Cell Surface Topography of Candida and Leucosporidium Yeasts as ...

Vol. 130, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 312-317 Copyright © 1977 American Society for Microbiology

Cell Surface Topography of Candida and Leucosporidium Yeasts as Revealed by Scanning Electron Microscopy KENNETH WATSON* AND HELEN ARTHUR

Department of Chemistry and Biochemistry, James Cook University, Townsville 4811, Australia Received for publication 22 December 1976

The cell surface topography of the following yeast strains was examined by scanning electron microscopy: Candida slooffii, C. lipolytica, Leucosporidium frigidum, and L. nivalis. Multipolar and lateral budding were observed in the Candida yeasts in contrast to bipolar budding in the Leucosporidium species. The cell surface topography and the morphology of the bud and birth scars in these yeasts differed markedly. Apart from the bud and birth scars, the cells of C. slooffii showed a relatively smooth topography. The bud scars were seen as a circular ridge of wall material surrounding a markedly convex scar plug. Birth scars were raised, rounded structures, which appeared to distend upon cell growth. In contrast, bud scars of C. lipolytica were platelike, lacked a distinct annulus of wall material, and were much less protuberent than those of C. slooffii. Birth scars were a more permanent feature of these cells. The topography of Leucosporidium yeasts was characterized by the presence of numerous protrusions on the cell surface. In some cases, the entire cell surface was covered by these protrusions. There appeared to be some correlation between the age of the cell and the extent of surface protrusions and degree of surface convolution. The bud scars in these yeasts were seen as a circular, broken collar of material surrounding a slightly convex scar plug. Birth scars were not unlike those seen in C. lipolytica. If the varied appearances of birth and bud scars are considered together with scanning electron micrographs showing bud-parent junctions before bud release, three different modes of budding are suggested. scar in S. cerevisiae (18). This result was not unexpected, since the mechanisms of budding in R. glutinis and S. cerevisiae, as noted by transmission electron microscopy, are reported to differ (13). We describe here a scanning electron microscopy study of the cell surface topography, with particular respect to the morphology of bud and birth scars, of Candida and Leucosporidium yeasts. The species examined included: C. slooffii, a thermophilic yeast; C. lipolytica, a mesophile; and two Leucosporidium yeasts, L. frigidum and L. nivalis, both obligate psychrophiles (2). Our results show that, depending on the species, three morphologically distinct types of bud scars are observed in these yeasts.

There have been numerous investigations of budding in yeast cells, particularly the genus Saccharomyces. These include studies using light (4), fluorescence (16, 17) and electron (1, 13) microscopy. In Saccharomyces, an ascomycete, the cell walls of the parent and the bud are continuous at all stages of budding as seen by transmission electron microscopy (9, 13). On the other hand, bud formation in basidiomycetous yeasts is reported to be different in that the new bud wall is continuous only with the inner layers of the parent cell wall. Basidiomycetous yeasts that have been reported to bud in this manner include Rhodotorula glutinis (9, 11, 13), Sporobolomyces (11, 14), and Leucosporidium scottii (11). The morphology of budding in yeast cells has also recently been examined by scanning electron microscopy. Such studies have indicated that the morphology of bud scars in Saccharomyces yeasts (5, 18) is analogous to those described for Candida albicans (3) and Metschnikowia krissii (18). It was noteworthy, however, that the bud scar in R. glutinis was platelike in contrast to the protuberant character of the bud

MATERIALS AND METHODS Yeast cultures. The following organisms were examined: L. frigidum Fell et al. CBS 5270, formerly C. frigida (7, 8); L. nivalis Fell et al., strain 3AH17 received from di Menna (7), formerly C. nivalis (7, 8); C. lipolytica (Harrison) Diddens and Lodder, NCYC 153; C. slooffii van Uden et al., CBS 4068 and CBS 2419. Maintenance of yeast cultures. Stock cultures were maintained on agar slopes containing (per liter 312

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of distilled water): glucose, 20 g; yeast extract, 10 g; peptone, 5 g; and agar, 15 g; pH 6.1. The psychrophilic yeasts L. frigidum and L. nivalis were maintained at 4°C. The mesophilic yeast C. lipolytica was incubated for 2 to 3 days at 25°C before transferral to 4°C. The respiratory-deficient thermophile C. slooffli was incubated overnight at 34°C and then maintained at 25°C. Growth conditions. All organisms were grown in 250-ml conical flasks containing 100 ml of medium consisting of 1% yeast extract, 0.5% peptone, and mineral salts (20), with 2% (wt/vol) glucose as carbon source. In the case of C. slooffii, the basal medium was supplemented with required vitamins (20). Cultures were inoculated from a starter culture grown to the end of the exponential growth phase. Flasks were placed on orbital shakers (180 rpm), and cells were grown at -0.5°C (L. frigidum and L. nivalis), 28°C (C. lipolytica), and 37°C (C. slooffii). Cells were harvested at the end of the exponential growth phase and then processed for examination by scanning electron microscopy. Scanning electron microscopy. Cells were fixed for 1 h in 3% glutaraldehyde buffered with 0.05 M Veronal-acetate (pH 7.2), dehydrated in a graded series of acetone solutions, and then dried by the critical-point method. Dried cells were coated with gold-palladium and examined in an Hitachi HHS-2R scanning electron microscope at an accelerating voltage of 10 kV.

