Abstract. The polyamine contents of the yeast and mycelial forms of seven dimorphic fungi were examined to determine if a relationship existed between ...
CURRENT MICROBIOLOGY, Vol. 2 (1979), pp. 187-190
Current Microbiology An International Journal
Polyamines in Dimorphic Fungi Michael Marshall,J- Geraldine Russo,'[" James Van Etten,j-* and Kenneth Nickerson:[: J-Department of Plant Pathology and :[:School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68583, USA
Abstract. The polyamine contents of the yeast and mycelial forms of seven dimorphic fungi were examined to determine if a relationship existed between polyamine composition and fungal morphology. Although the polyamine contents differed, no consistent patterns emerged. Evidently, the observed differences in polyamine composition were not related to fungal dimorphism but rather to the growth conditions used to achieve those morphological forms.
Until recently, it was generally accepted that all eukaryotic organisms, including the fungi, both contained and synthesized the three common polya m i n e s putrescine, spermidine, a n d spermine, whereas the prokaryotes were unable to synthesize spermine [2,4,15]. However, results from several laboratories have led to a reexamination of this generalization. Investigators have been unable to detect spermine in the slime mold Physan~m polycephalum [ 10], the protozoan Tetrahyrnena pyriformis [ 12,16], four trypanosomatids [I], the alga Scenedesmus [13], the water mold Blastocladiella emersonii [9], and the wheat rust fungus Puccinia graminis F. sp. tritici [8]. Subsequently, we [11] examined the polyamine content in several fungi. We were unable to detect spermine in 15 species of filamentous fungi even though it was readily detected in three yeast species. In light of this apparent dichotomy in polyamine composition between yeasts and filamentous fungi, we thought it profitable to examine the polyamine content of several dimorphic fungi. These fungi can exist in either a yeast or mycelial form depending on the chemical and physical environment in which they are grown. In the present investigation, we have examined seven species of dimorphic fungi to determine if there is a relationship between polyamine composition and morphology, i.e., yeast versus mycelial. Materials and Methods Organisms and cultural conditions. The yeast and mycelial forms of Mucor bacilliformis and Histoplasrna cap.rutamm were kindly provided by Roger Storck (Rice University) and George Bogus*To whom offprint requests should be addressed.
lawski (University of Kansas), respectively. Mycotypha microspora, Ceratocystis ulmL and Ceratocystis fagacearum were obtained from the culture collection, Department of Plant Pathology and Bacteriology, West Virginia University. Yeast cells of C. ulmi were grown in liquid shake culture (25~ for 4 days) in the basal medium of Hindal and MacDonald [71, while the yeast forms of Mycotypha microspora and C. fagacearum were produced as previously described [3,14]. Fusarium moniliforme, from the culture collection, Department of Plant Pathology, University of Nebraska, was grown in the yeast form under the same culture conditions in Czapek's broth (Difco Laboratories, Detroit, Michigan). The mycelial forms of each of the above fungi were grown (25~ for 9 days) on their respective agar media (15 g agar/liter) overlayed with dialysis tubing; the mycelial mats were then peeled off the agar surface. The yeastlike and mycelial forms of Aspergillus parasiticus (NRRL 2999) were grown as described by Detroy and Ciegter [5]. Even though A. parasiticus exhibits a yeast-mycelial dimorphism, it is not a true dimorphic fungus, since its yeast phase does not multiply by budding [5]. The three true (i.e., nondimorphic) yeasts used in this study, Saccharomyces cerevisiae, Saccharomycopsis lipolytica (NRRL YB-423), and Wickeramiafluorescens (NRRL YB-4819), were also grown in both solid and liquid culture utilizing the medium cited for Mycotypha microspora [14]. Fungal dry weights were obtained by heating the cells to 80~ until they reached a constant weight. Polyamine assays. Two grams of cells (wet weight) were sus pended in 10 mt of 6% perchloric acid (PCA), broken in a Waring blender for 2 rain, homogenized in a Potter-Elvehjem hand homogenizer, and stirred overnight. Following centrifugation to remove cellular debris, the PCA extracts were dansylated and the dansylated polyamines were extracted as described previously [11]. Precoated Silica Gel G thin-layer chromatography (TLC) plates (250 ffm; Analtech, Inc., Newark, Delaware) were spotted with 20-bd portions of extract, and the dansylated polyamines were separated by two-dimensional chromatography. The plates were first developed in cyclohexane-ethyl acetate (2:3 vol/vol) and then in the same solvents (3:2 vol/vol). Five microliters of a mixture of putrescine, spermidine, and spermine standards (each at 3.33 x 10 - 4 M) were run in the margins for each dimension. The plates 0343-8651/79/0002-0187 $01.00 9 1979 Springer-Verlag New York Inc.
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Results
Fig. I. Separation of polyamine standards by two-dimensional thin-layer chromatography as described in Materials and Methods. The arrows indicate the first and second dimensions. Symbols: putrescine (P), spermidine (SD), spermine (S), ammonium (NH3), dansyl hydroxide (DN). The photograph was obtained by placing the thin-layer chromatography plate on an ultraviolet light source (Ultraviolet Products, San Gabriel, California, model C62) and photographing through an orange no. 2 filter with a Polaroid MP-4 camera containing 665 Polaroid film.
were subsequently sprayed and quantitated by fluorometric scanning [11]. Fig. 1 shows the separation of dansylated spermine, spermidine, putrescine, and ammonia achieved by the two-dimensional TLC system, which is superior to the one-dimensional system used previously [11].
