JOURNAL OF BACTERIOLOGY, May 1975, p. 710-718 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 2 Printed in U.S.A.
Morphological Change in the Early Stages of the Mating Process of Rhodosporidium toruloides KEIKO ABE, IWAO KUSAKA, AND SAKUZO FUKUI* The Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan
Received for publication 27 January 1975
The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.
In 1967, a sexual generation in Rhodotorula glutinis was discovered by I. Banno, and the yeast was classified as Rhodosporidium toruloides (3). His report describes the microorganism as taking a characteristic cell morphology which is dependent on mating or ploidy: the haploid strains (type A and a strains) are ovoid in cell form, and the diploid or dicaryon strain is filamentous. Our interest has been in the morphological alteration caused by cell-to-cell interaction. In this paper, therefore, we describe the morphological events which occur in the early stages of the mating process, i.e., cell-to-cell interaction between different mating type cells of Rhodosporidium toruloides.
Banno, types A and a represent the mating type for M-919 and M-1057, respectively. Media and cultivation. YS liquid medium (pH 6.4), which contained 10 g of yeast extract (Daigoeiyo, Osaka), 10 g of sucrose, and 2.5 g of NaCl per liter, was employed for shaking culture. YS agar medium was used for stock culture and microculture. Minimal agar medium (MM), which contained 30 g of sucrose, 3 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.1 g of NaCl, and 20 g of agar per liter, with pH adjusted to 6.5 with 1 N NaOH, was used as the selection medium for mated cells. For respective selection of M-919 and M-1057 from mixed cultures of these strains, methionine-pantothenate MM, containing 0.1 g of L-methionine and 0.4 mg of calcium pantothenate per liter of MM, and p-aminobenzoate MM, containing 0.2 mg of p-aminobenzoic acid per liter of MM, were used. The preculture was carried out in a test tube (16 by 180 mm) containing 8.0 ml of MATERIALS AND METHODS YS liquid medium with reciprocal shaking (320 Microorganisms. Rhodosporidium toruloides IFO strokes per min, 2-cm width) at 27 C by inoculation 0559-M-919 (haploid, mating type A, Met-, Pan-, from stock culture. Determination of mating frequency. (i) Liquid ovoid cell form, orange-colored colony) and Rhodosporidium toruloides IFO 0880-M-1057 (haploid, mat- culture method. Both strains M-919 and M-1057 ing type a, PABA- long ovoid cell form, yellow-col- were separately cultured in YS liquid medium in the ored colony), which were kindly provided by I. Banno, test tube which was described above. A small amount Institute for Fermentation, Osaka, Japan, were desig- of the culture of M-919 at a middle log growth phase nated as M-919 and M-1057, respectively, and used (107 cells per ml) was mixed with the culture of throughout this paper. Mated cells which were de- M-1057 (107 cells per ml) to give a cell number ratio of rived from M-919 and M-1057 show filamentous form 1:103 (104 M-919 cells per ml/107 M-1057 cells per ml) and give brown-colored colony. Phenotype abbrevia- and vice versa. The mixed culture in the test tube was tions used are as follows: Met-, Pan-, and PABA-, continuously incubated at 30 C with shaking at 140 requiring methionine, pantothenate, and p- strokes per min, and a small amount of the culture aminobenzoate, respectively, for growth. Colonies of was sampled at appropriate intervals to determine the M-919, M-1057, and mated cells are distinguishable percentage of mated cells on selection plates (methiofrom each other by their color and by their nutritional nine-pantothenate MM or p-aminobenzoate MM requirements. According to the description of I. plate). After a 3-day cultivation of the plates, the 710
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CELL MORPHOLOGY IN EARLY STAGES OF MATING
brown- and yellow- (or orange-) colored colonies formed were counted as mated and unmated cells, respectively. Mating frequency was presented as a ratio of the brown-colony number to the total colony number on the selection plate for the minority strain. (ii) Membrane-filter microculture method. Cul-
covered with a piece of dialysis membrane of cellophane (Visking, Co.). Cells of M-919 were spread onto the cellophane membrane, with a cell population of 104 cells per cm2. Then incubation was performed at 30 C overnight. Nutrients in YS medium are permeable through the membrane, so the cells of both M-919 tures of M-919 and M-1057 in YS liquid medium (107 and M-1057 were able to grow separately on and under cells per ml, respectively) were mixed to give a cell the membrane. Under the phase-contrast microscope, number ratio of 1:1 and immediately collected on cell morphology in microcolony of M-919 and M-1057 membrane filters (Millipore Corp., Bedford, Mass.; was independently observed by control of focus. HA type; pore size 0.45 Am) for high frequency of Detection of inducing substance for initiation of cell-to-cell contact. The membrane filters were placed mating tube formation. YS agar film, on which the on an MM agar plate (cells were on the upper side of cells of M-919 or M-1057 were previously spread with the filter), and incubation