Folia Microbiologica

, 49:277

Physiological and morphological changes in autolyzingAspergillus nidulans cultures



Physiological and morphological changes in carbon-limited autolyzing cultures ofAspergillus nidulans were described. The carbon starvation arrested conidiation while the formation of filamentous and “yeast-like” hyphal fragments with profoundly altered metabolism enabled the fungus to survive the nutritional stress. The morphological and physiological stress responses, which maintained the cellular integrity of surviving hyphal fragments at the expense of autolyzing cells, were highly concerted and regulated. Moreover, sublethal concentrations of the protein synthesis inhibitor cycloheximide or the mitochondrial uncoupler 2,4-dinitrophenol completely blocked the autolysis. In accordance with the propositions of the free-radical theory of ageing reactive oxygen species accumulated in the surviving fragments with a concomitant increase in the specific superoxide dismutase activity and a continuous decrease in cell viability. Glutathione was degraded extensively in carbon-starving cells due to the action of γ-glutamyltranspeptidase, which resulted in a glutathione-glutathione disulfide redox imbalance during autolysis.







dry cell mass


glucose-6-phosphate dehydrogenase


glutathione peroxidase


glutathione reductase




glutathione disulfide


glutathione transferase




isocitrate dehydrogenase (NADP+)


3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide


reactive oxygen species


superoxide dismutase


Cu,Zn-superoxide dismutase


Mn-superoxide dismutase


  1. Anderson M.E.: Determination of glutathione and glutathione disulfide in biological samples.Meth.Enzymol. 113, 548–555 (1985).PubMedCrossRefGoogle Scholar
  2. Ashok B.T., Ali R.: The aging paradox: free radical theory of aging.Exp.Gerontol. 34, 293–303 (1999).PubMedCrossRefGoogle Scholar
  3. Bahr J.T., Bonner W.D. Jr.: Cyanide-insensitive respiration. I. The steady states of skunk cabbage spadix and bean hypocotyl mitochondria.J.Biol.Chem. 248, 3441–3445 (1973).PubMedGoogle Scholar
  4. Barratt R.W., Johnson G.B., Ogata W.N.: Wild-type and mutant stocks ofAspergillus nidulans.Genetics 52, 233–246 (1965).PubMedGoogle Scholar
  5. Bruinenberg P.M., van Dijken J.P., Scheffers W.A.: An enzymic analysis of NADPH production and consumption inCandida utilis.J.Gen.Microbiol. 129, 965–971 (1983).PubMedGoogle Scholar
  6. Cheng J., Park T.S., Chio L.C., Fischl A.S., Ye X.S.: Induction of apoptosis by sphingoid long-chain bases inAspergillus nidulans.Mol.Cell.Biol. 23, 163–177 (2003).PubMedCrossRefGoogle Scholar
  7. Chiu D.T.Y., Stults F.H., Tappel A.L.: Purification and properties of rat lung soluble glutathione peroxidase.Biochim.Biophys.Acta 445, 558–566 (1976).PubMedGoogle Scholar
  8. Costa V., Moradas-Ferreira P.: Oxidative stress and signal transduction inSaccharomyces cerevisiae: insights into ageing, apoptosis and disease.Mol.Aspects Med. 22, 217–246 (2001).PubMedCrossRefGoogle Scholar
  9. Dunn-Coleman N., Prade R.G.: Toward a global filamentous fungus genome sequencing effort.Nature Biotechnol. 16, 5 (1998).CrossRefGoogle Scholar
  10. Emri T., Bartók G., Szentirmai A.: Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase inPenicillium chrysogenum.FEMS Microbiol.Lett. 117, 67–70 (1994).CrossRefGoogle Scholar