RESULTS Candida. Scanning electron micrographs of

C. slooffii cells (Fig. 1) show the presence of numerous budding cells with prominent birth and bud scars. These can be readily distinguished on the basis of their different surface topographies. A bud scar is seen as a distinct annulus of cell material surrounding a markedly convex scar plug (Fig. 1 through 3). By comparison, the birth scar is relatively smooth with a raised, rounded topography. Once formed, the bud scar appears to be a permanent feature of the cell surface topography, again in contrast to the birth scar, which appears to diffuse during cell growth and enlargement (Fig. 2 and 3). It is noteworthy that budding and, hence, bud scars frequently occur on areas previously occupied by a birth scar. On the other hand, bud scars themselves do not overlap. These points are well illustrated in micrographs showing bud scars (three to four in some cases) formed on birth scars that have been distended by growth (Fig. 2 through 4). Multipolar budding is clearly evident in these cells, but lateral budding in one strain of C. slooffii (CBS 4068) is rare. C. slooffli yeasts are capable of lateral budding as illustrated in Fig. 4, which shows evidence of frequent lateral budding in a different strain of C. slooffii (CBS 2419). Features of the birth and bud scars are characteristic of the genus. The overall cell sur-

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FIG. 1. Scanning electron micrograph of intact cells of C. slooffii CSB 4068. The overall cell surface topography is smooth. Many cells show bud andlor birth scars. (b) Bud scar; (s) birth scar. Note smooth bud-parentjunction (arrow). Bar represents 1 ,um.

face topography of C. slooffii cells, apart from the birth and bud scars, is relatively smooth. The birth scar, bud scar, and cell surface topography of C. lipolytica are quite distinct from those observed in cells of C. slooffii. Although both yeasts characteristically exhibit multipolar and lateral budding, C. lipolytica shows the presence of a few protrusions both on the cell surface (Fig. 5) and on the bud scars (Fig. 6). The morphology of the bud scars of this yeast also differs from that of C. slooffii bud scars in that the scars have a less-defined annulus of wall material and the scar plugs show considerably less protuberance. The birth scars also contrast. They appear to be less extendible and do not appear to diffuse to the same extent on cell aging as those in C. slooffii. The birth scars are thus a more permanent feature of the cell surface topography in C. lipolytica. In this yeast, bud scars are never observed to form on a site previously occupied by a birth scar, again

in contrast to C. slooffli. Leucosporidium. Multipolar budding and lateral budding are not observed in L . frigidum and L. nivalis yeasts. Budding in these yeasts occurs almost exclusively by a bipolar mechanism. The presence of adjacent bud scars or two

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FIG. 2. C. slooffli CBS 4068 cells illustrating multipolar budding and prominent bud scars (b). The distinct annulus (a) of cell wall material forms a circular ridge around the markedly convex scar plug (p). The bud scars have been formed on a site previously occupied by a birth scar (s). Bar represents 1 ,um.

buds at the same pole is an extremely rare event. On the other hand, cells with multiple collars of wall material, indicative of budding at the same pole, are frequently observed (Fig. 7). A characteristic collar of material is always present at the junction of parent and bud cell. The bud scars in L. frigidum and L. nivalis are substantially different from those observed in the other yeasts described in this report. The bud scar is seen as a circular, broken collar of material surrounding a slightly convex scar plug (Fig. 8). The birth scars in L. frigidum cells (Fig. 7 and 8) are not unlike those of C. lipolytica (Fig. 5 and 6) and are raised, slightly rounded structures. Birth scars in L. nivalis are sometimes difficult to distinguish. The bipolar budding and the elongated nature of the cells contribute to this difficulty. Scanning electron microscopy also illustrates differences in the surface topographies of bud and parent. The latter is rough, and in many of the cells the surface is exten-

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FIG. 3. C. slooffii CBS 4068 cells illustrating bud scars. Bud scars are frequently observed on sites previously occupied by a birth scar that has been distended by cell growth. Bar represents 1 gm.

