Skip to main content
SpringerLink
Log in

Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48–72 h; fraction 2, 24–48 h; fraction 3, 0–24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.

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. 1A, B
Fig. 2A, B
Fig. 3A–D
Fig. 4A–G
Fig. 5A–E
Fig. 6A–E
Fig. 7A–D
Fig. 8

Similar content being viewed by others

References

  • Amor C, Dominguez AI, Lucas JRD, Laborda F (2000) The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes. Arch Microbiol. 174:59–66

  • Atkin AL (1999) Preparation of yeast cells for confocal microscopy. Methods Mol Biol 122:131–139

    CAS  PubMed  Google Scholar 

  • Binder M, Hartig A, Sata T (1996) Immunogold labeling of yeast cells: an efficient tool for the study of protein targeting and morphological alterations due to overexpression and inactivation of genes. Histochem Cell Biol 106:115–130.

    Article  CAS  PubMed  Google Scholar 

  • Calvo AM, Wilson RA, Bok JW, Keller NP (2002) Relationship between secondary metabolism and fungal development. Microbiol Mol Biol Rev 66:447–459

    Article  CAS  PubMed  Google Scholar 

  • Cary JW, Ehrlich KC, Wright MS, Chang P-K, Bhatnagar D (2000) Generation of aflR disruption mutants of Aspergillus parasiticus. Appl Microbiol Biotechnol. 53:680–684

  • Cary JW, John MD, Ehrlich KC, Wright MS, Liang S-H, Linz JE (2002) Molecular and functional characterization of a second copy of the aflatoxin regulatory gene, AflR, from Aspergillus parasiticus. Biochim Biophys Acta 1576:316–323

    Article  CAS  PubMed  Google Scholar 

  • CAST (2003) Council for Agricultural Science and Technology. Mycotoxins: risks in plant, animal, and human systems. Report 139. Council for Agricultural Science and Technology, Ames, Iowa

  • Chang P-K, Yu J, Bhatnagar D, Cleveland TE (1998) Abstract. The USDA-ARS Aflatoxin Elimination Workshop, October 25–27, 1998, St. Louis, Missouri

  • Chiou C-H, Miller M, Wilson DL, Trail F, Linz J (2002) Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus. Appl Environ Microbiol 68:306–315

    Article  CAS  PubMed  Google Scholar 

  • Cleveland TE, Lax AR, Lee LS, Bhatnagar D (1987) Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures. Appl Environ Microbiol 53:1711–3

    CAS  PubMed  Google Scholar 

  • Eaton DL, Groopman JD (eds.) (1994) The toxicology of aflatoxins: human health, veterinary, and agricultural significance. Academic, San Diego, California

    Google Scholar 

  • Keller NP, Dischinger HC, JR, Bhatnagar D, Cleveland TE, Ullah AHJ (1993) Purification of a 40-kDa methyltransferase activity in the aflatoxin biosynthetic pathway. Appl Environ Microbiol 36:270–273

    Google Scholar 

  • Klionsky DJ (1997) Protein transport from the cytoplasm into vacuole. J Membr Biol 157:105–115

    Article  CAS  PubMed  Google Scholar 

  • Klionsky DJ, Herman PK, EMR SD (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54:266–292

    CAS  PubMed  Google Scholar 

  • Lee L-W, Chiou C-H, Linz JE (2002) Function of native OmtA in vivo and expression and distribution of this protein in colonies of Aspergillus parasiticus. Appl Environ Microbiol 68:5718–5727

    CAS  PubMed  Google Scholar 

  • Liang S-H (1996) The function and expression of the ver-1 gene and localization of the Ver-1 protein involved in aflatoxin B1 biosynthesis in Aspergillus parasiticus. PhD Dissertation, Michigan State University, East Lansing

  • Liang S-H, Wu T-S, Lee R, Chu FS, Linz JE (1997) Analysis of mechanism regulating expression of the ver-1 gene, involved in aflatoxin biosynthesis. Appl Environ Microbiol 63:1058–1065

    Google Scholar 

  • Liu BH, Keller NP, Bhatnagar D, Cleveland TE, Chu FS (1993) Production and characterization of antibodies against sterigmatocystin O-methyltransferase. Food Agric Immunol 5:155–164

    CAS  Google Scholar 

  • Luchese RH, Harrigan WF (1993) Biosynthesis of aflatoxin- the role of nutritional factors. J Applied Bacteriol 74:5–14

    CAS  Google Scholar 

  • Müller WH, van der Krift TP, Knoll G, Smaal EB, Verkleij AJ (1991) A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastuctural and immuno-electron microscopical studies. J Microsc 164:29–41

    PubMed  Google Scholar 

  • Ohsumi K, Arioka M, Nakajima H, Kitamoto K (2002) Cloning and characterization of a gene (avaA) from Aspergillus nidulans encoding a small GTPase involved in vacuole biogenesis. Gene 291:77–84

    Article  CAS  PubMed  Google Scholar 

  • Paul GC, Kent CA, Thomas CR (1994) Hyphal vacuolation and fragmentation in Penicillium chrysogenum. Biotechnol Bioeng 44:655–660

    Google Scholar 

  • Payne GA, Brown MP (1998) Genetics and physiology of aflatoxin biosynthesis. Annu Rev Phytopathol 36:329–362

