Skip to main content
  • Short Communication
  • Published:

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Abstract

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.

Abbreviations

dCTP:

deoxycytidine triphosphate

Moco:

molybdenum cofactor

niaD :

nitrate reductase-encoding gene

niiA :

nitrite reductase-encoding gene

OMPD:

orotidine-5′-monophosphate decarboxylase

ORF:

open reading frame

pyrG:

orotidine-5′-monophosphate decarboxylase-encoding gene

References

  1. Hjört, C.M. Production of food additives using filamentous fungi. in: Genetically Engineered Food (Heller, K.J., Ed.), Wiley-VCH Verlag Gmbh & Co. KgaA, Weinheim, 2003, 86–99.

    Chapter  Google Scholar 

  2. Yamaguchi, S., Mase, T. and Takeuchi, K. Cloning and structure of the mono- and diacylglycerol lipase-encoding gene from Penicillium camembertii U-150. Gene 103 (1991) 61–67.

    Article  PubMed  CAS  Google Scholar 

  3. Dupont, J., Magnin, S., Marti, A. and Brousse, M. Molecular tools for identification of Penicillium starter cultures used in the food industry. Int. J. Food Microbiol. 49 (1999) 109–118.

    Article  PubMed  CAS  Google Scholar 

  4. Boualem, K., Waché, Y., Garmyn, D., Karbowiak, T., Durand, A., Gervais, P. and Cavin, J.F. Cloning and expression of genes involved in conidiation and surface properties of Penicillium camemberti grown in liquid and solid cultures. Res. Microbiol. 159 (2008) 110–117.

    Article  PubMed  CAS  Google Scholar 

  5. Campbell, W.H. Structure and function of eukaryotic NAD(P)H:nitrate reductase. Cell. Mol. Life Sci. 58 (2001) 194–204.

    Article  PubMed  CAS  Google Scholar 

  6. Wittmann, J.G., Heinrich, D., Gasow, K., Frey, A., Diederichsen, U. and Rudolph, M.G. Structures of the human orotidine-5′-monophosphate decarboxylase support a covalent mechanism and provide a framework for drug design. Structure 16 (2008) 82–92.

    Article  PubMed  CAS  Google Scholar 

  7. Fierro, F., Laich, F., Garcia-Rico, R.O. and Martin, J.F. High efficiency transformation of Penicillium nalgiovense with integrative and autonomously replicating plasmids. Int. J. Food Microbiol. 90 (2004) 237–248.

    Article  PubMed  CAS  Google Scholar 

  8. De Maeseneire, S.L., De Groeve, M.R., Dauvrin, T., De Mey, M., Soetaert, W. and Vandamme, E.J. Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5′-monophosphate decarboxylase gene. FEMS Microbiol. Lett. 261 (2006) 262–271.

    Article  PubMed  Google Scholar 

  9. Pereira, J.F., de Queiroz, M.V., Lopes, F.J.F., Rocha, R.B. and de Araujo, E.F. Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation. Can. J. Microbiol. 50 (2004) 891–900.

    Article  PubMed  CAS  Google Scholar 

  10. Lubertozzi, D. and Keasling, J.D. Marker and promoter effects on heterologous expression in Aspergillus nidulans. Appl. Microbiol. Biotechnol. 72 (2006) 1014–1023.

    Article  PubMed  CAS  Google Scholar 

  11. Garcia-Rico, R.O., Fierro, F. and Martin, J.F. Heterotrimeric Gα protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism. Biochem. Cell Biol. 86 (2008) 57–69.

    Article  PubMed  CAS  Google Scholar 

  12. Bainbridge, B.W., Spreadbury, C.L., Scalise, F.G. and Cohen, J. Improved methods for the preparation of high molecular weight DNA from large and small-scale cultures of filamentous fungi. FEMS Microbiol. Lett. 54 (1990) 113–117.

    Article  PubMed  CAS  Google Scholar 

  13. Sambrook, J. and Russell, D.W. Molecular cloning: a laboratory manual (Third edition). Cold Spring Harbor Laboratory Press (2001).

  14. Yelton, M.M., Hamer, J.E. and Timberlake, W.E. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81 (1984) 1470–1474.

    Article  CAS  Google Scholar 

  15. Fischer, K., Barbier, G.G., Hecht, H.-J, Mendel, R.F., Campbell, W.H. and Schwarz, G. Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. Plant Cell 17 (2005) 1167–1179.

    Article  PubMed  CAS  Google Scholar 

  16. Gómez, D., Garcia, I., Scazzocchio, C. and Cubero, B. Multiple GATA sites: protein binding and physiological relevance for the regulation of the proline transporter gene of Aspergillus nidulans. Mol. Microbiol. 50 (2003) 277–289.

    Article  PubMed  Google Scholar 

  17. Wong, K.H., Hynes, M.J. and Davis, M.A. Recent advances in nitrogen regulation: a comparison between Saccharomyces cerevisiae and filamentous fungi. Eukaryot. Cell 7 (2008) 917–925.

    Article  PubMed  CAS  Google Scholar 

  18. Bernreiter, A., Ramon, A., Fernandez-Martinez, J., Berger, H., Araujo-Bazan, L., Espeso, E.A., Pachlinger, R., Gallmetzer, A., Anderl, I., Scazzocchio, C. and Strauss, J. Nuclear export of the transcription factor NirA is a regulatory checkpoint for nitrate induction in Aspergillus nidulans. Mol. Cell. Biol. 27 (2007) 791–802.

    Article  PubMed  CAS  Google Scholar 

  19. Berger, H., Basheer, A., Böck, S., Reyes-Dominguez, Y., Dalik, T., Altmann, F. and Strauss, J. Dissecting individual steps of nitrogen transcription factor cooperation in the Aspergillus nidulans nitrate cluster. Mol. Microbiol. 69 (2008) 1385–1398.

    Article  PubMed  CAS  Google Scholar 

  20. Slot, J.C. and Hibbett, D.S. Horizontal transfer of a nitrate assimilation gene cluster and ecological transitions in fungi: a phylogenetic study. PLoS ONE 2 (2007), e1097.

    Article  PubMed  Google Scholar 

  21. Haas, H. and Marzluf, G. NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters. Curr. Genet. 28 (1995) 177–183.

    Article  PubMed  CAS  Google Scholar 

  22. Ellegren, H. Comparative genomics and the study of evolution by natural selection. Mol. Ecol. 17 (2008) 4586–4596.

    Article  PubMed  Google Scholar 

  23. Traut, T.W. and Temple, B.R.S. The chemistry of the reaction determines the invariant amino acids during the evolution and divergence of orotidine 5′-monoposphate decarboxylase. J. Biol. Chem. 275 (2000) 28675–28681.

    Article  PubMed  CAS  Google Scholar 

  24. Haas, H., Marx, F., Graessle, S. and Stöffler, G. Sequence analysis and expression of the Penicillium chrysogenum nitrate reductase encoding gene (niaD). Biochim. Biophys. Acta 1306 (1996) 81–84.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Renato Chávez.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Navarrete, K., Roa, A., Vaca, I. et al. Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers. Cell Mol Biol Lett 14, 692–702 (2009). https://doi.org/10.2478/s11658-009-0028-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-009-0028-y

Key words