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Universidade Metodista de São Paulo, CP 5002, São, Bernardo do Campo, SP 09735-460, Brazil
Glenn Freshour
The University of Georgia, Complex Carbohydrate, Research Center, 315 Riverbend Road, Athens, Georgia 30602-4712
Rita de Cássia L. Figueiredo-Ribeiro
Instituto de Botânica, Seção de Fisiologia e Bioquímica, de Plantas, CP 4005, São Paulo, SP 01061-970, Brazil
Michael G. Hahn
The University of Georgia, Complex Carbohydrate, Research Center, 315 Riverbend Road, Athens, Georgia 30602-4712
Marcia R. Braga 1
Instituto de Botânica, Seção de Fisiologia e Bioquímica, de Plantas, CP 4005, São Paulo, SP 01061-970, Brazil
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ABSTRACT |
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Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.
Key words: carbohydrates, electron transmission microscopy, filamentous fungus, inulin, inulinases, savanna, structural polysaccharides, sucrose, tropical fungus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Polysaccha-rides are the main constituents of fungal cell walls and some of them, such as ß-1,3-D-glucans that also contain some ß-1,6-linked branches, and chitin, a linear polymer of N-acetyl-glucosamine, are distributed widely in many species of different genera (Leal et al 1997, Santos et al 2000). Proteins also are present in the cell wall, mainly associated with hetero- or homopolymers of sugars (Fontaine et al 1997).
Cell-wall polysaccharides have been used for classification of filamentous fungi (Bartinicki-Garcia 1968), identification of yeasts (Gorin and Spencer 1970) and as possible aids in the classification and identification of lichen mycobionts (Carbonero et al 2001). In addition alkali-extractable water-soluble cell-wall polysaccharides can be used as chemotaxonomic markers at the genus or subgenus level with both teleomorphic and anamorphic species, as reported for Penicillium (Leal et al 1995, Prieto et al 1997).
Some studies have focused on the role of the fungal cell wall in chemical sensing and processing of environmental signals that control growth and cell morphology of microorganisms and synthesis and secretion of extracellular enzymes (Terenzi et al 1992, Silva et al 1994).
Protein secretion is a process of major importance to filamentous fungi. Secreted proteins have attracted interest as a valuable source of industrial enzymes (Wallis et al 1997). Among these enzymes are fructosyl-hydrolases, such as inulinases and invertases. Microbial inulinases play an important role in the hydrolysis of inulin for production of high-fructose syrups, inulo-oligosaccharides and ethanol (Skowronek and Fiedurek 2003).
Penicillium janczewskii is a widespread, filamentous soil fungus. Isolates obtained from the rhizosphere of Asteraceae from the Brazilian cerrado (a savanna-like area) are efficient microorganisms for the production of extracellular inulinases and grow rapidly on media containing sucrose or inulin as sole carbon sources (Pessoni et al 1999). Maintenance of P. janczewskii on inulin medium induces secretion of proteins with higher inulinase activity compared to a sucrose-containing medium but results in a mycelium that easily collapses and breaks. We describe the influence of inulin on cell-wall ultrastructure and composition and discuss possible reasons for the differences in cell-wall properties arising from growing the fungus on sucrose or inulin as carbon sources.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Culturing.— – The fungus was grown on medium containing the following components (g/L): NaNO3 (3), KH2PO4 (1), KCl (0.5), MgSO40.7H2O (0.5), FeSO40.7H2O (0.01) and either inulin (1%, w/v) from Helianthus tuberosus (Sigma) or sucrose (1%, w/v) as sole carbon sources in liquid or solid cultures (agar 1.5%, w/v). Cultures were incubated 5–18 d at 28 C in the dark. Some cultures were grown on PDA (potato-dextrose agar, Difco) for inoculum production.
Growth curve of P. janczewskii grown on solid medium.— – Samples of 5 mm diam were taken from the outer actively growing edge of pure cultures cultivated 6 d on PDA medium and were placed in the center of Petri dishes (six plates for each day) on solid-culture media containing either inulin or sucrose as sole carbon sources described above. The plates were incubated inverted at 28 C and the diameter of each colony was measured daily.
