1 Laboratoire de Physiologie et Ecologie Microbienne, Faculté des Sciences, Université Libre de Bruxelles, c/o Institut Pasteur, 642 Rue Engeland, B-1180 Brussels, Belgium
2 Laboratory of Actinomycetes and Fungi imperfecti, National Institute of Hygiene, Warsaw, Poland
3 Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, Debrecen, Hungary
Correspondence
M. J. Penninckx
upemulb{at}resulb.ulb.ac.be
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ABSTRACT |
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INTRODUCTION |
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The pellets are formed by the development of a spore inoculum into agglomerates of hyphae trapped together during germination (Gerin et al., 1993). The exact mechanism behind pellet formation is not known. The morphology of pellets may depend on many factors including the inoculum level, genetic factors, medium composition, addition of surfactants, shearing forces, etc. (Metz & Kossen, 1977
).
Cultivation in the form of pellets was proposed for the industrial production of some secondary metabolites and enzymes (Braun & Vecht-Lifshitz, 1991). Because fungal morphology affects the rheological properties of the fermentation broth, control of morphology is highly desired in industrial fungal fermentation (Park et al., 2002
). Phanerochaete chrysosporium grown in agitated liquid culture typically forms pellets, and produces MnP under nitrogen limitation in the presence of Mn2+. The size of the pellets was found to be a crucial factor for LiP and MnP production (Jaspers et al., 1994
; Jiménez-Tobon et al., 1997
). In order to learn more about the interrelation between fungal architecture and MnP production, we investigated the ultrastructure of P. chrysosporium hyphae from pellets in submerged liquid cultures. The enzyme production was found to be principally associated with chlamydospore-like spherical cells, produced by differentiation of subapical cells located in the outer and middle zones of the pellet. These structures could operate as an enzyme reservoir, delivering their content into the surrounding medium.
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METHODS |
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Extracellular chitinase (EC 3.2.1.14) was estimated with colloidal crab shell chitin (Xia et al., 2001). One unit (U) of chitinase activity is defined as the amount of enzyme catalysing the release of reducing sugars corresponding to 1 µmol N-acetylglucosamine min-1.
Extracellular protease activity (EC 3.4..) was measured with Azocoll as the substrate (Dosoretz et al., 1990). One enzyme unit (U) is defined as the amount of enzyme which catalyses the release of azo dye causing an A520 change of 0·001 min-1.
Procedure for microscopy.
Pellet formation and growth of mycelium was monitored by phase-contrast microscopy (Docuval microscope, Carl-Zeiss).
For electron microscopy ultrathin sections of hyphae from the periphery and central parts of pellets were prepared by the procedure of Strunk (1978) modified by Kurzatkowski et al. (1991)
. Immunolabelling on ultrathin sections was carried out basically according to Kurzatkowski et al. (1991)
. For the immunodetection of MnP, a rabbit antiserum obtained according to Jiménez-Tobon (1999)
was raised against a highly purified preparation of the H3 enzyme isoform (Orth et al., 1994
). Preimmune serum was obtained from the same rabbit before immunization. Western blot experiments have shown that the antibodies obtained react with purified MnP, although some faint cross-reaction was observed with H8 LiP isoenzyme (Jiménez-Tobon, 1999
).
Antigenantibody complexes were visualized with goat anti-rabbit (IgG)15 nm gold conjugate (BioCell). Controls were included either by omitting the antibody against MnP, or by adding pre-immune serum (dilution 1 : 400 in PBG; Jiménez-Tobon, 1999), followed by goat anti-rabbit IgG15 nm gold conjugate. It was checked that the immunomarker was found only at locations where the antigen (MnP)antibody reaction was effective. The ultrathin sections were examined with a JEM 100 C (JEOL) transmission electron microscope at 80 kV.