was performed at 30 C to a cell population of 104 cells per cm2, was covered with give enough time for cell fusion. At 0-, 1-, and 2-h a cover glass (10 mm square), followed by fixation of incubations, cells on the filter were suspended in the glass at four corners with paraffin. Then, 5 gl of liquid MM (about 106 cells per ml) with mild vibra- the culture fluid (clear supernatant), which was tion for good separation of individual cells and fused prepared by centrifugation from YS liquid culture pairs. Then the suspension was spread on MM agar medium of M-919 or M-1057 at an appropriate film (3 mm square by 0.3 mm thick) to give a cell incubation time, was added in the space surrounding population of approximately 104 cells per cm2 (aver- the agar film. After a 1-h incubation at 30 C to age cell-to-cell distance was 100 Am). Microculture diffuse the fluid into the agar film, another supplewas performed at 30 C. Morphology and behavior of ment of 5 ,ul of supernatant was added. Then the -the individual cells were followed for 4 h under a microculture system was sealed with paraffin, folphase-contrast microscope (Chiyoda Kogaku Co. lowed by cultivation at 30 C for 5 h. During the Ltd., Tokyo). Mating frequency was presented as a cultivation morphological changes were traced; picpercentage of the coupled cells which formed a tures of the cells were taken at appropriate incubation filamentous tube in the 4-h microculture per total times. When formation of filamentous tubes is inducicells at the beginning of the microculture. After the ble, the fluid used is presented as inducing substance cultivation, coupled cells, tube-formed cells without positive. fusion with partner cells, and bud-formed cells were observed. These cells were distinguishable from each RESULTS other by their morphological character. Microculture method for follow-up survey of Mating frequency. When the number of morphology and behavior of individual cells in the mating process. The microculture was carried out as M-919 cells was limited in the liquid culture described below. Strains M-919 and M-1057 were method (see Materials and Methods), the orgaseparately cultured in YS liquid medium with shak- nism showed a mating frequency of 89% in a 5-h ing, and the cultures of both strains at a middle log culture, and M-1057, which was limited, gave a growth phase (107 cells per ml) were mixed to give a frequency of 41%. By employing the membranecell number ratio of 1:1, followed by further cultiva- filter microculture method, a frequency of 62% tion for 1 to 2 h. Then cells were spread on YS agar was obtained. When the process of cell collecfilm for microculture with a cell population of 105 cells tion on the membrane filter was omitted, the per cm2. Microculture was performed at 30 C for 6 h. frequency was less than 0.1%. The results sugDuring the microculture, morphology and behavior of gest that cell-to-cell proximity and time for individual cells were traced under phase-contrast interaction facilitate the induction of the matmicroscope. Identification of the strain first showing the ing response. Details are summarized in Tables 1 and 2. response to opposite-typed cell in mating. To idenThe high frequency of occurrence of the tify the strain which first alters its morphology in mating phenomenon indicates that the strains response to the strain having the opposite mating type, two modified microculture methods-the line-up culture method and the cellophane-sandwich culture method-were employed. The tube formed by the mutual relation of strains M-919 and M-1057 is designated as the mating tube in this paper. (i) Line-up culture method. Cells of M-919 and M-1057 were separately spread on YS agar films (3 by 5 mm) for microculture with a cell population of 104
cells
cm2. These two films were arranged edge to edge. Then incubation was performed at 30 C overnight. Cell morphology in microcolony thus formed was observed under the phase-contrast microscope. (ii) Cellophane-sandwich culture method. YS agar film, on which cells of M-1057 were previously spread with a cell population of 104 cells per cm2, was per
TABLE 1. Mating frequency in the liquid culture methoda Strain which is limited in cell no.
0
1
2
3
4
5
M-919 M-1057
5
15 15
50 31
71 33
80
4
89 41
Mating frequency (%) after incubation of mixed culture for (h):
38
a The viable cell number of the limited strain (total of unmated and mated cells) was constant during the mixed culture.
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TABLE 2. Mating frequency in membrane-filter microculture methoda Frequency (%) after incubation on membrane filter for (h):
Cell type 0
Mated cellsc Unmated cellsd
7 93 (15e + 78f)
21
39 61 (lle + 50')
62 38 (5e + 33')
Ob
0.1 99.9
aWhen the organisms were incubated for 3 h on the membrane filter, they were hardly separated from the membrane. b Omission of collecting process on membrane filter, viz., M-919 and M-1057 were mixed and immediately spread on MM agar film for microculture. c Mated cells which were mated after 4-h incubation (see text) are counted as two cells. d Unmated cells are a total of the tube-forming cells without partners and the bud-forming cells. eTube-forming cells without partners. f Bud-forming cells.