Transmission electron microscopy (TEM).— –
A 1 mm3 plug was cut from the actively growing margin of the colony of each fungal solid culture using a razorblade and initially fixed at room temperature in a solution containing 2.5% (v/v) glutaraldehyde, 2.5% (v/v) paraformaldehyde and 5 mM CaCl2 in 0.05 M cacodylate buffer pH 7.2. After 1 h the material was washed with three changes (10 min each) of the same buffer and postfixed 1 h in 0.05 M cacodylate buffer supplemented with 5 mM CaCl2 containing 1% (w/v) OsO4. The fixed material subsequently was transferred to 1% (w/v) uranyl acetate and left in this solution overnight. Samples were dehydrated with a graded aqueous acetone series (30, 50, 70, 90, 100, 100, 100% [v/v], 10 min each step). The dehydrated material first was infiltrated with Spurr’s resin in 1% (w/v) uranyl acetate (1:1) for 5 h and then in pure Spurr’s resin overnight. The infiltrated tissue was transferred to templates containing 100% Spurr’s resin. Polymerization was carried out at 60 C for 24 h. Thin sections (<100 nm) were cut with an MT 6000-XL ultramicro-tome and collected on Formvar-coated, gilded copper slot grids, placed on Formvar bridges to dry (Rowley and Moran 1975) and poststained 30 s with 2% (w/v) lead citrate (Reynolds 1963). The sections were examined at 80 kV with a Zeiss EM 902A electron microscope.
Scanning electron microscopy (SEM).— – Colonies grown in solid medium containing sucrose or inulin were fixed in 2% (w/v) OsO4, for 24 h. A 1 mm3 plug was cut from peripheral mycelial parts of colonies and postfixed in 2% (w/v) OsO4 for 24 h. The fixed material was sputter-coated with gold for 180 s. The material was examined and photographed with a Zeiss DSM 940 scanning electron microscope.
Cell-wall extraction.— – Cell walls were obtained from mycelium grown 5–18 d at 28 C in the dark in liquid medium at 120 rpm in an orbital incubator. At the end of each incubation period, the mycelium from each flask was vacuum filtered onto glass-fiber filters (Whatman), washed with distilled water to remove traces of medium and stored at –18 C. Cell walls were obtained from mycelia by homogenization in a Virtis homogenizer (three times, 10 min each). The homogenate was centrifuged at 8000 g for 15 min, the supernatant discarded and the pellet resuspended in water and sonicated for 1 h. The precipitates were collected after centrifuging at 8000 g for 15 min. This procedure was repeated until the cell walls were free of cytoplasmic contamination as judged by microscopic examination. The cell walls were lyophilized and stored in a desiccator.
Cell-wall analysis.— –
Neutral sugar assay..
1 mg of freeze-dried cell wall was incubated with 1 mL concentrated H2SO4 at room temperature for 2 h. The neutral sugars were evaluated by the phenol-sulfuric method (Dubois et al 1956), using glucose as standard.
Soluble protein assay..
Proteins from cell walls were extracted with 1M NaOH (1 mg cell wall/mL) at room temperature for 2 h, and the protein content was determined colorimetrically by the method of Lowry (Lowry et al 1951) with BSA as standard.
Glycosyl composition.— –
Cell-wall samples were hydrolyzed with 2 M H2SO4 at 100 C for 5 h. Neutral sugars were converted into their corresponding alditol acetates, identified, and quantified by GC-MS in a Hewlett Packard 5985 GC-MS system, as described by Guest and Momany (2000).
Glycosyl linkage analysis.— –
The samples were permethylated, depolymerized, reduced and acetylated. An aliquot of the samples was permethylated by the method of Ciucanu and Kerek (1984), by treating with sodium hydroxide and methyl iodide in dry DMSO. The permethylation was repeated twice to obtain complete methylation of the polymer. After permethylation, the material was hydrolyzed by 2 M TFA (2 h in sealed tube at 121 C), reduced with NaBD4 and acetylated using acetic anhydride/TFA. Partially methylated alditol acetates (PMAAs) were separated on a 30 m Supelco 2330 bonded phase fused silica capillary column, and the eluting peaks analyzed by gas chromatography-mass spectrometry (GC-MS), as described by York et al (1985), on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode).
Fractionation.— –
Cell-wall fractionation was performed as reported by Domenech et al (1999) and Carbonero et al (2001). Freeze-dried cell walls were extracted with 1M NaOH at room temperature for 12 h, followed by centrifugation at 10 000 g for 30 min at 20 C. The pellet was discarded. The soluble material was neutralized with HCl, dialyzed against distilled water, centrifuged and the supernatant freeze-dried and weighed, constituting the first alkali-extractable fraction (FI). The precipitate obtained after HCl neutralization was dissolved in 0.5% (w/v) KOH at 50 C, for 12 h and neutralized with HCl as described above. After centrifugation the pellet was discarded and the new supernatant was dialyzed, freeze-dried and weighed, yielding the second alkali-extractable fraction (FII). Aliquots of both fractions were dissolved in citrate-phosphate buffer 50 mM, pH 5.2 and applied to a Sepharose CL 6B column (1.5 x 120.0 cm), eluted with the same buffer for evaluation of the molecular weight of the polysaccharides extracted. Fractions were monitored for carbohydrate by the phenol-sulfuric acid method (Dubois et al 1956). The column was calibrated using dextrans (Sigma) of 9.3, 39.2, and 156 kDa.