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RESULTS |
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In a transmission electron microscopy analysis (not illustrated here) we have observed that the wall of young apical cells present in the outer region of the pellet was typically composed of one thin layer with an electron-opaque cytoplasm densely packed with ribosomes. A large number of elongated mitochondria possessing numerous flat cristae were also present in the cytoplasm. Nuclei in young, growing hyphae were often elongated parallel to the long axis of the hyphae. Nuclear pores were observed in the nuclear envelope. At the nuclear envelope the extensive membrane system of an endoplasmic reticulum was present. Small vacuoles were characteristic for the young cells and the cross-walls were not closed. The cytoplasm of subapical cells was electron-opaque and contained a large number of mitochondria and nuclei. The cell wall was composed of two layers. Small vacuoles surrounded by a tonoplast could also be seen. In ageing cells present in the inner part of the pellet, the cell wall was composed of several sublayers. The cytoplasm was electron-transparent and large vacuoles surrounded by a tonoplast were noted. The developed cross-walls were accompanied by Woronin bodies.
At day 2 of cultivation, some subapical regions of hyphae apparently started a process of differentiation into spherical cells of about 10 µm diameter morphologically similar to chlamydospores (Fig. 2a, b). These intercalary chlamydospore-like cells grew in number to culminate between days 3 and 4 of cultivation. Disappearance of these structures apparently started between days 4 and 5 and was nearly complete after 7 days of cultivation. Chlamydospore-like cells have also been observed in cultures not producing MnP but apparently never engaged in an autolytic process without the addition of extra Mn2+, which is necessary for enzyme induction (not shown).
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Immunolocalization of the sites of MnP production
The sites of MnP production were localized at different stages of cultivation. MnP-reacting antibodies were found concentrated mainly in the chlamydospore-like cells and secondarily at the apical ends (Fig. 3a, b). In chlamydospore-like cells from 3 and 4 day cultures, the immunomarker was found to be predominantly concentrated in the Golgi-derived vesicles located at the peripheral part of the cytoplasm (Figs 3b and 4a
). In chlamydospore-like cells from 6 day cultures the immunomarker density was lower and distributed in approximately equal proportions between the cell wall and the Golgi-derived vesicles (Figs 3b and 4b
). A much lower density of the immunomarker was always detected in the other cell organelles and in the cytoplasm. Cells in the senescent central core of the pellet exhibited only a very faint immunoreaction (Fig. 3c
).
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DISCUSSION |
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MnP production in P. chrysosporium chlamydospore-like cells was found mainly associated with Golgi-derived vesicles produced from the endoplasmic reticulum and located in the peripheral part of the cytoplasm. Mitochondria and nuclei were characteristically co-located with the vesicles, which suggests a cooperative metabolic activity. LiP and MnP were also found mainly associated with Golgi-derived vesicles in apical cells of secondary growing hyphae (Bonnarme et al., 1994).
Release of MnP in the culture supernatant coincided with the phase of disappearance of the chlamydospore-like cells from the mycelial pellets of P. chrysosporium, and with maximal activity of extracellular chitinase and protease. Chitinases and proteases in fungi were identified as autolytic enzymes involved in a variety of functions (Gooday, 1997a; Rao et al., 1998
), including disruption of organelle and cell wall structure (Gooday, 1997b
; White et al., 2002
; Pocsi et al., 2003
). Production of proteases is a common feature among fungi including Basidiomycetes (Rao et al., 1998
). Chitinases have apparently been reported only occasionally in Basidiomycetes (Tracey, 1955
; Ohtakara, 1988
) but, as far we are aware, not previously in P. chrysosporium. The presence of protease and chitinase activities in the culture supernatant of P. chrysosporium might reflect their participation in hyphal autolysis steps, possibly including the disappearance of chlamydospore-like cells. For example, age-related extracellular chitinases have been shown to play a crucial role in both autolysis and fragmentation of Penicillium chrysogenum (Sami et al., 2001
). In this context, the first peak of extracellular protease produced by P. chrysosporium could be involved in combination with chitinases (and possibly other enzymes) in the lysis of the chlamydospore-like cells, whereas the protease produced during late idiophase would play a role in the decline of MnP, as was suggested for LiP (Dosoretz, 1990; Dass, 1995
). The exact role of these lytic enzymes has to be demonstrated in future in vivo enzyme inhibition experiments using for example allosamidin (Sami et al. 2001
). From all the various data collected in this study, we conclude the chlamydospore-like structures of P. chrysosporium apparently show the characteristics of metabolically active entities acting as an enzyme reservoir and delivering their content into the surrounding medium by an autolytic process.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 April 2003;
revised 22 July 2003;
accepted 22 August 2003.
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