used in this paper are useful in investigating the mating process from the viewpoint of biochemistry. The tube formation as an early event in the mating process. Whole steps in the process of mating between type A and a strains of Rhodosporidium toruloides were schematically presented by Banno (3), but the events occurring in the early stages of the mating process were not analyzed in detail. We, therefore, investigated the events, such as morphological alteration and behavior, in the early stages. In this paper, we tentatively define the early stages and the early events of the mating process as follows: the early stages are the period from initiation of mating to cell-to-cell fusion, and the early events are the events which occur during the early stages. Morphology and behavior of individual cells in the early stages. Results of the follow-up survey (see Materials and Methods) of cell morphology in the early stages are shown in Fig. 1, which includes six micrographs, A, B, C, D, E, and F. In each micrograph, five examples (cell pairs 1, 2, 3, 4, and 5) indicating the morphological alteration caused by cell-to-cell interaction are pointed out. As seen in examples 1, 2, and 3, the mating tube elongates to the polar zone of a mating partner cell, which does not form the tube, followed by cell-to-cell fusion between the partners. After the fusion filamentous cell appears, and then migration of cytoplasmic materials occurs from the mating tubeformed cell to the newly formed filamentous part of cell through the tube. During the migration the mixing of cytoplasmic materials of mating partners might be taking place. In examples 4 and 5, cell-to-cell fusion is observed at tips of the tubes formed in mating partners. Morphology of microcolonies in the early stages. The mutual relation in the morphology of the microcolony between M-919 and M-1057 was microscopically examined by the microcul-
ture method with some modification as described below. Cells of both strains which were separately cultured in YS liquid medium were mixed at a cell number ratio of approximately 1:1, diluted immediately with fresh YS medium, and spread on YS agar film giving a cell population of about 104 cells per cm2. Then incubation was carried out at 30 C overnight. Individual cells grew from single cells to microcolonies having round forms with and without tubes (Fig. 2). When M-919 and M-1057 were separately cultured on YS agar film, microcolonies without tubes were formed exclusively. Identification of the strain that responds first to the opposite mating type. To identify the strain which forms a round colony with tubes in the mixed culture, both the line-up culture and cellophane-sandwich culture methods were employed. In the line-up culture, M-1057 exclusively showed the characteristic morphology of the microcolony (Fig. 3). The tube formation was induced with the cells in M-1057 colonies which were located in an area close to M-919 colony. The tubes which were formed elongated toward the nearest colony of M-919. These findings indicate that direct contact of type A and a cells is not required for tube formation, and suggest that the initiation of tube formation and determination of tube-elongating direction are controlled by the substance which was secreted from the cells of M-919. When the cellophane-sandwich culture was employed under the conditions of cell number ratio 1:1 and cell population of 104 per cm2, the tube formation was demonstrated by every colony of M-1057 after 18-h cultivation, but there was no occurrence with M-919 (Fig. 4). Direction of tube elongation was random in this culture. When cell number ratio of M-1057 to M-919 was adjusted to 1:100 (on cellophane, 104 M-1057 cells per cm2/under cellophane, 106 M-919 cells per cm2) and incubated by this method, M-1057
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cells formed tubes after 3-h incubation. At a cell number ratio of 100:1 (on cellophane, 106 M-1057 cells per cm2/under cellophane, 104 M-919 cells per cm2), however, no tube formation was observed. Thus the inducing substance, which was produced by M-919 for induction of mating tube formation in M-1057, might be concluded to be water soluble, cellophane membrane permeable, and agar diffusible and was designated as A factor.
713
On the other hand, in the follow-up survey, it was found that cell-to-cell fusion occurs at the tips of the mating tubes (Fig. 1, examples 4 and 5). This phenomenon suggests that M-919 is able to secondly form mating tube in response to the opposite-typed strain in mating. However, in both the line-up and cellophane-sandwich cultures, M-1057 exclusively formed mating tube. The tube formation in M-919 might be induced by the inducing substance which was secreted from the tube-formed cells of M-1057.
I
FIG. 1. Cell morphology and cell behavior in the mating process of Rhodosporidium toruloides in microculture. A, B, C, D, E, and F are the micrographs taken at 70-, 90-, 110-, 160-, 250- and 360-min incubations respectively. 1, 2, 3, 4 and 5 in each micrograph are the same partners, indicating cell-to-cell interaction. Bar = 50 ,um.
J. BACTERIOL.
ABE, KUSAKA, AND FUKUI
714
_ _ _ _
_
_
_
_
_
_
_
1-
_~~~~~~~~~~~
-
* .~7
I
r
I
FIG. 2. Microcolonies in microculture of the mixed-cell suspension of M-919 and M-1057. Each colony was a single cell at 0-h incubation. Bar = 100 um.
Ls
.
171n.
d
o w
*
FIG. 3. Microcolonies in the line-up culture of M-919 (right) and M-1057 (left). The central zone in the micrograph is the connecting line of two agar films. Bar = 100 ,um.
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FIG. 4. Microcolonies in the cellophane-sandwich culture of M-919 and M-1057. (A) Micrograph of M-919 (on cellophane membrane); (B) micrograph of M-1057 (under cellophane membrane), both at 17-h incubation. Bar = 100 Am.
Formation of the inducing substance for mating tube formation. Detection of the inducing substance for mating-tube formation was performed with culture fluids which were prepared at appropriate growth phases in YS liquid medium. Culture fluids of M-919 during middle and late log growth phases were inducing substance (A factor) positive, whereas the fluids at early and stationary growth phases were A factor negative. A factor was constitutively formed by M-919 in YS medium and exclusively induced morphological alteration, i.e., mating tube formation, in M-1057 (Fig. 5). In the above section, we described a possibility of the presence of another extracellular inducing substance (a factor) which induces the
formation of mating tube in M-919. The possibility was examined with the culture fluids which were prepared from an 8-h culture of M-1057 in YS liquid media with and without addition of partially purified A factor (K. Abe et al., unpublished data). As seen in Fig. 6, a factor was formed by M-1057 cells grown in A factorcontaining YS medium, whereas the factor was not formed in the medium in the absence of A factor. Consequently, A factor might be required as an inducer for the formation of a factor by M-1057. It should be noted that the culture fluid of the mixed culture of both strains M-919 and M-1057 in YS liquid medium was negative in both A and a factors by the detection methods used.
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FIG. 5. Mating tube formation in M-1057 cells by supplement of A factor, the culture fluid of M-919. (A) and (B) are micrographs of M-1057 cells in microculture on the YS agar films supplemented with the culture fluids at log growth phase of M-1057 and M-919, respectively. Bar = 100 ,gm.
