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Folia Microbiologica

, 50:415 | Cite as

Agrobacterium tumefaciens-mediated transformation ofMucor circinelloides

  • I. Nyilasi
  • K. Ács
  • T. Papp
  • E. Nagy
  • C. Vágvölgyi
Article

Abstract

TheAgrobacterium tumefaciens-mediated transformation of the zygomycetous fungusMucor circinelloides is described. A method was also developed for the hygromycin B-based selection ofMucor transformants. Transformation with the hygromycin B phosphotransferase gene ofEscherichia coli controlled by the heterologousAspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.

Keywords

Hygromycin Induction Medium Crown Gall Rose Bengal Putative Transformants 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Amey R.C., Athey-Pollard A., Burns C., Mills P.R., Bailey A., Foster G.D.: PEG-mediated andAgrobacterium-mediated transformation in the mycopathogenVerticillium fungicola.Mycol.Res. 106, 4–11 (2002).CrossRefGoogle Scholar
  2. Anaya N., Roncero M.I.G.: Transformation of a methionine auxotrophic mutant ofMucor circinelloides by direct cloning of the corresponding wild-type gene.Mol.Gen.Genet. 230, 449–455 (1991).PubMedCrossRefGoogle Scholar
  3. Arnau J., Stroman P.: Gene replacement and ectopic integration in the zygomyceteMucor circinelloides.Mol.Gen.Genet. 23, 542–546 (1993).Google Scholar
  4. Arnau J., Jepsen L.P., Stroman P.: Integrative transformation by homologous recombination in the zygomyceteMucor circinelloides.Mol.Gen.Genet. 225, 193–198 (1991).PubMedCrossRefGoogle Scholar
  5. Benito E.P., Díaz-Minguez J.M., Iturriaga E.A., Campuzano V., Eslava A.P.: Cloning and sequence analysis of theMucor circinelloides pyrG gene encoding orotidine-5′-monophosphate decarboxylase: use ofpyrG for homologous transformation.Gene 225, 59–67 (1992).CrossRefGoogle Scholar
  6. Benito E.P., Campuzano V., López-Matas M.A., de Vicente J.I., Eslava A.P.: Isolation, characterization and transformation by autonomous replication ofMucor circinelloides OMPdecase-deficient mutants.Mol.Gen.Genet. 248, 126–135 (1995).PubMedCrossRefGoogle Scholar
  7. Bundock P., Hooykaas P.J.J.: Integration ofAgrobacterium tumefaciens T-DNA in theSaccharomyces cerevisiae genome by illegitimate recombination.Proc.Nat.Acad.Sci.USA 93, 15272–15275 (1996).PubMedCrossRefGoogle Scholar
  8. Bundock P., Dendulkras A., Beijersbergen A., Hooykaas P.J.J.: Transkingdom T-DNA transfer fromAgrobacterium tumefaciens toSaccharomyces cerevisiae.EMBO J. 14, 3206–3214 (1995).PubMedGoogle Scholar
  9. Campoy S., Perez F., Martin J.F., Gutierrez S., Liras P.: Stable transformants of the azophilone pigment-producingMonascus purpureus obtained by protoplast transformation andAgrobacterium-mediated DNA transfer.Curr.Genet. 43, 447–452 (2003).PubMedCrossRefGoogle Scholar
  10. Covert S.F., Kapoor P., Lee M., Briley A., Nairn C.J.:Agrobacterium tumefaciens-mediated transformation ofFusarium circinatum.Mycol.Res. 105, 259–264 (2001).CrossRefGoogle Scholar
  11. Dickinson L., Harboe M., van Heeswijk R., Stroman P., Jepsen L.P.: Expression of activeMucor miehei aspartic protease inMucor circinelloides.Carlsberg Res.Commun. 52, 243–252 (1987).CrossRefGoogle Scholar
  12. Godtfredsen S.E.: Microbial lipases, pp. 255–274 in W.M. Fogarty, C.T. Kelly (Eds):Microbial Enzymes and Biotechnology, 2nd ed. Elsevier, London 1990.Google Scholar
  13. Gooday G.W.: Hormones in mycelial fungi, pp. 401–411 in J.G.H. Wessels, F. Meinhardt (Eds):The Mycota, Vol. 1. Springer-Verlag, Berlin 1994.Google Scholar
  14. de Groot M.J.A., Bundock P., Hooykaas P.J.J., Beijersbergen A.G.M.:Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnol. 16, 839–842 (1998).CrossRefGoogle Scholar
  15. van Heeswijck R., Roncero M.I.G.: High frequency transformation ofMucor with recombinant plasmid DNA.Carlsberg Res.Commun. 49, 691–702 (1984).CrossRefGoogle Scholar
  16. Hocking A.D.: Improved media for enumeration of fungi in foods.CSIRO Food Res.Quart. 41, 7–11 (1981).Google Scholar
  17. Hood E.E., Helmer G.L., Fraley R.T., Chilton M.D.: The hypervirulence ofAgrobacterium tumefaciens A281 is encoded in the region pTiBo542 outside the T-DNA.J.Bacteriol. 168, 1291–1301 (1986).PubMedGoogle Scholar
  18. Iturriaga E.A., Díaz-Minguez J.M., Benito E.P., Alvarez M.I., Eslava A.P.: Heterologous transformation ofMucor circinelloides with thePhycomyces blakesleeanus leuI gene.Curr.Genet. 21, 215–223 (1992).PubMedCrossRefGoogle Scholar
  19. Iturriaga E.A., Velayos A., Eslava A.P.: Structure and function of the genes involved in the biosynthesis of carotenoids in theMucorales.Biotechnol.Bioproc.Eng. 5, 263–274 (2000).Google Scholar