  11. Emri T., Pócsi I., Szentirmai A.: Glutathione metabolism and protection against oxidative stress caused by peroxides inPenicillium chrysogenum.Free Radical Biol.Med. 23, 809–814 (1997).CrossRefGoogle Scholar
  12. Emri T., Pócsi I., Szentirmai A.: Analysis of the oxidative stress response ofPenicillium chrysogenum to menadione.Free Radical Res. 30, 125–132 (1999a).CrossRefGoogle Scholar
  13. Emri T., Sámi L., Szentirmai A., Pócsi I.: Co-ordination of the nitrate and nitrite assimilation, the glutathione and free radical metabolisms, and the pentose-phosphate pathway inPenicillium chrysogenum.J.Basic Microbiol. 39, 109–115 (1999b).CrossRefGoogle Scholar
  14. Harman D.: Free radical involvement in aging.Drug Aging 3, 60–80 (1993).CrossRefGoogle Scholar
  15. Harvey L.M., McNeil B., Berry D.R., White S.: Autolysis in batch cultures ofPenicillium chrysogenum at varying agitation rates.Enzyme Microb.Technol. 22, 446–458 (1998).CrossRefGoogle Scholar
  16. Islam M.S., Nessa A.: Cell-biology of ageing, III. Malondialdehyde as an index of free radical reactions in the early senescent mutants ofNeurospora crassa and study of the effect of free radical scavengers on malondialdehyde contents.Cell Biol.Internat.Rep. 8, 373–377 (1984).CrossRefGoogle Scholar
  17. Jakubowski W., Biliński T., Bartosz G.: Oxidative stress during aging of stationary cultures of the yeastSaccharomyces cerevisiae.Free Radical Biol.Med. 28, 659–664 (2000).CrossRefGoogle Scholar
  18. Jüsten P., Paul G.C., Nienow A.W., Thomas C.R.: Dependence ofPenicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity.Biotechnol.Bioeng. 59, 762–775 (1998).CrossRefGoogle Scholar
  19. Karaffa L., Vaczy K., Sandor E., Biro S., Szentirmai A., Pócsi I.: Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels inAcremonium chrysogenum.Free Radical Res. 34, 405–416 (2001).CrossRefGoogle Scholar
  20. Lahoz R., Reyes F., Perez-Leblic M.I.: Lytic enzymes in the autolysis of filamentous fungi.Mycopathologia 60, 45–49 (1976).PubMedCrossRefGoogle Scholar
  21. Leary N.O., Pembroke A., Duggan P.F.: Improving accuracy of glucose oxidase procedure for glucose determinations on discrete analyzers.Clin.Chem. 38, 298–302 (1992).PubMedGoogle Scholar
  22. Lee D.G., Shin S.Y., Maeng C.Y., Jin Z.Z., Kim K.L., Hahm K.S.: Isolation and characterisation of a novel antifungal peptide fromAspergillus niger.Biochem.Biophys.Res.Commun. 263, 646–651 (1999).CrossRefGoogle Scholar
  23. Leiter E., Emri T., Gyemant G., Nagy I., Pocsi I., Winkelmann G., Pocsi I.: Penicillin V production byPenicillium chrysogenum in the presence of Fe(III) and in low-iron culture medium.Folia Microbiol. 46, 183–186 (2001).CrossRefGoogle Scholar
  24. Longo V.D., Gralla E.B., Valentine J.S.: Superoxide dismutase activity is essential for stationary phase survival inSaccharomyces cerevisiae.J.Biol.Chem. 271, 12275–12280 (1996).PubMedCrossRefGoogle Scholar
  25. Lorin S., Dufour E., Boulay J., Begel O., Marsy S., Sainsard-Chanet A.: Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant ofPodospora anserina.Mol.Microbiol. 42, 1259–1267 (2001).PubMedCrossRefGoogle Scholar
  26. McIntyre M., Berry D.R., McNeil B.: Response ofPenicillium chrysogenum to oxygen starvation in glucose- and nitrogen-limited chemostat cultures.Enzyme Microb.Technol. 25, 447–454 (1999).CrossRefGoogle Scholar