Some properties of morphological alteration caused by the inducing substances (A and a factors). A and a factors induced the morphological alteration with strains M-1057 and M-919, respectively. In this alteration it is interesting that: (i) formation and elongation of the mating tube occur with suppression of bud growth, (ii) the loci of tube-forming sites in a cell are usually polar or subpolar, and (iii) the number of tubes per cell is 1 to 2, sometimes 3. DISCUSSION In this paper we demonstrate that strains M-919 and M-0157 of Rhodosporidium toruloides produce the extracellular diffusible substances inducing the formation of mating tubes in the opposite mating-typed cells, M-1057 and M-919, respectively. Thus, the
substances are concluded to be sex hormones. Investigation of microbial differentiation has increased recently (1). Some kinds of chemical substances which control the sexual differentiation in fungi have been reported; trisporic acid is formed in many families of Mucorales (2), and antheridiol is formed in Achlva (4). For occurrence of cell-to-cell fusion, the close proximity of the mating partner cell is first required. Recently, agglutinine was proved to be the hormone agglutinating the cells of Hansenula wingei to achieve the close proximity (6), and sirenin as male gamete-attracting hormone of A llomyces (8). In Rhodosporidium toruloides, in addition to the inducing substances for mating tube formation, the directional growth of the tube toward the opposite mating-typed cell is also apparent, including recognition of
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the appropriate cell (or copulation tube) for fusion. According to the suggestion presented by Bucking-Throme et al. (5), synchronization of cell cycle between partner cells is required for occurrence of cell-to-cell fusion. In Saccharomyces cerevisiae, a factor (7) inhibits the initiation of deoxyribonucleic acid synthesis in type a cells, and subsequently synchronization of cell cycle is achieved (5). In Rhodosporidium toruloides, the inhibition of bud growth in M-919 (type A) and M-1057 (type a) was observed in the presence of a and A factors, respectively. The reason for these phenomena is obscure; therefore, investigation on purification and biochemical activities of the factors is in progress. In the early stages of the mating process of Rhodosporidium toruloides, the reciprocal information exchange, A factor from type A strain to type a strain and a factor from type a strain to type A strain, has sequentially taken place. A similar observation was reported with Achlya (4). Hormone B, which induces the formation of oogonium in the female strain of Achlya sp., is produced as an extracellular hormone by the male strain of the organism only in the presence
717
of antheridiol, which is constitutively secreted by the female strain. The events in the early stages of the mating process in Rhodosporidium toruloides can then be schematically represented (Fig. 7), the mating tubes, once formed, continuously elongate in the absence of mating partners. In the cellophane-sandwich culture M-1057 cells had many tubes, whereas M-919 cells failed to produce tubes. We suppose that a factor might be impermeable to cellophane membrane. However, it will be necessary to purify the factor for further investigation. No hormonal activity was detected in the supernatant fluid of the mixed culture. This phenomenon might be introduced by removal of both a and A factors from the medium, not by the presence of hormone-inactivating substance, because when A factor was mixed with M-1057 cells at 27 C, the factor could not be recovered in the supernatant fluid. ACKNOWLEDGMENTS We thank S. Tamura, A. Sakurai, and K. Yamamoto, who prepared the partially purified sample of A factor for us, and I. Banno, T. Hasegawa, K. Komagata, and A. Kusanagi for their helpful advice and discussion.
FIG. 6. Mating tube formation in M-919 cells by supplement of a factor, the culture fluid of the mating tube-formed M-1057. (A), (B), and (C) are micrographs of M-919 cells in microculture at 5-h incubation: (A), without supplement; (B) with supplement of the culture fluid of M-1057 (log growth phase) in the YS liquid medium without addition of A factor; (C) with supplement of the culture fluid of M-1057 (log growth phase) in the YS liquid medium with addition of the partially purified A factor equivalent to a 100-Al amount of the original fluid. Bar = 50 um.
718
ABE, KUSAKA, AND FUKUI
J. BACTERIOL.
A
*
mlw
0
s
A factor
:
afactor
A
*2 U~~ : mlw
a
m
3/aQ7 C:)j/
FIG. 7. Schematic representation of the events in the early stages of the mating process. (1) M-919 cells secrete A factor; (2) M-1057 cells receive A factor; (3) M-1057 cells form the mating tube in response to A factor; (4) the tube-forming M-1057 cells secrete a factor; (5) M-919 cells receive a factor; (6) M-919 cells form the mating tube in response to a factor; (7) the mating tubes elongate to reach the cells or tubes of mating partners; (8) growing tip of the tube recognizes the cells or tube of mating partner, followed by cell-to-cell fusion. Dotted line arrows indicate the movement of the extracellular factors. Solid line arrows indicate the behaviors of cells in response to A factor (heavy arrows) and the cell-recognizing factor (light arrows). LITERATURE CITED 1. Ashworth, J. M., and J. E. Smith. 1973. The Society for General Microbiology, symposium 23. Microbial differentiation. Cambridge University Press, New York. 2. Austin, D. J., J. D. Bu'Lock, and G. W. Gooday. 1969. Trisporic acids: sexual hormones from Mucor mucedo and Blakeslea trispora. Nature (London) 223:1178-1179. 3. Banno, I. 1967. Studies on sexuality of Rhodotorula. J. Gen. Appl. Microbiol. 13:167-196. 4. Barksdale, A. W. 1969. Sexual hormones of Achlya and other fungi. Science 116:831-837. 5. Bucking-Throme, E., W. Duntze, L. H. Hartwell, and T.