Table 1 reports the polyamine content for both the yeast and mycelial forms of seven dimorphic fungi. The data in Table 1 include only the commonly recognized polyamines, spermine, spermidine, and putrescine, even though several unidentified, and potentially polyamine, spots were present in some fungal extracts. Based on our previous observation that certain filamentous fungi lacked spermine, whereas spermine was present in yeasts [11], three relationships could be postulated for dimorphic fungi: (i) neither the yeast nor mycelial form contains spermine, (ii) both forms contain spermine, and (iii) the yeast form contains spermine whereas the mycelial form does not, i.e., spermine production could be developmentally regulated. Our results (Table 1) do not indicate a consistent polyamine pattern in the dimorphic fungi since each of the three possible situations with regards to spermine was obtained. Spermine was not detected in either the yeast or mycelial forms of Mucor bacilliformis and Mycotypha microspora even though both organisms contained abundant spermidine. In contrast, both morphological states of four fungi, Ceratocystis ulmi, C. fagacearum, Fusarium moniliforme, and Histoplasma capsulatum contained spermine. Finally, spermine was detected in the yeast form ofAspergillusparasiticus but not in the mycelial form. In five of the seven fungi, the yeast forms contained higher spermidine levels than the mycelial forms. In several of the dimorphic fungi, the mycelial form was produced by growing the organism on an agar surface, whereas the yeast form was grown in
Table 1. Polyamine concentration of dimorphic fungi. Micromoles polyamine/gm dry weight Fungus
Mucor baciliformis Mycotypha microspora Ceratocystis ulmi Ceratocystisfagacearum Fusarium moniliforme Aspergillusparasiticus Histoplasma capsulatum
Form M Y M Y M Y M Y M Y M (high mn) Y (low mn) M Y
"Not detected, i.e. _< 0.01 #mol/g dry weight.
Putrescine
Spermidine
Spermine
Total
0.47 1.73 1.14 0.25 ND a ND 0.27 0.6:7 1.14 0.18 ND 0.44 0.19 0.37
8.46 9.98 0.70 1.46 2.3 1.8 1.47 10.5 2.98 0.60 0.49 3.01 0.75 0.84
ND ND ND ND 0.09 0.06 0.20 0.59 0.18 0.16 ND 0.19 0.15 1.7
8.93 11.71 1.84 1.71 2.39 1.86 1.94 11.76 4.3 0.94 0.49 3.73 1.09 2.91
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M. Marshall et al.: Polyamines in Fungi
Table 2. Polyamine concentration o f several yeast species grown on agar plates or in liquid shake culture. Micromoles p o l y a m i n e / g m dry weight Fungus
Saccharomyces cerevisiae Saccharomycopsis lipolytica Wickerhamia fluorescens
Growth Conditions
Putrescine
Spermidine
Spermine
Total
Plate Liquid Plate Liquid Plate Liquid
0.11 0.33 0.67 0.13 1.42 1.97
0.66 4.4 0.84 0.88 0.8 4.60
0.38 0.73 0.16 0.75 0.53 2.20
1.15 5.46 1.67 1.76 2.75 8.77
liquid shake culture. In order to distinguish the relative effects of the morphological change per se as opposed to the culture conditions used to achieve that morphological form, we examined the influence of plate versus shake culture on the polyamine content of three yeast species (Table 2). Saccharomyces cerevisiae and Wickeramia fluorescens only grow in the yeast form, whereas Saccharomycopsis lipolytica forms a pseudomycelium when grown on solid agar surfaces. In all three cases, the spermidine and spermine contents were higher for the yeasts grown in liquid shake culture. Significantly, the magnitude of these differences (Table 2) approximate those observed between some of the dimorphic forms (Table 1). Discussion This study is concerned with two questions: (i) Do the dimorphic fungi contain spermine? And (ii) do the polyamines have a functional role in fungal dimorphism? The question of whether spermine is present in dimorphic fungi was originally posed by our observation [i 1] that true yeasts contained and synthesized spermine whereas sperrnine was not detected in the filamentous fungi. However, the validity of this conclusion was questioned by Hart, Winther, and Stevens I6], who showed that certain filamentous fungi did indeed contain spermine, and of course, in the present study we have detected spermine in the filamentous forms of several of the dimorphic fungi. The spermine levels detected by Hart, Winther, and Stevens [6], approximately 10-30% of the dominant polyamine spermidine, were high enough that we would certainly have detected them had they been present in our original samples [11]. The combination of these two sets of data constitutes an apparent contradiction regarding the presence of spermine in filamentous fungi. However, it now appears that both groups may be correct. The answer is that the sper-
mine levels seem to be physiologically regulated, e.g., by temperature, magnesium concentration, or manganese concentration (K. Nickerson and C. L. Kramer, unpublished data). Presumably, the growth conditions chosen by Hart, Winther, and Stevens [6] accentuated spermine production while those employed by us [11] did not. Our second question concerns the functional role of the polyamines in fungal dimorphism. Some quantitative differences in polyamine composition certainly exist between the morphological forms (Table 1). However, no dominant patterns are evident, and the differences observed can be explained in a large part by the agar plate versus liquid shake culture data seen in Table 2. We conclude that the observed differences in polyamine composition are probably not related to fungal dimorphism but instead are manifestations of the growth conditions used to produce those morphological forms, e.g., manganese concentration, temperature, and solid versus liquid media.
ACKNOWLEDGMENTS We thank Bob Hill for use of a scanning fluorometer, and Roger Storck, George Boguslawski, Cletus Kurtzman, and Date Hindal for providing some of the dimorphic cells and cultures used in this study. This investigation was supported in part by Public Health Service grant AI-08057 from the National Institute of Allergy and Infectious Diseases. K. N. is a National Institute of ttealth Career Development Awardee (AI 00327-TMP). The paper is no. 5748, Journal Series, Nebraska Agricultural Experiment Station, and was conducted under Project no. 21-17.
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