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The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms

Abstract

The bacterial reverse mutation test, commonly called Ames test, is used worldwide. In Japan, the genetically modified organisms (GMOs) are regulated under the Cartagena Domestic Law, and organisms obtained by self-cloning and/or natural occurrence would be exempted from the law case by case. The strains of Salmonella typhimurium and Escherichia coli recommended for use in the bacterial reverse mutation test (OECD guideline 471), have been considered as non-GMOs because they can be constructed by self-cloning or naturally occurring bacterial strains, or do not disturb the biological diversity. The present article explains the reasons why these tester strains should be classified as non-GMOs.

Definition of genetically modified organisms

Genetically modified organisms (GMOs) are defined as an organism “in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination, without using modern recombinant DNA technology” [1]. Accordingly, organisms are considered to be a non-GMO if they are made by the transfer of genetic material through bacterial conjugation between same/different species. For example, the transfer of the antibiotic resistance genes naturally occurs by bacterial conjugation in a broad host range [2]. It is also known that bacteria gain the property of antibiotic resistance through the genetic mutations and horizontal transfer of the antibiotic resistance genes under selective pressures [3]. Therefore, non-GMOs are not considered to disturb the biological diversity.

Gene mutations of the Ames tester strains recommended in OECD guideline 471

The strains which are used in the bacterial reverse mutation test (OECD guideline 471) [4] are derivatives of S. enterica serovar Typhimurium (S. typhimurium) LT2 or E. coli B strain [57]. All the Ames tester strains recommended for use in the bacterial reverse mutation test are listed in Table 1. The Salmonella tester strains harbor different mutations (hisD3052, hisG46, hisC3076, hisG428, hisD6610 and hisO1242) in the genes of the histidine operon of S. typhimurium. The Salmonella strains originated from S. typhimurium LT2 are histidine auxotrophs which are the result from treatment with mutagens or radiation [5, 6, 813]. In addition, the all Salmonella tester strains carry an rfa (deep rough) mutation for permeation of test chemicals, and the strains except for TA102 have a deletion mutation of uvrB gene to keep adducts generated with test chemicals as well as gal, chl, and bio genes [5, 6, 14]. The two tester strains of E. coli carry a terminating ochre mutation in the trpE gene as well as a uvrA mutation [15, 16]. Thus, the genetic background changes (mutations) in the Ames tester strains can naturally occur without using modern recombinant DNA technology.

Table 1 Strains recommended for use in the bacterial reverse mutation test (OECD guideline 471)

pKM101 plasmid can naturally occur and self-transmissible

As shown in Table 1, the five strains of S. typhimurium (TA98, TA100, TA102, TA97 and TA97a) and one strain of E. coli (WP2uvrA/pKM101) harbor plasmid pKM101. Plasmid pKM101 carries an ampicillin resistance gene and mucAB genes encoding analogs of UmuD/C proteins of E. coli, which are involved in error-prone DNA repair [6, 17]. pKM101 (35.4 kb) is derived from its clinically isolated parent R46 plasmid by an in vivo 14-kb deletion [18]. R46 plasmid contains four drug-resistance genes, while pKM101 dose not contain the other three drug-resistance genes with the exception of the ampicillin resistance gene [19, 20]. In addition, since R plasmids have a self-transmissible nature, pKM101 is normally present in the members of the family Enterobacteriaceae including the genera Salmonella and Escherichia [6, 17]. Taken together, plasmid pKM101 is considered to be a derivative of a naturally occurring plasmid, and self-transmittable.

pAQ1 plasmid in the Ames tester strains does not disturb the biological diversity

The S. typhimurium TA102 strain harbors plasmids pAQ1 in addition to pKM101. The pAQ1 is a derivative of pBR322 and carries the target DNA sequence for reversion, hisG428, a part of the histidine biosynthetic operon originated from S. typhimurium. Thus, hisG428 is a self-cloned gene. The vector pBR322 consists of the following DNA segments assembled in vitro; the tetracycline resistance gene, ampicillin resistance gene, and the replicator regions derived from colicin plasmid, pMB1 [21, 22]. The two drug resistance genes are derived from transposons, Tn10 and Tn3, respectively [22, 23]. Transposons are known to be transferred between related bacteria [24]. So, the drug resistance genes can be naturally introduced into the genome of S. typhimurium. The hisG428 gene is inserted into the ampicillin resistance gene. It was also reported that S. typhimurium LT2 strains are inherently non-colicinogenic, but the strain was shown to have an ability to receive colicin plasmids from E. coli through conjugation [17, 25, 26]. Thus S. typhimurium has a possibility to have the plasmid pMB1 as well as toxin colicin naturally in their cells even without introducing pAQ1. Therefore, the introduction of pAQ1 into the Ames tester strains does not disturb the biological diversity of S. typhimurium, and pAQ1 plasmid can be generated via self-cloning technology and transferred to S. typhimurium LT2.

Conclusion

In Japan, the Cartagena Domestic Law regulates living organisms resulting from modern biotechnology including recombinant DNA technology, and probable exemptions for microorganisms obtained by self-cloning and/or “natural occurrence” are assessed and decided case by case (for each produced organism) [27, 28]. Based on the following stated reasons, we conclude that all the Ames tester strains recommended for use in the bacterial mutation test [4] can be certified as non-GMOs;

  1. 1)

    Genetic backgrounds of the nine strains recommended for use in the bacterial mutation test [4] can be generated spontaneously, or by radiation or chemicals.