R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 6. Crandall, M., L. H. Lawrence, and R. T. Saunders. 1974. Molecular complimentality of yeast glycoprotein mating factors. Proc. Natl. Acad. Sci. U.S.A. 71:26-29. 7. Duntze, W., D. Stotzler, E. Bucking-Throme, and S. Kalbitzer. 1973. Purification and partial characterization of a-factor, a mating-type specific inhibitor of cell reproduction from Saccharomyces cerevisiae. Eur. J. Biochem. 35:357-365. 8. Nutting, W. H., H. Rapoport, and L. Machlis. 1968. The structure of sirenin. J. Am. Chem. Soc. 90:6434-6438.
Vol. 122, No. 2 Printed in U.S.A.
Morphological Change in the Early Stages of the Mating Process of Rhodosporidium toruloides KEIKO ABE, IWAO KUSAKA, AND SAKUZO FUKUI* The Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan
Received for publication 27 January 1975
The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.
In 1967, a sexual generation in Rhodotorula glutinis was discovered by I. Banno, and the yeast was classified as Rhodosporidium toruloides (3). His report describes the microorganism as taking a characteristic cell morphology which is dependent on mating or ploidy: the haploid strains (type A and a strains) are ovoid in cell form, and the diploid or dicaryon strain is filamentous. Our interest has been in the morphological alteration caused by cell-to-cell interaction. In this paper, therefore, we describe the morphological events which occur in the early stages of the mating process, i.e., cell-to-cell interaction between different mating type cells of Rhodosporidium toruloides.
Banno, types A and a represent the mating type for M-919 and M-1057, respectively. Media and cultivation. YS liquid medium (pH 6.4), which contained 10 g of yeast extract (Daigoeiyo, Osaka), 10 g of sucrose, and 2.5 g of NaCl per liter, was employed for shaking culture. YS agar medium was used for stock culture and microculture. Minimal agar medium (MM), which contained 30 g of sucrose, 3 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.1 g of NaCl, and 20 g of agar per liter, with pH adjusted to 6.5 with 1 N NaOH, was used as the selection medium for mated cells. For respective selection of M-919 and M-1057 from mixed cultures of these strains, methionine-pantothenate MM, containing 0.1 g of L-methionine and 0.4 mg of calcium pantothenate per liter of MM, and p-aminobenzoate MM, containing 0.2 mg of p-aminobenzoic acid per liter of MM, were used. The preculture was carried out in a test tube (16 by 180 mm) containing 8.0 ml of MATERIALS AND METHODS YS liquid medium with reciprocal shaking (320 Microorganisms. Rhodosporidium toruloides IFO strokes per min, 2-cm width) at 27 C by inoculation 0559-M-919 (haploid, mating type A, Met-, Pan-, from stock culture. Determination of mating frequency. (i) Liquid ovoid cell form, orange-colored colony) and Rhodosporidium toruloides IFO 0880-M-1057 (haploid, mat- culture method. Both strains M-919 and M-1057 ing type a, PABA- long ovoid cell form, yellow-col- were separately cultured in YS liquid medium in the ored colony), which were kindly provided by I. Banno, test tube which was described above. A small amount Institute for Fermentation, Osaka, Japan, were desig- of the culture of M-919 at a middle log growth phase nated as M-919 and M-1057, respectively, and used (107 cells per ml) was mixed with the culture of throughout this paper. Mated cells which were de- M-1057 (107 cells per ml) to give a cell number ratio of rived from M-919 and M-1057 show filamentous form 1:103 (104 M-919 cells per ml/107 M-1057 cells per ml) and give brown-colored colony. Phenotype abbrevia- and vice versa. The mixed culture in the test tube was tions used are as follows: Met-, Pan-, and PABA-, continuously incubated at 30 C with shaking at 140 requiring methionine, pantothenate, and p- strokes per min, and a small amount of the culture aminobenzoate, respectively, for growth. Colonies of was sampled at appropriate intervals to determine the M-919, M-1057, and mated cells are distinguishable percentage of mated cells on selection plates (methiofrom each other by their color and by their nutritional nine-pantothenate MM or p-aminobenzoate MM requirements. According to the description of I. plate). After a 3-day cultivation of the plates, the 710
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CELL MORPHOLOGY IN EARLY STAGES OF MATING
brown- and yellow- (or orange-) colored colonies formed were counted as mated and unmated cells, respectively. Mating frequency was presented as a ratio of the brown-colony number to the total colony number on the selection plate for the minority strain. (ii) Membrane-filter microculture method. Cul-