  27. Medvedev Z.A.: An attempt at a rational classification of theories of aging.Biol.Rev. 65, 375–398 (1990).PubMedCrossRefGoogle Scholar
  28. Mehdi K., Penninckx M.J.: An important role for glutathione and γ-glutamyl transpeptidase in the supply of growth requirements during nitrogen starvation of the yeastSaccharomyces cerevisiae.Microbiology 143, 1885–1889 (1997).PubMedGoogle Scholar
  29. Mehdi K., Thierie J., Penninckx J.: γ-Glutamyl transpeptidase in the yeastSaccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione.Biochem.J. 359, 631–637 (2001).PubMedCrossRefGoogle Scholar
  30. Nagy M., Emri T., Fekete E., Sandor E., Springael J.Y., Penninckx M.J., Pocsi I.: Glutathione metabolism ofAcremonium chrysogenum in relation to cephalosporin C production: is γ-glutamyltranspeptidase in the center?Folia Microbiol. 48, 149–155 (2003).Google Scholar
  31. Nestelbacher R., Laun P., Vondrakova D., Pichova A., Schüller C., Breitenbach M.: The influence of oxygen toxicity on yeast mother cell specific aging.Exp.Gerontol. 35, 63–70 (2000).PubMedCrossRefGoogle Scholar
  32. Oberley L.W., Spitz D.R.: Assay of superoxide dismutase activity in tumour tissue.Meth.Enzymol. 105, 457–464 (1984).PubMedCrossRefGoogle Scholar
  33. Paul G.C., Kent C.A., Thomas C.R.: Hyphal vacuolation and fragmentation inPenicillium chrysogenum.Biotechnol.Bioeng. 44, 655–660 (1994).PubMedCrossRefGoogle Scholar
  34. Pena-Muralla R., Ayoubi P., Graminha M., Martinez-Rossi N.M., Rossi A., Prade R.A.: Antifungal target selection inAspergillus nidulans. Using bioinformatics to make a difference, pp. 215–230 in K.J. Shaw (Ed.):Pathogen Genomics: Impact on Human Health. Humana Press, Totowa (USA) 2002.CrossRefGoogle Scholar
  35. Penninckx M.J.: A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses.Enzyme Microb.Technol. 26, 737–742 (2000).PubMedCrossRefGoogle Scholar
  36. Penninckx M.J., Elskens M.T.: Metabolism and functions of glutathione in micro-organisms.Adv.Microb.Physiol. 34, 239–301 (1993).PubMedCrossRefGoogle Scholar
  37. Perez-Leblic M.I., Reyes F., Martinez M.J., Lahoz R.: Cell wall degradation in the autolysis of filamentous fungi.Mycopathologia 80, 147–155 (1982).PubMedCrossRefGoogle Scholar
  38. Peterson G.L.: Determination of total protein.Meth.Enzymol. 91, 86–105 (1983).Google Scholar
  39. Pinto M.C., Mata A.M., Lopez-Barea J.: Reversible inactivation ofSaccharomyces cerevisiae glutathione reductase under reducing conditions.Arch.Biochem.Biophys. 228, 1–12 (1984).PubMedCrossRefGoogle Scholar
  40. Pócsi I., Pusztahelyi T., Bogati M.S., Szentirmai A.: The formation ofN-acetyl-β-d-hexosaminidase is repressed by glucose inPenicillium chrysogenum.J.Basic Microbiol. 33, 259–267 (1993).CrossRefGoogle Scholar
  41. Pocsi I., Pusztahelyi T., Sámi L., Emri T.: Autolysis ofPenicillium chrysogenum — a holistic approach.Indian J.Biotechnol. 2, 293–301 (2003).Google Scholar
  42. Pusztahelyi T., Pócsi I., Szentirmai A.: Aging ofPenicillium chrysogenum cultures under carbon starvation. II. Protease andN-acetyl-β-d-hexosaminidase production.Biotechnol.Appl.Biochem. 25, 87–93 (1997a).Google Scholar
  43. Pusztahelyi T., Pócsi I., Kozma J., Szentirmai A.: Aging ofPenicillium chrysogenum cultures under carbon starvation. I. Morphological changes and secondary metabolite production.Biotechnol.Appl.Biochem. 25, 81–86 (1997b).Google Scholar
