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Nutrient limitation promotes pigment production in Aureobasidium pullulans.


Aureobasidium pullulans ATCC 42023 is an environmental fungus found in soil and on plant leaves that produces both melanin-like pigments and exopolysaccharides such as pullulan. Industrial processes remove the melanin before pullulan can be used in consumer products such as breath freshner strips. The melanin-like pigments are also known to enhance fungal virulence. Thus, control of pigmentation and pullulan production is important in industrial and medical applications. The goals of this project were to determine if nutrient limitation or ultraviolet (UV) light exposure impacted pigmentation production. Results indicate that nutrient deficiencies of salt and potassium phosphate may contribute to pigment production. Exposure to UV-light for up to eight minutes does not appear to play a significant role in reducing pigmentation or polysaccharide production.

Keywords: Aureobasidium, melanin, pigment, ultraviolet

1. Introduction

The polymorphic fungus Aureobasidium pullulans produces pullulan and the black pigment melanin (Cooke 1961). The fungus is commonly found in the environment on apple tree leaves, in the soil, and in ventilation ducts. Pigment and polysaccharide synthesis occurs in fungal cell walls (Campbell et al. 2004; Shingel 2004). Pigmentation is seen in organisms frequently exposed to the sun and is thought to protect the cells from UV-rays (Jacobson 2000). Melanin normally is bleached or removed from pullulan for commercial applications (Gibson and Coughlin 2002) which is expensive (Tarabasz-Szymanska and Galas 1993). Effects of UV-rays on Aureobasidium have been investigated by Vasilevskaia et al. (2003) and experiments by Tarabasz-Szymanska and Galas (1993) utilized a 15 minute UV-exposure to reduce the population count to 10% of the original inoculum. Studies to determine polysaccharide and pigment synthesis regulation have been performed, and attempts to mutate colonies to reduce melanin-like pigment production and increase pullulan yields have met with moderate success (Pollock et al. 1992).

The fungus is also implicated in human diseases such as peritonitis and nosocomial infections (Caporale et al. 1996; Bolignano and Criseo 2003). Invasive fungi often produce pigmentation, such as melanin, and are referred to as dematiaceous (Jacobson 2000). Aureobasidium pullulans var. melanigenum is considered among this group of dematiaceous fungi (Bolignano and Criseo 2003). The polymorphism of A. pullulans complicates studies because observations of pigmentation or polysaccharide production may not be consistent due to variations in ingredients or manufacturers of media. Morphological stages responsible for pigmentation and pullulan elaboration have been widely studied with conflicting results (Badr-Eldin et al. 1994; Buliga and Brant 1987; Campbell et al. 2004; Catley 1971; Seviour et al. 1992).

The purpose of this study was to determine if nutrient limitation promotes pigmentation by A. pullulans, as well as to determine the response of fungal pigmentation to varying times of UV-light exposure.

2. Materials and Methods

Aureobasidium pullulans ATCC 42023 was purchased from the American Type Culture Collection and grown on modified Ueda medium. Traditional Ueda medium contains 0.5% [K.sub.2]HP[O.sub.4], 0.1% NaCl, 0.02% MgS[O.sub.4]x7[H.sub.2]O, 0.06% [(N[H.sub.4]).sub.2]S[O.sub.4] and 10% (weight/volume) sucrose per liter (Ueda et al. 1963). The modified recipe halves each ingredient except for sucrose, which remains the same. To determine specific nutrient deficiencies which affect melanin synthesis, four 150 ml broth cultures, each lacking one ingredient of the modified Ueda media, were prepared in duplicate. Traditional Ueda broth served as a control for melanin synthesis, and modified Ueda broth served as a control containing all nutrients. Cultures were grown in a shaker incubator for 9 days at 28[degrees]C at 175 rpm. Samples (1 mL) were aseptically removed each day and placed in sterile 1.5 mL microcentrifuge tubes and centrifuged to pellet the cells. The supernatant was removed and ethanol was added to the pellet. Slides were prepared to determine microscopic melanin-like pigment production.

Traditional Ueda and modified Ueda plates were inoculated with a lawn of growth (200 [micro]l of a log-phase broth culture) and immediately subjected to ultraviolet radiation (LabConco, 30W) approximately 45 cm above the agar surface ranging from 0 seconds up to eight minutes. Cultures were allowed to grow at 28[degrees]C for up to seven days. Photographs were taken on day four with a Nikon 990E digital camera, and no changes were seen after day four.

3. Results

Traditional Ueda broth cultures remained white in color throughout the experiment, whereas modified Ueda and the ingredient-deficient broth cultures (except the magnesium sulfate deficient cultures) eventually produced melanin. Cultures grown on modified Ueda began producing melanin at day two, whereas the salt and [K.sub.2]HP[O.sub.4] deficient broth cultures turned color on days three to four. The ammonium sulfate deficient culture was the slowest, beginning pigment synthesis on day five, and the magnesium sulfate deficient broth did not show any culture growth.

Results of the UV-exposure showed that traditional Ueda colonies will remain white, even when subjected to long periods of UV-light (Figure 1). Modified Ueda colonies, subjected to the same UV-light exposure times, produced pigmentation from the time growth was observed at forty-eight hours (Figure 2). Colonies on traditional Ueda only began to show slight pigmentation at day seven. Both figures also show a decrease of colonies by plate G in comparison with the time zero control plate. Based upon UV experiments by Tarabasz-Szymanska and Galas (1993), approximately half of the original inoculum count should be present by plate G. A polysaccharide sheen was also seen on the traditional Ueda plates, although it was not as prominent as the sheen observed on the pigmented colonies on modified Ueda (Figure 2). As UV-light exposure increased, it appeared that the polysaccharide sheen was more prominent than the time zero control plates. Since the organisms were grown on agar plates, and pullulan precipitation requires an ethanol precipitation step, the polysaccharide was not quantified.


