31071486).

Author information

Authors and Affiliations

  1. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai, People’s Republic of China

    Yong-Sheng Tian, Jing Xu, Jing Han, Wei Zhao, Xiao-Yan Fu, Ri-He Peng & Quan-Hong Yao

Authors
  1. Yong-Sheng Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jing Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiao-Yan Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ri-He Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Quan-Hong Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ri-He Peng or Quan-Hong Yao.

Additional information

Yong-Sheng Tian and Jing Xu are contributed equally to the article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 847 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tian, YS., Xu, J., Han, J. et al. Complementary screening, identification and application of a novel class II 5-enopyruvylshikimate-3-phosphate synthase from Bacillus cereus . World J Microbiol Biotechnol 29, 549–557 (2013). https://doi.org/10.1007/s11274-012-1209-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-012-1209-9

Keywords

Search

Navigation