  2. 2)

    pKM101 harbored in the tester strains TA97, TA97a, TA98, TA100, TA102, and WP2uvrA/pKM101 is a naturally occurring plasmid and self-transmittable.

  3. 3)

    pAQ1 plasmid which the strain TA102 carries, can be generated via self-cloning technology and transferred to S. typhimurium LT2 by conjugation.

References

  1. European Commission. Directive 2001/18/EC of the European Parliament and the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC, L106. Official J Eur Communities. 2001: 1-38.

  2. Courvalin P. Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Antimicrob Agents Chemother. 1994;38:1447.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Mazel D, Davies J. Antibiotic resistance in microbes. Cell Mol Life Sci. 1999;56:742–54.

    Article  PubMed  CAS  Google Scholar 

  4. OECD guideline for the testing of chemicals 471. Bacterial Reverse Mutation Test. 1997.

  5. Ames BN, Lee FD, Durstonv WE. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A. 1973;70:782–6.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113:173–215.

    Article  PubMed  CAS  Google Scholar 

  7. Mortelmans K, Riccio ES. The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Mutat Res. 2000;455:61–9.

    Article  PubMed  CAS  Google Scholar 

  8. Ames BN, Durston WE, Yamasaki E, Lee FD. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A. 1973;70:2281–5.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Gee P, Maron DM, Ames BN. Detection and classification of mutagens: a set of base-specific Salmonella tester strains. Proc Natl Acad Sci U S A. 1994;24:11606–10.

    Article  Google Scholar 

  10. Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000;455:29–60.

    Article  PubMed  CAS  Google Scholar 

  11. Tejs S. The Ames test: a methodological short review. Environ Biotechnol. 2008;4:7–14.

    Google Scholar 

  12. Hartman PE, Hartman Z, Stahl RC, Ames BN. Classification and mapping of spontaneous and induced mutations in the histidine operon of Salmonella. Adv Genet. 1971;16:1–34.

    Article  PubMed  CAS  Google Scholar 

  13. Whitfield HJ, Martin RG, Ames BN. Classification of aminotransferase (C gene) mutants in the histidine operon. J Mol Biol. 1966;21:335–55.

    Article  PubMed  CAS  Google Scholar 

  14. O'Donovan M. The comparative responses of Salmonella typhimurium TA1537 and TA97a to a range of reference mutagens and novel compounds. Mutagenesis. 1990;5:267–74.

    Article  PubMed  Google Scholar 

  15. Green M, Muriel W. Mutagen testing using Trp+ reversion in Escherichia coli. Mutat Res. 1976;38:3–32.

  16. Parry JM, Parry EM. Genetic toxicology: principles and methods. Methods Mol Biol. 2012;817:21–34.

    Article  Google Scholar 

  17. Mortelmans K. Isolation of plasmid pKM101 in the Stocker laboratory. Mutat Res. 2006;612:151–64.

    Article  PubMed  CAS  Google Scholar 

  18. Winans SC, Walker GC. Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol. 1985;161:402–10.

    PubMed  CAS  PubMed Central  Google Scholar 

  19. Langer PJ, Walker GC. Restriction endonuclease cleavage map of pKM101: relationship to parental plasmid R46. Mol Gen Genet. 1981;182:268–72.

    Article  PubMed  CAS  Google Scholar 

  20. Langer PJ, Shanabruch WG, Walker GC. Functional organization of plasmid pKM101. J Bacteriol. 1981;145:1310–6.

    PubMed  CAS  PubMed Central  Google Scholar 

  21. Betlach MC, Hershfield V, Chow L, Brown W, Goodman HM, Boyer HW. A restriction endonuclease analysis of the bacterial plasmid controlling the EcoRI restricition and modification of DNA. Fed Proc. 1976;35:2035.

    Google Scholar 

  22. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, et al. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2:95–113.

    Article  PubMed  CAS  Google Scholar 

  23. Bukhari AI, Shapiro JA, Adhya SL. DNA insertion elements, plasmids and episomes. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory; 1977. p. 639–40.

    Google Scholar 

  24. de la Cruz F, Davies J. Horizontal gene transfer and the origin of species: lessons from bacteria. Trends Microbiol. 2000;8:128–33.

    Article  PubMed  Google Scholar 

  25. Levin DE, Hollstein M, Christman MF, Schwiers EA, Ames BN. A new Salmonella tester strain (TA102) with A• T base pairs at the site of mutation detects oxidative mutagens. Proc Natl Acad Sci U S A. 1982;79:7445–9.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Sutcliffe J. Complete nucleotide sequence of the Escherichia coli plasmid pBR322, Cold Spring Harb Symp Quant Biol. 1979;43 Pt 1: 77-90.

  27. The Cartagena Protocol on Biosafety to the Convention on Biological Diversity, Montreal, 2000. URL: https://www.cbd.int/doc/legal/cartagena-protocol-en.pdf . Accessed 11 Jan 2016.

  28. The Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms (Act No 97 of 2003).

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Acknowledgements

The authors are grateful to the members of JEMS (The Japanese Environmental Mutagen Society)/BMS (The Bacterial Mutagenicity Study Group) for their valuable comments and helpful discussion on these contents.

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Correspondence to Atsushi Hakura.

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KS drafted the manuscript. MY, AH, and TA critically reviewed on the manuscript. All authors read and approved the final manuscript.

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Sugiyama, Ki., Yamada, M., Awogi, T. et al. The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms. Genes and Environ 38, 2 (2016). https://doi.org/10.1186/s41021-016-0030-3

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