Chitin analysis.— –
2 mg of freeze-dried cell walls were hydrolyzed with 1 mL of 6 M HCl at 90 C for 48 h. After cooling to room temperature the hydrolyzate was filtered through a glass microfiber filter and the flow-through evaporated at 50 C under reduced pressure. The dry hydrolyzate was dissolved in de-ionized water (Nilsson and Bjurman 1998). The concentration of glucosamine hydrochloride in the hydrolyzate was determined colorimetrically according to Chen and Johnson (1983), modified as follows. The dilute hydrolyzate solution (1 mL) was added to 0.25 mL 4% ace-tylacetone solution (4% v/v acetylacetone in 1.25 M sodium carbonate) and heated 1 h at 90 C in a test tube with a tape-lined screw cap. After cooling in a water bath to room temperature 2 mL of ethanol were added under shaking to dissolve the precipitate. About 0.25 mL of Ehrlich reagent (1.6 g of N-N-dimethyl-p-aminobenzaldehyde in 60 mL of a 1:1 mixture of ethanol and concentrated HCl) was added and the absorbance was measured at 530 nm. Chitin content, expressed as micrograms of glucosamine hydrochloride per mg dry weight of fungal cell wall was calculated from a standard curve of glucosamine hydrochloride at five concentrations (5, 10, 15, 20 and 30 µg/mL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Colonies were reddish when grown in media containing sucrose (FIG. 2A) and greenish when cultivated on inulin (FIG. 2B). Spores were observed in both media.
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), 12 and 18 d old (data not shown) fungal cultures revealed that hyphae from fungus grown on inulin-containing medium were much thinner than those cultivated on sucrose; they appeared better defined, swollen and turgid.
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) for hyphae grown on inulin or sucrose. The ultrastructure of the hyphal cells showed a normally structured cytoplasm with Woronin bodies similarly distributed regardless of which carbon source was used in the medium as previously reported (Pessoni et al 2002).
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). Chitin content varied from 2.1 to 4.6% of the cell-wall dry weight, being higher in the fungus grown on inulin-containing medium. An increase in the relative proportion on a dry-weight basis of chitin with the age of the culture was observed in the cell walls of the fungus grown in this medium (TABLE I).
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). Glycosyl-linkage composition analysis also indicated the absence of 2,3-linked-glucosyl residues in the walls isolated from cultures grown on sucrose.
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). Gel permeation elution profiles of these fractions on a Sepharose CL 6B column were quite similar, regardless of the carbon source on which the fungus was grown. The molecular masses of the eluting polysaccharides range was 70.0–9.3 kDa (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It already is known that changes in the carbon sources or micro-nutrient composition of the culture medium can affect growth, sporulation, colony morphology and polysaccharide storage in microorganisms, as shown for Streptomyces brasiliensis (Rueda et al 2001) and Neurospora crassa (Bhanoori and Venkateswerlu 2000). In Penicillium janczewskii, no differences in growth rates and sporulation were observed when the fungus was grown on medium containing either sucrose or inulin (FIG. 1
). However, colony morphology varied depending on the carbon source (FIG. 2). Hyphae from P. janczewskii grown on inulin-containing medium were more delicate than those cultivated on sucrose and were more easily damaged, as reported by Pessoni et al (2002). These differences seem to be related to the thickness of the hyphal wall, which is thinner in the mycelia grown on inulin compared to those grown on sucrose (FIGS. 3–4).
Cell walls determine the shape of fungal cells and are essential for their integrity. Fungal walls consist mainly of carbohydrates in the form of polysaccharides, some of which are linked to proteins (Santos et al 2000). The contents of carbohydrates and proteins of the cell walls of P. janczewskii do not differ significantly in relation to the carbon source added to the culture media (FIG. 5). Cell-wall sugar composition of P. janczewskii indicates that glucose, ga-lactose and mannose are the main monosaccharide constituents (TABLE I). These compositional data are in general agreement with those reported by Leal et al (1997) for 44 species of Penicillium and are independent of the carbon source supplied to the culture. Variations in the proportion of sugars of P. janczewskii were observed only with the age of the culture, which together with other factors, such as nutrients and temperature, are known to cause marked differences in fungal cell-wall composition (Ruiz-Herrera 1992).