(b) and birth (s)

sively covered with protrusions. (Fig. 7 and 8). By contrast, the surface topography of the bud is characterized by a paucity of these protrusions. A comparison of the topography of the parent with that of young and mature buds indicates that the extent of surface protrusions is a factor of the age of the cell. This phenomenon is particularly well illustrated in the surface topography of budding cells (Fig. 7). The surface topography of very young buds is invariably smooth; as the bud approaches maturity, there is a development of surface protrusions and convolutions. One never observes buds that have moreextensive surface protrusions than those of the attached parent. On the other hand, in some cases both parent and bud cell may be sparsely covered with surface protrusions (Fig. 7). Young and mature buds of C. lipolytica also follow a pattern similar to that observed for cells of L. frigidum. Young buds are smoother and have fewer protrusions than mature buds and parent cells (Fig. 5). It was difficult to assess whether a similar phenomenon occurred

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in cells of C. slooffii, since the parent cells themselves are relatively smooth.

DISCUSSION In the present studies, the application of scanning electron microscopy has given new insights into the cell surface topography of budding in yeasts belonging to the genera Candida and Leucosporidium. Multipolar budding and lateral budding were clearly identified in the ascomycete yeasts C. slooffii and C. lipolytica, and only bipolar budding was identified in the basidiomycete yeasts L. frigidum and L. nivalis. The bipolar budding and the multiple scars seen on the cells of Leucosporidium yeasts (Fig. 7 and 8) correspond to the situation, as studied by fluorescence microscopy, of bud formation in apiculate yeasts (19). FIG. 4. C. slooffii CBS 2419 showing lateral budding (lb). The bud (b) and birth scars (s) are characteristic of C. slooffli. Note the bud scar formed on a birth scar (arrow). Bar indicates 1 ,um.

FIG. 5. C. lipolytica illustrating multipolar budding (mb) and cell surface topography. Young buds (y) have a smoother morphology than mature buds (mi). Note elongated bud-parent junction (arrow). (M) Parent cell; (b) bud scar. Bar represents 1 um. FIG. 6. C. lipolytica illustrating lateral budding (lb). Note protrusions on bud scars (b). (s) Birth scar. Bar indicates 1 ,um.

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FIG. 7. Scanning electron micrograph illustrating cell surface topography of L. frigidum cells. Note the protrusions on the cell surfaces and the characteristic collar of material at the junction of parent and bud cells (arrow). Multiple collars (cs), indicative of budding at the same pole, are seen on some cells. Young buds (y) have a smooth morphology as compared to mature buds (m) and parent cells (M). Bar represents 1 pm.

FIG. 8. L. frigidum cells illustrating typical bud (b) and birth scars (s). Note the raised, broken collar of wall material of the bud scar (arrow). Bar represents 1 pm.

The yeasts could be further differentiated on the basis of their bud scars. Bud scars in C. slooffii were essentially identical to those reported in recent scanning electron micrograph studies on S. cerevisiae (5, 18), M. krissii (18), and C. albicans (3). On the other hand, the much flatter bud scars of C. lipolytica resemble those seen in a micrograph published by Talens et al. (18) of bud scars in R. glutinis (Fig. 18 of reference 18). In the case of L. frigidum and L. nivalis, the morphology of the bud scars is unique and has not been reported previously. However, it is consistent with the concept, developed from transmission electron microscopy studies (9, 11), that in these yeasts only the inner wall layers of parent and bud are continuous. The outer layers of parental cell wall rupture and peel back on bud release. By contrast, the morphology of bud scars in R. glutinis, as seen by scanning electron microscopy (18), is not consistent with such a mechanism. Talens et al. (18) were unable to detect any indication of the ruptured outer parental cell wall layers at any

poridium species, although both basidiomycetous yeasts, differs since the morphology of their bud scars differs. If the varied appearances of bud scars are considered together with scanning electron micrographs showing bud-parent junctions before bud release, three different modes of budding are suggested by the present studies. Bud-parent junctions in C. slooffii were short and smooth (Fig. 1), whereas in C. lipolytica the junctions were distinctly elongated (Fig. 5). In L. frigidum a characteristic collar of material was always observed in bud-parent junctions (Fig. 7). Examination of other species of Candida has also shown differences in bud scar

stage of budding. This observation is puzzling in view of the fact that budding in R. glutinis, as studied by transmission electron microscopy (9, 12), resembles that of Leucosporidium species (9, 11). The possibility is raised, therefore, that budding in Rhodosporidium and Leucos-

morphology (H. Arthur and K. Watson, unpublished data). For example, bud scars in C. freyschussii and C. blankii are morphologically similar to those observed in C. slooffli and C. lipolytica, respectively. It was difficult to differentiate the Candida and Leucosporidium yeasts on the basis of their birth scars, which were a less prominent and, in the case of C. slooffii cells, a less permanent