  20. Iturriaga E.A., Velayos A., Eslava A.P., Alvarez M.I.: The genetics and molecular biology of carotenoid biosynthesis inMucor.Rec.Res.Dev.Genet. 1, 79–92 (2001).Google Scholar
  21. Jung M.K., Ovechkina Y., Prigozhina N., Oakley C.E., Oakley B.R.: The use of β-d-glucanase as a substitute for Novozym 234 in immunofluorescence and protoplasting.Fungal Genet.Newslett. 47, 65–66 (2000).Google Scholar
  22. Kado C.I.: Molecular mechanisms of crown gall tumorigenesis.Crit.Rev.Plant Sci. 10, 1–32 (1991).CrossRefGoogle Scholar
  23. King A.D. Jr.,Hocking A.D., Pitt J.I.: Dichloran-Rose Bengal medium for enumeration and isolation of molds from foods.Appl.Environ.Microbiol. 37, 959–964 (1979).PubMedGoogle Scholar
  24. Lazo G.R., Stein P.A., Ludwig R.A.: A DNA transformation-competentArabidopsis genomic library inAgrobacterium.Biotechnology 9, 963–967 (1991).PubMedCrossRefGoogle Scholar
  25. Leach J., Finkelstein D.B., Rambosek J.A.: Rapid miniprep of DNA from filamentous fungi.Fungal Genet.Newslett. 33, 32–33 (1991).Google Scholar
  26. Malonek S., Meinhardt F.:Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomyceteCalonectria morganii.Curr.Genet. 40, 152–155 (2001).PubMedCrossRefGoogle Scholar
  27. Michielse C.B., Salim K., Ragas P., Ram A.F.J., Kudla B., Jarry B., Punt P.J., van den Hondel C.A.M.J.J.: Development of a system for integrative and stable transformation of the zygomyceteRhizopus oryzae byAgrobacterium-mediated DNA transfer.Mol.Genet.Genomics 271, 499–510 (2004).PubMedCrossRefGoogle Scholar
  28. Mikosh T.S.P., Lavrijssen B., Sonnenberg A.S.M., van Griensven L.J.L.D.: Transformation of the cultivated mushroomAgaricus bisporus (Lange) using T-DNA fromAgrobacterium tumefaciens.Curr.Genet. 39, 35–39 (2001).CrossRefGoogle Scholar
  29. Monfort A., Cordero L., Maicas S., Polaina J.: Transformation ofMucor miehei results in plasmid deletion and phenotypic instability.FEMS Microbiol.Lett. 224, 101–106 (2003).PubMedCrossRefGoogle Scholar
  30. Mullins E.D., Chen X., Romaine P., Raina R., Geiser D.M., Kang S.:Agrobacterium-mediated transformation ofFusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer.Phytopathology 91, 173–180 (2001).CrossRefPubMedGoogle Scholar
  31. Obraztsova I.N., Prados N., Holzmann K., Avalos J., Cerdá-Olmedo E.: Genetic damage following introduction of DNA inPhycomyces.Fungal Genet.Biol. 41, 168–180 (2003).CrossRefGoogle Scholar
  32. Orlowsky M.:Mucor dimorphism.Microbiol.Rev. 55, 234–258 (1991).Google Scholar
  33. Outtrup H., Boyce C.O.L.: Microbial proteinases and biotechnology, pp. 227–254 in W.M. Fogarty, C.T. Kelly (Eds):Microbial Enzymes and Biotechnology, 2nd ed. Elsevier, London 1990.Google Scholar
  34. Roncero M.I.G., Jepsen L.P., Stroman P., van Heeswijck R.: Characterization of aleuA gene and anARS element fromMucor circinelloides.Gene 84, 335–343 (1989).PubMedCrossRefGoogle Scholar
  35. Ruiz-Herrera J.: Dimorphism inMucor species, pp. 257–265 in H. van den Bossche, F.C. Odds, D. Herridge (Eds):Dimorphic Fungi in Biology and Medicine. Plenum Press, New York 1993.Google Scholar
  36. Ruiz-Hidalgo M.J., Eslava A.P., Alvarez M.I., Benito E.P.: Heterologous expression of thePhycomyces blakesleanus phytoene dehydrogenase gene (carB) inMucor circinelloides.Curr.Microbiol. 39, 259–264 (1999).PubMedCrossRefGoogle Scholar
  37. Sambrook J., Fritsch E.F., Maniatis T.:Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.Google Scholar
  38. Skaar I., Stenwig H.: Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage.Appl.Environ.Microbiol. 62, 3614–3619 (1996).PubMedGoogle Scholar
  39. Velayos A., Alvarez M.I., Eslava A.P., Iturriaga E.A.: Interallelic complementation at thepyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) inMucor circinelloides.Mol.Gen.Genet. 260, 251–260 (1998).PubMedCrossRefGoogle Scholar
  40. Wolf A.M., Arnau J.: Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes inMucor circinelloides (syn.racemosus) and use of thegpáI promoter in recombinant protein production.Fungal Genet.Biol. 35, 21–29 (2002).CrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic 2005

Authors and Affiliations

  • I. Nyilasi
    • 1
  • K. Ács
    • 2
  • T. Papp
    • 2
  • E. Nagy
    • 1
  • C. Vágvölgyi
    • 2
  1. 1.HAS-USZ Microbiological Research Group, Department of MicrobiologyUniversity of SzegedSzegedHungary
  2. 2.Department of Microbiology, Faculty of ScienceUniversity of SzegedSzegedHungary

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