Chitin and its linkages with glucans are considered essential for making the fungal wall rigid and for decreasing the solubility of cell-wall polysaccharides in alkali (Fontaine et al 1997). Variations in the relative amounts of chitin in the cell walls have been described for Neolentinus lepideus and Phialophora sp., depending on the nutrient content of the medium and the growth temperature (Nilsson and Bjurman 1998). The chitin content of P. janczewskii hyphae was similar to those reported for other fungi (Boyle and Kropp 1992). The higher chitin content observed in the thinner cell wall of P. janczewskii grown on inulin (TABLE I) was not accompanied by differences in the solubility of the cell-wall polysaccharides because the yield of alkali-extractable polysaccharides was similar for walls prepared from fungal cultures grown on the two carbon sources (TABLE III). Furthermore the higher proportion of chitin in the walls of the inulin-grown fungus did not contribute to a strengthening of the cell walls in P. janczewskii grown on this medium. These results indicate that, at least in P. janczewskii, changes in the chitin content of mycelial walls led to changes in wall properties that are different from those predicted by Fontaine et al (1997).
Alpha and beta (1–3)-glucans or the beta-glucan-chitin complex are the main constituent of fungal cell walls and have been used as chemotaxonomic markers for Penicillium (Leal et al 1997). The alkali-extractable glucans from the cell wall of P. janczewskii represented about 20% of the mycelial dry weight. This alkali-extractable glucan content is much higher than values reported for Penicillium vermoesenii (Ahrazem et al 1999) and Paecilomyces (Domenech et al 1999), which varied from 8.2% to 10.7% and 3% to 6% of the dry weight of the cell wall, respectively. The value found in P. janczewskii was close to the alkali-extractable glucan content of the walls of Gliocladium viride at 25% (Gómez-Miranda et al 1990).
Remarkable differences were observed in the proportion of 3-linked glucosyl residues in cell walls of P. janczewskii grown on the two carbon sources. In the fungus grown on inulin, 3-linked glucans are the main components (TABLE II), whereas in the sucrose medium, a 3-linked galactosyl-containing polymer also was detected as suggested by the equal proportions of both 3-linked glucosyl and 3-linked galactosyl residues. Our results are the first report of the presence of a 3-linked galactosyl polymer in Penicillium species. Instead the main component, in addition to glucans and chitin, of the walls of some Penicillium and Eupenicillium species has been described as a linear beta (1–5)-galactofuranan (Leal et al 1997). In P. allahabadense and P. zacinthae the same authors also reported the presence of a rare beta (1–2)(1–3)-gal-acturonan. Glycosyl linkage analysis of P. janczewskii indicate the presence of 3-, 4- and 2,3-linked galactopyranosyl residues, suggesting that the poly-saccharides produced by this fungus differ from those described so far for other species of Penicillium.
Alterations in the abundance and composition of neutral and amino sugars can affect the organization of the cell wall and in turn have an influence on fungal morphogenesis (Ghfir et al 1997). In the present work we observed differences between the walls of P. janczewskii cultivated in medium containing inulin or sucrose as sole carbon sources. The observed differences are primarily in the chitin content and the structure of at least some of the other polysaccharides present in the walls. The mycelial walls of the fungus grown on inulin contain a higher proportion of chitin, and relatively more 3-linked glucan and less 3-linked galactan when compared with the walls of the sucrose-grown fungus. Therefore, the difference in the thickness of the cell walls of P. janczewskii grown on sucrose or inulin observed by SEM and TEM (FIGS. 3, 4) and the associated differences in wall strength could be related to the molecular composition and the degree of branching of the polysaccharides synthesized by the fungus when grown on the two different carbon sources.
More detailed characterization of the cell-wall polysaccharides from P. janczewskii grown on the different carbon sources will be necessary to confirm the presence of unexpected polymers (e.g., galactans) in the walls and to better understand how the observed structural differences in cell-wall polysaccharides lead to the observed differences in wall properties. Furthermore, it will be of great interest to understand how the different carbon sources lead to the observed differences in wall composition and whether these differences are related to the high secretion of inulinases observed only when the fungus is grown in inulin-containing medium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author: Instituto de Botânica, Seção de Fisiologia e Bioquímica de Plantas, CP 4005, São Paulo, SP 01061-970, Brazil. E-mail: bragamr{at}canalvip.com.br
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) or inulin (•). Mean ± Standard Deviation (n = 6).