covered with a piece of dialysis membrane of cellophane (Visking, Co.). Cells of M-919 were spread onto the cellophane membrane, with a cell population of 104 cells per cm2. Then incubation was performed at 30 C overnight. Nutrients in YS medium are permeable through the membrane, so the cells of both M-919 tures of M-919 and M-1057 in YS liquid medium (107 and M-1057 were able to grow separately on and under cells per ml, respectively) were mixed to give a cell the membrane. Under the phase-contrast microscope, number ratio of 1:1 and immediately collected on cell morphology in microcolony of M-919 and M-1057 membrane filters (Millipore Corp., Bedford, Mass.; was independently observed by control of focus. HA type; pore size 0.45 Am) for high frequency of Detection of inducing substance for initiation of cell-to-cell contact. The membrane filters were placed mating tube formation. YS agar film, on which the on an MM agar plate (cells were on the upper side of cells of M-919 or M-1057 were previously spread with the filter), and incubation was performed at 30 C to a cell population of 104 cells per cm2, was covered with give enough time for cell fusion. At 0-, 1-, and 2-h a cover glass (10 mm square), followed by fixation of incubations, cells on the filter were suspended in the glass at four corners with paraffin. Then, 5 gl of liquid MM (about 106 cells per ml) with mild vibra- the culture fluid (clear supernatant), which was tion for good separation of individual cells and fused prepared by centrifugation from YS liquid culture pairs. Then the suspension was spread on MM agar medium of M-919 or M-1057 at an appropriate film (3 mm square by 0.3 mm thick) to give a cell incubation time, was added in the space surrounding population of approximately 104 cells per cm2 (aver- the agar film. After a 1-h incubation at 30 C to age cell-to-cell distance was 100 Am). Microculture diffuse the fluid into the agar film, another supplewas performed at 30 C. Morphology and behavior of ment of 5 ,ul of supernatant was added. Then the -the individual cells were followed for 4 h under a microculture system was sealed with paraffin, folphase-contrast microscope (Chiyoda Kogaku Co. lowed by cultivation at 30 C for 5 h. During the Ltd., Tokyo). Mating frequency was presented as a cultivation morphological changes were traced; picpercentage of the coupled cells which formed a tures of the cells were taken at appropriate incubation filamentous tube in the 4-h microculture per total times. When formation of filamentous tubes is inducicells at the beginning of the microculture. After the ble, the fluid used is presented as inducing substance cultivation, coupled cells, tube-formed cells without positive. fusion with partner cells, and bud-formed cells were observed. These cells were distinguishable from each RESULTS other by their morphological character. Microculture method for follow-up survey of Mating frequency. When the number of morphology and behavior of individual cells in the mating process. The microculture was carried out as M-919 cells was limited in the liquid culture described below. Strains M-919 and M-1057 were method (see Materials and Methods), the orgaseparately cultured in YS liquid medium with shak- nism showed a mating frequency of 89% in a 5-h ing, and the cultures of both strains at a middle log culture, and M-1057, which was limited, gave a growth phase (107 cells per ml) were mixed to give a frequency of 41%. By employing the membranecell number ratio of 1:1, followed by further cultiva- filter microculture method, a frequency of 62% tion for 1 to 2 h. Then cells were spread on YS agar was obtained. When the process of cell collecfilm for microculture with a cell population of 105 cells tion on the membrane filter was omitted, the per cm2. Microculture was performed at 30 C for 6 h. frequency was less than 0.1%. The results sugDuring the microculture, morphology and behavior of gest that cell-to-cell proximity and time for individual cells were traced under phase-contrast interaction facilitate the induction of the matmicroscope. Identification of the strain first showing the ing response. Details are summarized in Tables 1 and 2. response to opposite-typed cell in mating. To idenThe high frequency of occurrence of the tify the strain which first alters its morphology in mating phenomenon indicates that the strains response to the strain having the opposite mating type, two modified microculture methods-the line-up culture method and the cellophane-sandwich culture method-were employed. The tube formed by the mutual relation of strains M-919 and M-1057 is designated as the mating tube in this paper. (i) Line-up culture method. Cells of M-919 and M-1057 were separately spread on YS agar films (3 by 5 mm) for microculture with a cell population of 104
cells
cm2. These two films were arranged edge to edge. Then incubation was performed at 30 C overnight. Cell morphology in microcolony thus formed was observed under the phase-contrast microscope. (ii) Cellophane-sandwich culture method. YS agar film, on which cells of M-1057 were previously spread with a cell population of 104 cells per cm2, was per
TABLE 1. Mating frequency in the liquid culture methoda Strain which is limited in cell no.
0
1
2
3
4
5
M-919 M-1057
5
15 15
50 31
71 33
80
4
89 41
Mating frequency (%) after incubation of mixed culture for (h):
38
a The viable cell number of the limited strain (total of unmated and mated cells) was constant during the mixed culture.
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TABLE 2. Mating frequency in membrane-filter microculture methoda Frequency (%) after incubation on membrane filter for (h):
Cell type 0
Mated cellsc Unmated cellsd
7 93 (15e + 78f)
21
39 61 (lle + 50')
62 38 (5e + 33')
Ob
0.1 99.9
aWhen the organisms were incubated for 3 h on the membrane filter, they were hardly separated from the membrane. b Omission of collecting process on membrane filter, viz., M-919 and M-1057 were mixed and immediately spread on MM agar film for microculture. c Mated cells which were mated after 4-h incubation (see text) are counted as two cells. d Unmated cells are a total of the tube-forming cells without partners and the bud-forming cells. eTube-forming cells without partners. f Bud-forming cells.