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feature of cell surface topography. Comparison with previous studies is not possible since there have been few studies on the morphology of birth scars in yeasts as examined by scanning electron microscopy (however, see references 5 and 18). One thing is clear: that the birth scar is not concave as indicated by the early work of Bradley (6) and Agar and Douglas (1). Scanning electron microscopy revealed further differences in the cell surface morphologies of the Candida and Leucosporidium yeasts. In Candida, particularly C. slooffii, cell surfaces were relatively smooth, but Leucosporidium species were characterized by the presence of numerous protrusions on the cell surface (Fig. 7 and 8). The exact nature and function of these protrusions are unknown. They appear to be dependent on the age of the cell, being less prominent or even absent in very young buds. The protrusions may represent a mucilaginous layer surrounding the cell wall. The yeasts examined may be classified into different thermal domains based on their temperature limits of growth. C. slooffii is an obligate thermophile, the Leucosporidium yeasts are obligate psychrophiles, and C. lipolytica is a typical mesophilic yeast (2). All three yeasts differed markedly in their cell surface topography and in the morphology of their bud and birth scars. However, at this stage, it would be premature to anticipate any relationship between cell surface topography and temperature adaptation in yeasts. ACKNOWLEDGMENTS This work was supported by a grant from the Australian Research Grants Committee. K.W. thanks the Australian National University for a short-term Visiting Fellowship in the Research School of Biological Sciences. H.A. acknowledges the receipt of an Australian Government Research

Scholarship. The technical assistance of K. Hopkinson is gratefully acknowledged. LITERATURE CITED 1. Agar, H. D., and H. C. Douglas. 1955. Studies of budding and cell wall structure of yeast. J. Bacteriol. 70:427-434. 2. Arthur, H., and K. Watson. 1976. Thermal adaptation in yeast: growth temperature, membrane lipid, and


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cytochrome composition of psychrophilic, mesophilic, and thermophilic yeasts. J. Bacteriol. 128:56-68. Barnes, W. B., A. Flesher, A. E. Berger, and J. D. Arnold. 1971. Scanning electron microscopic studies of Candida albicans. J. Bacteriol. 106:276-280. Barton, A. A. 1950. Some aspects of cell division in Saccharomyces cerevisiae. J. Gen. Microbiol. 4:84-86. Belin, J. M. 1972. A study of the budding of Saccharomyces uvarum Beijerinck with the scanning electron microscope. Antonie van Leeuwenhoek J. Microbiol. Serol. 38:341-349. Bradley, D. E. 1956. A carbon replica technique for microbiological speciments applied to the study of the division of Saccharomyces cerevisiae with the electron microscope. J. R. Microsc. Soc. 75:254-261. di Menna, M. E. 1966. Three new yeasts from Antarctic soils: Candida nivalis, Candida gelida and Candida frigida. Antonie van Leeuwenhoek J. Microbiol. Serol. 32:25-28. Fell, J. W., A. C. Statzell, I. L. Hunter, and H. J. Phaff. 1969. Leucosporidium gen. nov., the heterobasidiomycetous stage of several yeasts of the genus Candida. Antonie van Leeuwenhoek J. Microbiol Serol. 35:433-462. Kreger-van Rij, N. J., and M. Veenhuis. 1971. A comparative study of the cell wall structure of basidiomycetous and related yeasts. J. Gen. Microbiol. 68:8795. McClary, D. O., and W. D. Bowers. 1965. The integrity of the cell wall during bud formation in yeasts. Can. J. Microbiol. 11:447-452. McCulley, E. K., and C. E. Bracker. 1972. Apical vesicles in growing bud cells of heterobasidiomycetous yeasts. J. Bacteriol. 109:922-926. Marchant, R., and D. G. Smith. 1967. Wall structure and bud formation in Rhodotorula glutinis. Arch. Mikrobiol. 58:248-256. Marchant, R., and D. G. Smith. 1968. Bud formation in Saccharomyces cerevisiae and a comparison with the mechanism of cell division in other yeasts. J. Gen. Microbiol. 53:163-169. Prusso, D. C., and K. Wells. 1967. Sporobolomyces roseus. 1. Ultrastructure. Mycologia 59:337-347. Sinclair, N. A., and J. L. Stokes. 1965. Obligate psychrophilic yeasts from the polar regions. Can. J. Microbiol. 11:259-269. Streiblova, E., and K. Beran. 1963. Demonstration of yeast scars by fluorescence microscopy. Exp. Cell Res. 30:603-605. Streiblova, E., K. Beran, and V. Pokorvy. 1964. Multiple scars, a new type of yeast scar in apiculate yeasts. J. Bacteriol. 88:1104-1111. Talens, L. T., M. Miranda, and M. W. Miller. 1973. Electron micrography of bud formation in Metshnikowia krissii. J. Bacteriol. 114:413-423. Travassos, L. R. R. G., and A. Cury. 1971. Thermophilic enteric yeasts. Annu. Rev. Microbiol. 25:49-74. Wickerham, L. J. 1946. A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J. Bacteriol. 52:293-301.