Molecular Genetics and Genomics

, Volume 289, Issue 2, pp 215–223 | Cite as

Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria

  • A. Wensing
  • M. Gernold
  • S. Jock
  • R. Jansen
  • K. GeiderEmail author
Original Paper


We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.


Gene expression Biosynthesis HPLC/MS analysis E. coli inhibition 



We thank David. L. Coplin for valuable comments on the manuscript.


  1. Coyne S, Chizzali C et al (2013) Biosynthesis of the antimetabolite 6-thioguanine in Erwinia amylovora plays a key role in fire blight pathogenesis. Angew Chem Int Ed Engl 52:10564–10568. doi: 10.1002/anie.201305595 PubMedCrossRefGoogle Scholar
  2. Deo SS, Tseng WC et al (1985) Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt. Biochim Biophys Acta 839:233–239PubMedCrossRefGoogle Scholar
  3. Elgemeie GH (2003) Thioguanine, mercaptopurine: their analogs and nucleosides as antimetabolites. Curr Pharm Des 9:2627–2642PubMedCrossRefGoogle Scholar
  4. Falkenstein H, Zeller W et al (1989) The 29 kb plasmid, common in strains of Erwinia amylovora, modulates development of fireblight symptoms. J Gen Microbiol 135:2643–2650Google Scholar
  5. Feistner GJ (1988) (l)-2,5-Dihydrophenylalanine from the fireblight pathogen Erwinia amylovora. Phytochemistry 27:3417–3422CrossRefGoogle Scholar
  6. Feistner G, Staub CM (1986) 6-Thioguanine from Erwinia amylovora. Curr Microbiol 13:95–101. doi: 10.1007/bf01568289 CrossRefGoogle Scholar
  7. Gehring I, Geider K (2012) Differentiation of Erwinia amylovora and E. pyrifoliae strains with single nucleotide polymorphisms and by synthesis of dihydrophenylalanine. Curr Microbiol 65:73–84. doi: 10.1007/s00284-00012-00116-00285 PubMedCrossRefGoogle Scholar
  8. Jakovljevic V, Jock S et al (2008) Hypersensitive response and acyl-homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae. Microb Biotechnol 1:416–424. doi: 10.1111/j.1751-7915.2008.00043.x PubMedCentralPubMedCrossRefGoogle Scholar
  9. Jock S, Donat V et al (2002) Following spread of fire blight in Western, Central and Southern Europe by molecular differentiation of Erwinia amylovora strains with PFGE analysis. Environ Microbiol 4:106–114PubMedCrossRefGoogle Scholar
  10. Larsen RA, Wilson MM et al (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178:193–201. doi: 10.1007/s00203-002-0442-2 PubMedCrossRefGoogle Scholar
  11. Luvisi A, Panattoni A et al (2011) Thiopurine prodrugs for plant chemotherapy purposes. J Phytopath 159:390–392. doi: 10.1111/j.1439-0434.2010.01779.x CrossRefGoogle Scholar
  12. Mandel HG, Latimer RG et al (1965) The actions of thioguanine in Bacillus cereus. Biochem Pharmacol 14:661–682PubMedCrossRefGoogle Scholar
  13. McLennan AG (2006) The Nudix hydrolase superfamily. Cell Mol Life Sci 63:123–143. doi: 10.1007/s00018-005-5386-7 PubMedCrossRefGoogle Scholar
  14. Mohammadi M, Geider K (2007) Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria. FEMS Microbiol Lett 266:34–41. doi: 10.1111/j.1574-6968.2006.00510.x PubMedCrossRefGoogle Scholar
  15. Pratt D, Subramani S (1983) Nucleotide sequence of the Escherichia coli xanthine–guanine phosphoribosyl transferase gene. Nucl Acids Res 11:8817–8823PubMedCentralPubMedCrossRefGoogle Scholar
  16. Sartorelli AC, Lepage GA (1958) Metabolic effects of 6-thioguanine. II. Biosynthesis of nucleic acid purines in vivo and in vitro. Cancer Res 18:1329–1335PubMedGoogle Scholar
  17. Scannel JP, Pruess DL et al (1971) Antimetabolites produced by microorganisms. 3. 2-aminopurine-6-thiol (thioguanine). J Antibiot (Tokyo) 24:328–329CrossRefGoogle Scholar
  18. Schwartz T, Bernhard F et al (1991) Diversity of the fire blight pathogen in production of dihydrophenylalanine, a virulence factor of some Erwinia amylovora strains. Phytopathology 81:873–878CrossRefGoogle Scholar
  19. Vora A, Mitchell CD et al (2006) Toxicity and efficacy of 6-thioguanine versus 6-mercaptopurine in childhood lymphoblastic leukaemia: a randomised trial. Lancet 368:1339–1348. doi: 10.1016/S0140-6736(06)69558-5 PubMedCrossRefGoogle Scholar
  20. Vos S, Parry RJ et al (1998) Structures of free and complexed forms of Escherichia coli xanthine–guanine phosphoribosyltransferase. J Mol Biol 282:875–889. doi: 10.1006/jmbi 1998.2051PubMedCrossRefGoogle Scholar
  21. Wang H, Wang Y (2009) 6-Thioguanine perturbs cytosine methylation at the CpG dinucleotide site by DNA methyltransferases in vitro and acts as a DNA demethylating agent in vivo. Biochemistry 48:2290–2299. doi: 10.1021/bi801467z PubMedCentralPubMedCrossRefGoogle Scholar
  22. Yuan B, Wang Y (2008) Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid. J Biol Chem 283:23665–23670. doi: 10.1074/jbc.M804047200 PubMedCentralPubMedCrossRefGoogle Scholar
  23. Zhang Y, Jock S et al (2000) Genes of Erwinia amylovora involved in yellow color formation and release of a low-molecular-weight compound during growth in the presence of copper ions. Mol Gen Genet 264:233–240. doi: 10.1007/s004380000290 PubMedCrossRefGoogle Scholar
  24. Zhang Y, Jock S et al (2001) Genes of Erwinia amylovora involved in yellow color formation and release of a low-molecular-weight compound during growth in the presence of copper ions (correction). Mol Gen Genet 264:732–733. doi: 10.1007/s004380000425 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • A. Wensing
    • 1
  • M. Gernold
    • 1
  • S. Jock
    • 1
  • R. Jansen
    • 2
  • K. Geider
    • 1
    Email author
  1. 1.Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and ViticultureDossenheimGermany
  2. 2.Department Microbial DrugsHelmholtz Centre for Infection ResearchBrunswickGermany

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