used in this paper are useful in investigating the mating process from the viewpoint of biochemistry. The tube formation as an early event in the mating process. Whole steps in the process of mating between type A and a strains of Rhodosporidium toruloides were schematically presented by Banno (3), but the events occurring in the early stages of the mating process were not analyzed in detail. We, therefore, investigated the events, such as morphological alteration and behavior, in the early stages. In this paper, we tentatively define the early stages and the early events of the mating process as follows: the early stages are the period from initiation of mating to cell-to-cell fusion, and the early events are the events which occur during the early stages. Morphology and behavior of individual cells in the early stages. Results of the follow-up survey (see Materials and Methods) of cell morphology in the early stages are shown in Fig. 1, which includes six micrographs, A, B, C, D, E, and F. In each micrograph, five examples (cell pairs 1, 2, 3, 4, and 5) indicating the morphological alteration caused by cell-to-cell interaction are pointed out. As seen in examples 1, 2, and 3, the mating tube elongates to the polar zone of a mating partner cell, which does not form the tube, followed by cell-to-cell fusion between the partners. After the fusion filamentous cell appears, and then migration of cytoplasmic materials occurs from the mating tubeformed cell to the newly formed filamentous part of cell through the tube. During the migration the mixing of cytoplasmic materials of mating partners might be taking place. In examples 4 and 5, cell-to-cell fusion is observed at tips of the tubes formed in mating partners. Morphology of microcolonies in the early stages. The mutual relation in the morphology of the microcolony between M-919 and M-1057 was microscopically examined by the microcul-
ture method with some modification as described below. Cells of both strains which were separately cultured in YS liquid medium were mixed at a cell number ratio of approximately 1:1, diluted immediately with fresh YS medium, and spread on YS agar film giving a cell population of about 104 cells per cm2. Then incubation was carried out at 30 C overnight. Individual cells grew from single cells to microcolonies having round forms with and without tubes (Fig. 2). When M-919 and M-1057 were separately cultured on YS agar film, microcolonies without tubes were formed exclusively. Identification of the strain that responds first to the opposite mating type. To identify the strain which forms a round colony with tubes in the mixed culture, both the line-up culture and cellophane-sandwich culture methods were employed. In the line-up culture, M-1057 exclusively showed the characteristic morphology of the microcolony (Fig. 3). The tube formation was induced with the cells in M-1057 colonies which were located in an area close to M-919 colony. The tubes which were formed elongated toward the nearest colony of M-919. These findings indicate that direct contact of type A and a cells is not required for tube formation, and suggest that the initiation of tube formation and determination of tube-elongating direction are controlled by the substance which was secreted from the cells of M-919. When the cellophane-sandwich culture was employed under the conditions of cell number ratio 1:1 and cell population of 104 per cm2, the tube formation was demonstrated by every colony of M-1057 after 18-h cultivation, but there was no occurrence with M-919 (Fig. 4). Direction of tube elongation was random in this culture. When cell number ratio of M-1057 to M-919 was adjusted to 1:100 (on cellophane, 104 M-1057 cells per cm2/under cellophane, 106 M-919 cells per cm2) and incubated by this method, M-1057
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CELL MORPHOLOGY IN EARLY STAGES OF MATING
cells formed tubes after 3-h incubation. At a cell number ratio of 100:1 (on cellophane, 106 M-1057 cells per cm2/under cellophane, 104 M-919 cells per cm2), however, no tube formation was observed. Thus the inducing substance, which was produced by M-919 for induction of mating tube formation in M-1057, might be concluded to be water soluble, cellophane membrane permeable, and agar diffusible and was designated as A factor.
713
On the other hand, in the follow-up survey, it was found that cell-to-cell fusion occurs at the tips of the mating tubes (Fig. 1, examples 4 and 5). This phenomenon suggests that M-919 is able to secondly form mating tube in response to the opposite-typed strain in mating. However, in both the line-up and cellophane-sandwich cultures, M-1057 exclusively formed mating tube. The tube formation in M-919 might be induced by the inducing substance which was secreted from the tube-formed cells of M-1057.
I
FIG. 1. Cell morphology and cell behavior in the mating process of Rhodosporidium toruloides in microculture. A, B, C, D, E, and F are the micrographs taken at 70-, 90-, 110-, 160-, 250- and 360-min incubations respectively. 1, 2, 3, 4 and 5 in each micrograph are the same partners, indicating cell-to-cell interaction. Bar = 50 ,um.
J. BACTERIOL.
ABE, KUSAKA, AND FUKUI
714
_ _ _ _
_
_
_
_
_
_
_
1-
_~~~~~~~~~~~
-
* .~7
I
r
I
FIG. 2. Microcolonies in microculture of the mixed-cell suspension of M-919 and M-1057. Each colony was a single cell at 0-h incubation. Bar = 100 um.
Ls
.
171n.
d
o w
*
FIG. 3. Microcolonies in the line-up culture of M-919 (right) and M-1057 (left). The central zone in the micrograph is the connecting line of two agar films. Bar = 100 ,um.
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CELL MORPHOLOGY IN EARLY STAGES OF MATING
715
FIG. 4. Microcolonies in the cellophane-sandwich culture of M-919 and M-1057. (A) Micrograph of M-919 (on cellophane membrane); (B) micrograph of M-1057 (under cellophane membrane), both at 17-h incubation. Bar = 100 Am.
Formation of the inducing substance for mating tube formation. Detection of the inducing substance for mating-tube formation was performed with culture fluids which were prepared at appropriate growth phases in YS liquid medium. Culture fluids of M-919 during middle and late log growth phases were inducing substance (A factor) positive, whereas the fluids at early and stationary growth phases were A factor negative. A factor was constitutively formed by M-919 in YS medium and exclusively induced morphological alteration, i.e., mating tube formation, in M-1057 (Fig. 5). In the above section, we described a possibility of the presence of another extracellular inducing substance (a factor) which induces the
formation of mating tube in M-919. The possibility was examined with the culture fluids which were prepared from an 8-h culture of M-1057 in YS liquid media with and without addition of partially purified A factor (K. Abe et al., unpublished data). As seen in Fig. 6, a factor was formed by M-1057 cells grown in A factorcontaining YS medium, whereas the factor was not formed in the medium in the absence of A factor. Consequently, A factor might be required as an inducer for the formation of a factor by M-1057. It should be noted that the culture fluid of the mixed culture of both strains M-919 and M-1057 in YS liquid medium was negative in both A and a factors by the detection methods used.