4. Discussion and Conclusions

Aureobasidium pullulans exhibits pigmentation on varying (two to five) days after inoculation, depending upon the ingredients in the media. Since modified Ueda almost always induces pigmentation and traditional Ueda only does so with an aged culture, it appears that nutrient limitation promotes the protective response of a melanin-like pigment. Pigmentation production also appears to be linked to nutrient limitations of [K.sub.2]HP[O.sub.4] and salt, with ammonium sulfate a secondary limitation. This supports similar observations by Calvo et al. (2002) that Aspergillus flavus inhibits sclerotia development when grown on nitrate or ammonium medium. Since chlamydospores are often observed with a pigmented cell wall in A. pullulans, perhaps ammonium is required for chlamydospore formation, particularly after other ingredients are depleted.

The results indicate that salt and [K.sub.2]HP[O.sub.4] are required in the production of melanin-like pigments and reduction in these compounds slows pigmentation production. Further, no growth observed on magnesium sulfate deficient medium precludes that magnesium sulfate is required for metabolic pathways and sustainability of the fungus. Molecularly, a G protein subunit has been shown to be responsible for melanin regulation in Cryptococcus neoformans and a mitogen activated protein (MAP) kinase stimulates filamentous growth in Saccharomyces cerevisiae when nutrients are depleted (Calvo et al. 2002). Molecular experiments would enhance the understanding of regulatory mechanisms in A. pullulans.

Exposure to UV-radiation does not appear to reduce visual pigmentation levels microscopically. Colonies on both media types showed polysaccharide production, which may be pullulan or a [beta]-glycan. Further analysis of the polysaccharide is needed. Traditional Ueda white colonies did not show decreased polysaccharide production from the controls on this media, nor were any pigmented mutants seen. Further, no mutated white colonies were seen after UV treatment on the modified Ueda medium. Without melanin protection, it is possible that the polysaccharide may prevent UV-light penetration and be protecting the cells. Future experiments are needed to determine if the polysaccharide is protecting the cells and to ensure that pullulan yields are not compromised if melanogenesis is inhibited.

5. Acknowledgements

Thanks to William Kirby for laboratory support of this project.

6. Literature Cited

Badr-Eldin, S.M., O.M. El-Tayeb, H.G. El-Masry, F.H.A. Mohamad, and O.A. Abd El-Rahman. 1994. Polysaccharide production by Aureobasidium pullulans: factors affecting polysaccharide production. World J. Microbiol. Biotechnol. 10:423-426.

Bolignano G. and G. Criseo. 2003. Disseminated nosocomial fungal infection by Aureobasidium pullulans var. melanigenum: a case report. J. Clin. Microbiol. 41:448.-4485.

Buliga, G. and D. Brant. 1987. Temperature and molecular weight dependence of the unperturbed dimensions of aqueous pullulan. Int. J. Biol. Macromol. 9:71-76.

Campbell, B.S., A.M. Siddique, B.M. McDougall, and R.J. Seviour. 2004. Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production? FEMS Microbiol. Lett. 232:225-228.

Catley, B.J. 1971. Utilization of carbon sources by Pullularia pullulans for the elaboration of extracellular polysaccharides. Appl. Microbiol. 22:641-649.

Calvo, A.M., R.A. Wilson, J.W. Bok, and N.P. Keller. 2002. Relationship between secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66:447-459.

Caporale, N.E., L. Calegari, D. Perex, and E. Gezuele. 1996. Peritoneal catheter colonization and peritonitis with Aureobasidium pullulans. Peritoneal. Dial. Int. 16:97-98.

Cooke, W.B. 1961. A taxonomic study in the "black yeasts". Mycopathologia et Mycologia Applicata. 17:1-43.

Gibson, L.H. and R.W. Coughlin. 2002. Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations. Biotechnol. Prog. 18:675-678.

Jacobson, E.S. 2000. Pathogenic roles for fungal melanins. Clin. Microbiol. Rev. 13:708-717.

Pollock, T., L. Thorne, and R. Armentrout. 1992. Isolation of new Aureobasidium strains that produce high-molecular-weight pullulan with reduced pigmentation. Appl. Environ. Microbiol. 58: 877-883.

Seviour, R.J., S.J. Statinopoulos, D.P. Auer, and P.A. Gibbs. 1992. Production of pullulan and other exopolysaccharides by filamentous fungi. Crit. Rev. Biotechnol. 12: 279-298.

Shingel, K.I. 2004. Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan. Carb. Res. 339:447-460.

Tarabasz-Szymanska, L. and E. Galas. 1993. Two-step mutagenesis of Pullularia pullulans leading to clones producing pure pullulan with high yield. Enz. Microb. Technol. 15:317-320.

Ueda, S., K. Fujita, K. Komatsu, and Z. Nakashima. 1963. Polysaccharide produced by the genus Pullularia. I. Production of polysaccharide by growing cells. Appl. Microbiol. 11:211-215.

Vasilevskaia, A.I., N.N. Zhdanova, A.T. Shkol'nyi, and L.V.Artyshkova. 2003. Survivability of some black yeast-like fungal species under V-irradiation. Mikrobiol. Z. 65:60-69

Anna R. Oller Department of Biology Central Missouri State University, WCM 306, Warrensburg, MO 64093
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Author:Oller, Anna R.
Publication:Transactions of the Missouri Academy of Science
Date:Jan 1, 2004
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