  44. Reyes F., Villanueva P., Alfonso C.: Nucleases in the autolysis of filamentous fungi.FEMS.Microbiol.Lett. 57, 67–72 (1990).PubMedCrossRefGoogle Scholar
  45. Roggenkamp R., Sahm H., Wagner F.: Microbial assimilation of methanol induction and function of catalase inCandida boidinii.FEBS Lett. 41, 283–286 (1974).PubMedCrossRefGoogle Scholar
  46. Sámi L., Pusztahelyi T., Emri T., Varecza Z., Fekete A., Grallert Á., Karányi Z., Kiss L., Pócsi I.: Autolysis and ageing ofPenicillium chrysogenum cultures under carbon starvation: chitinase production and antifungal effect of allosamidin.J.Gen.Appl.Microbiol. 47, 201–211 (2001a).PubMedCrossRefGoogle Scholar
  47. Sámi L., Emri T., Pócsi I.: Autolysis and ageing ofPenicillium chrysogenum cultures under carbon starvation: glutathione metabolism and formation of reactive oxygen species.Mycol.Res. 105, 1246–1250 (2001b).CrossRefGoogle Scholar
  48. Sámi L., Karaffa L., Emri T., Pócsi I.: Autolysis and ageing ofPenicillium chrysogenum under carbon starvation: respiration and glucose oxidase production.Acta Microbiol.Immunol.Hung. 50, 67–76 (2003).PubMedCrossRefGoogle Scholar
  49. Sigler K., Chaloupka J., Brozmanová J., Stadler N., Hófer M.: Oxidative stress in microorganisms — I. Microbialvs. higher cells — damage and defenses in relation to cell aging and death.Folia.Microbiol. 44, 587–624 (1999).CrossRefGoogle Scholar
  50. Sipiczki M., Takeo K., Yamaguchi M., Yoshida S., Miklós I.: Environmentally controlled dimorphic cycle in a fission yeast.Microbiology 144, 1319–1330 (1998).PubMedCrossRefGoogle Scholar
  51. Skulachev V.P.: The programmed death phenomena, aging, and the Samurai law of biology.Exp.Geront. 36, 995–1024 (2001).CrossRefGoogle Scholar
  52. Tomarelli R.M., Charney J., Harding M.L.: The use of azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity.J.Lab.Clin.Med. 34, 428–433 (1949).PubMedGoogle Scholar
  53. Trinci A.P.J., Righelato R.C.: Changes in constituents and ultrastructure of hyphal compartments during autolysis of glucose-starvedPenicillium chrysogenum.J.Gen.Microbiol. 60, 239–249 (1970).PubMedGoogle Scholar
  54. Yip J.Y., Vanlerberghe G.C.: Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of putrient uptake.Physiol.Plant. 112, 327–333 (2001).PubMedCrossRefGoogle Scholar
  55. Warholm M., Guthenberg C., von Bahr C., Mannervik B.: Glutathione transferases from human liver.Meth.Enzymol. 113, 499–504 (1985).PubMedCrossRefGoogle Scholar
  56. Wawryn J., Krzepilko A., Myszka A., Bilinski T.: Deficiency in superoxide dismutases shortens life span of yeast cells.Acta Biochim.Polon. 46, 249–253 (1999).PubMedGoogle Scholar
  57. White S., McIntyre M., Berry D.R., McNeil B.: The autolysis of industrial filamentous fungi.Crit.Rev.Biotechnol. 22, 1–14 (2002).PubMedCrossRefGoogle Scholar
  58. Wickens A.P.: Ageing and the free radical theory.Respir.Physiol. 128, 379–391 (2001).PubMedCrossRefGoogle Scholar
  59. Zhang Y., Herman B.: Ageing and apoptosis.Mech.Ageing Dev. 123, 245–260 (2002).PubMedCrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic 2004

Authors and Affiliations

  • T. Emri
    • 1
  • Z. Molnár
    • 1
  • T. Pusztahelyi
    • 1
  • I. Pócsi
    • 1
  1. 1.Department of Microbiology and Biotechnology, Faculty of ScienceUniversity of DebrecenDebrecenHangary

Personalised recommendations