Research Paper Ultrastructural Characteristics and Variability of Vegetative Reproduction in Fellomyces Penicillatus
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Journal of Basic Microbiology 2012, 52, 531 – 538 531 Research Paper Ultrastructural characteristics and variability of vegetative reproduction in Fellomyces penicillatus Marie Kopecká1, Wladyslav Golubev2, Vladimíra Ramíková1, Dobromila Klemová3 and Ladislav Ilkovics3 1 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 2 Russian Collection of Microorganisms, Institute for Biochemistry and Physiology of Microorganisms, Pushchino, Russia 3 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic The yeast strains VKM Y-2977 and VKM Y-2978, derived from the isolate Pa-202, were examined for their physiological properties and mycocin sensitivities and studied by light, phase-contrast, fluorescence, transmission and scanning electron microscopy. The cells of the first strain produced long stalk-like conidiophores, whereas the cells of the second one had the appearance of a typical budding yeast under the light microscope. Transmission and scanning electron microscopy showed the formation of stalk-like conidiophores and long necks in VKM Y-2977, similar in appearance to Fellomyces fuzhouensis. The actin cytoskeleton, microtubules and nuclei were similar as well, but due to presence of a capsule, they were not clearly visible. The second isolate, VKM Y-2978, had very short stalk-like conidiophores, and the neck, microtubules and actin cables were shorter as well. The actin patches, actin cables, and microtubules were similar in VKM Y-2977 and VKM Y-2978 and not clearly visible. The physiological character- istics and mycocin sensitivity patterns, together with the microscopic structures and ultra- structures, led us to conclude that both strains belong to Fellomyces penicillatus, even though they differ in the lengths of their stalk-like conidiophores and necks. Abbreviation: RhPh, rhodamine-phalloidin Keywords: Conidiogenesis / Sterigmata / Morphology / Cytoskeleton / Electron microscopy Received: August 11, 2011; accepted: September 26, 2011 DOI 10.1002/jobm.201100405 * Introduction ment of daughter cells on stalk-like conidiophores (Fel- lomyces Yamada et Banno). In some instances, however, The mode of vegetative reproduction is an important the mode of reproduction is liable to variation. For ex- characteristic in yeast taxonomy and identification. The ample, budding may be both on a narrow and a broad mode of reproduction is diagnostic not only of the ge- base in species of the genus Trichosporon Behrend, and nus but sometimes also of the family (e.g. Schizosac- nonpolar buds may occur in polar budding yeasts charomycetaceae). The majority of yeasts reproduce by (Schizoblastosporion Ciferri) [1]. multilateral budding (e.g. Saccharomyces Meyen ex Reess) During a survey of the yeast community colonizing whereas others do so by bipolar (Nadsonia Sydow) or dead needles of spruce litter, it has been found that monopolar budding on a broad base (Malassezia Baillon), several isolates produce new cells on stalk-like conidio- by fission (Schizosaccharomyces Lindner) or by develop- phores in addition to budding ones. The presence of different modes of vegetative reproduction in these Correspondence: Professor Marie Kopecká, MD, Ph.D., Department isolates can be attributed to contamination of these of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, A6, 625 00 Brno, Czech Republic cultures or to variance in daughter cell formation. By E-mail: [email protected] replating on malt extract agar, two different colony Phone: +420 54949 4778 forms were obtained from these isolates. Microscopi- Fax: +420 54949 1327 http://www.med.muni.cz/~mkopecka/publikations.php cally, one of them is largely characterized by budding, © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com 532 M. Kopecká et al. Journal of Basic Microbiology 2012, 52, 531 – 538 but another form generates stalk-like conidiophore with rhodamine-phalloidin (RhPh; Molecular Probes, Inc.) structures. The purpose of the current study is to exam- for 1 h and subsequently the cells were washed with PBS. ine cell reproduction in these two forms, and ultra- structural characteristics were compared with those Staining of nuclei found for Fellomyces fuzhouensis (Yue) Yamada et Banno Nuclei were stained with 4′,6-diamidino-2-phenyl-indo- [2] and Cryptococcus laurentii (Kuffrath) Skinner [3]. le dihydrochloride (DAPI) at a final concentration of 1 μg/ml of Vectashield mounting medium (Vector Labo- ratories, Inc., Burlingame, CA 94010, USA). Materials and methods Fluorescence microscopy Organisms and culture A Leica Laborlux S Leitz fluorescence microscope with The strains VKM Y-2977 and VKM Y-2978 were obtain- standard filter blocks for violet (355–425 nm), blue ed from the Russian Collection of Microorganisms (450–490 nm) and green (515–560 nm) emission light, (www.vkm.ru). They were derived from the isolate Pa- including a Plan Phaco 3100/1.25 objective and equip- 202 [4]. For