    Article  CAS  Google Scholar 

  • Prieto R, Woloshuk CP (1997) ord1, an oxidoreductase gene responsible for the conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus. Appl Environ Microbiol 63:1661–1666

    Google Scholar 

  • Saxena M, Allameh A, Mukerji KG, Raj HG (1991) Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate. Chem Biol Interactions 78:13–22

    Article  CAS  Google Scholar 

  • Schwencke J, De Robichon-Szulmajster H (1976) The transport of S-adenosyl-L-methionine in the isolated yeast vacuoles and spheroplasts. Eur J Biochem 65:49–60

    CAS  PubMed  Google Scholar 

  • Svihla G, Dainko JL, Schlenk F (1969) Ultraviolet micrography of penetration of exogenous cytochrome c into the yeast cell. J Bacteriol 100:498–504

    CAS  PubMed  Google Scholar 

  • Taylor KM, Kaplan CP, Gao X, Baker A (1996) Localization and targeting of isocitrate lyases in Saccharomyces cerevisiae. Biochem J 319:255–262

    CAS  PubMed  Google Scholar 

  • Thumm M (2000) Structure and function of the yeast vacuole and its role in autophagy. Microsc Res Tech 51:563–572

    Article  CAS  PubMed  Google Scholar 

  • Trail F, Chang P-K, Cary J, Linz JE (1994) Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxin by Aspergillus parasiticus. Appl Environ Microbiol 60:3315–3320

    Google Scholar 

  • Valenciano S, De Lucas JR, Van der Klei I, Veenhuis M, Laborda F (1998) Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy. Arch Microbiol 170:370–376

    Article  CAS  PubMed  Google Scholar 

  • van der Lende TR, van de Kamp M, Berg M, Sjollema K, Bovenberg RA, Veenhuis M, Konings WN, Driessen AJ (2002) δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine sythetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme. Fungal Genet Biol 37:49–55

    Article  PubMed  Google Scholar 

  • Watanabe CMH, Townsend CA (1998) The in vitro conversion of norsolorinic acid to aflatoxin B1. An improved method of cell-free enzyme preparation and stabilization. J Am Chem Soc 120:6231–6239

    Article  CAS  Google Scholar 

  • Weber RWS, Wakley GE, Thines E, Talbot NJ (2001) The vacuole as element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea. Protoplasma 216:101–112

    CAS  PubMed  Google Scholar 

  • Xu H, Mendgen K (1994) Endocytosis of 1,3-ß-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage). Planta 195:282–290

    CAS  Google Scholar 

  • Yabe K, Nakamura M, Hamasaki T (1999) Enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins. Appl Environ Microbiol 65:3867–3872

    CAS  PubMed  Google Scholar 

  • Yu, J, Cary JW, Bhatnagar D, Cleveland TE, Keller NP, Chu FS (1993) Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis. Appl Environ Microbiol 59:3564–71

    CAS  PubMed  Google Scholar 

  • Yu J, Chang P-K, Ehrlich KC, Cary JW, Bhatnagar D, Cleveland TE (1998) Characterization of the critical amino acids of Aspergillus parasiticus cytochrome P-450 monooxygenease encoded by ordA that is involved in the biosynthesis of aflatoxin B1, G1, B2 and G2. Appl Environ Microbiol 64:4834–4841

    Google Scholar 

  • Zhou R (1997) The function, accumulation, and localization of the Nor-1 protein involved in aflatoxin biosynthesis; the function of the fluP gene associated with sporulation in Aspergillus parasiticus. PhD Dissertation, Michigan State University, East Lansing

Download references

Acknowledgements

We thank Dr. Shirley Owens for help in quantification of fluorescence intensity and Dr. Xudong Fan in operating the jet-freezer at the Center of Advanced Microscopy at Michigan State University. This work was supported by the Michigan Agricultural Experiment Station (MSU), the National Food Safety and Toxicology Center (MSU), the Center for Environmental Toxicology (MSU), and the National Institutes of Health (RO1 CA52003-12).

Author information

Authors and Affiliations

  1. Department of Food Science and Human Nutrition, Michigan State University, 234B GM Trout Food Science and Human Nutrition Building, East Lansing, MI 48824, USA

    Li-Wei Lee, Ching-Hsun Chiou & John E. Linz

  2. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA

    John E. Linz

  3. National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824, USA

    John E. Linz

  4. Institute of Environmental Toxicology Center, Michigan State University, East Lansing, MI 48824, USA

    Li-Wei Lee, Ching-Hsun Chiou & John E. Linz

  5. Center for Advanced Microscopy, Michigan State University, East Lansing, MI 48824, USA

    Karen L. Klomparens

  6. Southern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, New Orleans, Louisiana , 70179, USA

    Jeffrey W. Cary

Authors
  1. Li-Wei Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ching-Hsun Chiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karen L. Klomparens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeffrey W. Cary
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John E. Linz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John E. Linz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, LW., Chiou, CH., Klomparens, K.L. et al. Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus . Arch Microbiol 181, 204–214 (2004). https://doi.org/10.1007/s00203-003-0643-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-003-0643-3

Keywords

Search

Navigation