their identification, standard methods cur- ment for phase contrast microscopy, was used. Photo- rently employed in yeast taxonomy were used [1]. The graphic images were taken on Ilford 400 film. procedure for determining mycocin sensitivity patterns has been described previously [5]. Cell preparation for Ultrathin sectioning phase contrast, fluorescent and scanning electron mi- Cells were fixed in combined 3% glutaraldehyde and croscopy has also been described previously [2, 6, 7]. For 1% paraformaldehyde in PBS for 3 h. After three microscopy, the strains were grown on agar containing 30 min washes with PBS, the cells were post-fixed in yeast extract (1%), peptone (2%) and dextrose (2%) at 1.5% KMnO4 for 20 min. The fixed cells were washed 23 °C for about 2 d. They were then inoculated into with five 10 min washes with distilled water until a liquid YEPD medium and cultivated on a shaker clear supernatant was achieved, and embedded in 2% (70 rpm) at 23 °C for 18 h. (w/v) agar. The cells were dehydrated in a graded alco- hol series (10–100%) and embedded in LR White resin. Fixation of cells First, the samples were incubated for 2 d in a refrigera- To visualize the cytoskeleton, the cells were fixed with tor, and then the LR White was allowed to polymerize 5.0% (w/v) paraformaldehyde in phosphate-buffered for 3 d at 60 °C. Subsequently, ultrathin sections were saline (PBS), supplemented with ethylene glycol-bis(β- produced. Some sections were contrasted with 2.5% aminoethyl-ether)-N,N,N′,N′-tetraacetic acid (EGTA) and uranyl acetate for 2 min and lead citrate for 1 min (see MgCl2, at pH 6.9 for 10–90 min. Subsequently, three Fig. 5d); other sections were used without any further washes with PBS were performed [2, 8]. contrasting (see Figs. 5a–c, e, 11–e). Visualisation of microtubules Transmission electron microscopy To stain the microtubules, a modified method as de- The ultrathin sections were viewed and photographed scribed in [8] was used. Immediately after fixation, the with a MORGAGNI 268D (100 kV) microscope. cells were exposed to lysing enzymes of Trichoderma Scanning electron microscopy harzianum (Sigma) at a concentration of 1 mg/ml for The cells were fixed with 5% paraformaldehyde [2, 7], 90 min at 37 °C. They were subsequently permeabilized dehydrated with a graded alcohol series, dried using a with 1% (v/v) Triton X-100 in PBS for 5 min, and then CPD 030 critical point dryer (BAL-TEC) and coated with 2.0% (w/v) bovine serum albumin was added for 30 min gold in a BALZERS SCD040 metal shadowing apparatus at 37 °C. For microtubule labeling, cells were incubated (5 min, 30 mA). The specimens were observed in Vega with the monoclonal anti-α-tubulin antibody TU 01 for TS5136 XM (TESCAN) using digital microscopy imaging 2–3 h at 37 °C [2]. After three washes with PBS at 5 min scanning equipment. intervals, swine anti-mouse IgG-fluorescein-isothiocya- nate (SwAM-FITC, Sigma) secondary antibody was added (2 h at 37 °C). Subsequently, the cells were washed Results three-times with PBS at 5 min intervals. Morphological and physiological characterization Visualisation of F-actin of the yeast strains The fixed cells were treated with 1.0% (v/v) Triton X-100 The appearance of a colony of the strain VKM Y-2977 is for 5 min. After two washes with PBS, actin was stained dull, white, rough, and dry. The cells in these colonies © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com Journal of Basic Microbiology 2012, 52, 531 – 538 Variability of vegetative reproduction in Fellomyces penicillatus 533 develop thin tubular stalk-like conidiophores, at the Table 1. Phenotypic characteristics of Fellomyces penicillatus cultures. end of which new ones are produced, separated by an end-break of the stalk. Capsules have not been observed VKM Y-2977 VKM Y-2978 in India ink preparations [9]. The strain VKM Y-2978 Fermentation – – has glistening, greyish, smooth, and slimy colonies Assimilation of carbon compounds composed of budding capsulated cells. These forms are Glucose + + unstable; they can produce streak cultures with sectors Inulin – – Sucrose + + of different cultural type upon long-term storage. Raffinose + + The cultures of both types were almost identical in Melibiose + s physiological characteristics. They are urease positive, Galactose + + Lactose + + nitrate and nitrite negative, assimilate i-inositol and Trehalose s s D-glucuronate, synthesize starch-like compounds and Maltose + + have no fermentative ability. They differed only in the Melezitose + + rate of utilization of some carbon sources (Table 1). In α-Methyl-D-glucoside – – Soluble starch w w addition, they have the same mycocin sensitivity pat- Cellobiose + + terns, i.e. they are sensitive to mycocins secreted by Salicin w w Bullera alba (Hanna) Derx, Cryptococcus perniciosus Arbutin w w Golubev et al., C. pinus Golubev et Pfeiffer, and Filo- L-Sorbose – – L-Rhamnose + + basidium capsuligenum Rodrigues de Miranda, but they D-Xylose + + are resistant to mycocins of B. hannae Hamamoto et L-Arabinose s s Nakase, B. sinensis Li var.