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ABE, KUSAKA, AND FUKUI
J. BACTERIOL.
FIG. 5. Mating tube formation in M-1057 cells by supplement of A factor, the culture fluid of M-919. (A) and (B) are micrographs of M-1057 cells in microculture on the YS agar films supplemented with the culture fluids at log growth phase of M-1057 and M-919, respectively. Bar = 100 ,gm.
Some properties of morphological alteration caused by the inducing substances (A and a factors). A and a factors induced the morphological alteration with strains M-1057 and M-919, respectively. In this alteration it is interesting that: (i) formation and elongation of the mating tube occur with suppression of bud growth, (ii) the loci of tube-forming sites in a cell are usually polar or subpolar, and (iii) the number of tubes per cell is 1 to 2, sometimes 3. DISCUSSION In this paper we demonstrate that strains M-919 and M-0157 of Rhodosporidium toruloides produce the extracellular diffusible substances inducing the formation of mating tubes in the opposite mating-typed cells, M-1057 and M-919, respectively. Thus, the
substances are concluded to be sex hormones. Investigation of microbial differentiation has increased recently (1). Some kinds of chemical substances which control the sexual differentiation in fungi have been reported; trisporic acid is formed in many families of Mucorales (2), and antheridiol is formed in Achlva (4). For occurrence of cell-to-cell fusion, the close proximity of the mating partner cell is first required. Recently, agglutinine was proved to be the hormone agglutinating the cells of Hansenula wingei to achieve the close proximity (6), and sirenin as male gamete-attracting hormone of A llomyces (8). In Rhodosporidium toruloides, in addition to the inducing substances for mating tube formation, the directional growth of the tube toward the opposite mating-typed cell is also apparent, including recognition of
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CELL MORPHOLOGY IN EARLY STAGES OF MATING
the appropriate cell (or copulation tube) for fusion. According to the suggestion presented by Bucking-Throme et al. (5), synchronization of cell cycle between partner cells is required for occurrence of cell-to-cell fusion. In Saccharomyces cerevisiae, a factor (7) inhibits the initiation of deoxyribonucleic acid synthesis in type a cells, and subsequently synchronization of cell cycle is achieved (5). In Rhodosporidium toruloides, the inhibition of bud growth in M-919 (type A) and M-1057 (type a) was observed in the presence of a and A factors, respectively. The reason for these phenomena is obscure; therefore, investigation on purification and biochemical activities of the factors is in progress. In the early stages of the mating process of Rhodosporidium toruloides, the reciprocal information exchange, A factor from type A strain to type a strain and a factor from type a strain to type A strain, has sequentially taken place. A similar observation was reported with Achlya (4). Hormone B, which induces the formation of oogonium in the female strain of Achlya sp., is produced as an extracellular hormone by the male strain of the organism only in the presence
717
of antheridiol, which is constitutively secreted by the female strain. The events in the early stages of the mating process in Rhodosporidium toruloides can then be schematically represented (Fig. 7), the mating tubes, once formed, continuously elongate in the absence of mating partners. In the cellophane-sandwich culture M-1057 cells had many tubes, whereas M-919 cells failed to produce tubes. We suppose that a factor might be impermeable to cellophane membrane. However, it will be necessary to purify the factor for further investigation. No hormonal activity was detected in the supernatant fluid of the mixed culture. This phenomenon might be introduced by removal of both a and A factors from the medium, not by the presence of hormone-inactivating substance, because when A factor was mixed with M-1057 cells at 27 C, the factor could not be recovered in the supernatant fluid. ACKNOWLEDGMENTS We thank S. Tamura, A. Sakurai, and K. Yamamoto, who prepared the partially purified sample of A factor for us, and I. Banno, T. Hasegawa, K. Komagata, and A. Kusanagi for their helpful advice and discussion.
FIG. 6. Mating tube formation in M-919 cells by supplement of a factor, the culture fluid of the mating tube-formed M-1057. (A), (B), and (C) are micrographs of M-919 cells in microculture at 5-h incubation: (A), without supplement; (B) with supplement of the culture fluid of M-1057 (log growth phase) in the YS liquid medium without addition of A factor; (C) with supplement of the culture fluid of M-1057 (log growth phase) in the YS liquid medium with addition of the partially purified A factor equivalent to a 100-Al amount of the original fluid. Bar = 50 um.
718
ABE, KUSAKA, AND FUKUI
J. BACTERIOL.
A
*
mlw
0
s
A factor
:
afactor
A
*2 U~~ : mlw
a
m
3/aQ7 C:)j/
FIG. 7. Schematic representation of the events in the early stages of the mating process. (1) M-919 cells secrete A factor; (2) M-1057 cells receive A factor; (3) M-1057 cells form the mating tube in response to A factor; (4) the tube-forming M-1057 cells secrete a factor; (5) M-919 cells receive a factor; (6) M-919 cells form the mating tube in response to a factor; (7) the mating tubes elongate to reach the cells or tubes of mating partners; (8) growing tip of the tube recognizes the cells or tube of mating partner, followed by cell-to-cell fusion. Dotted line arrows indicate the movement of the extracellular factors. Solid line arrows indicate the behaviors of cells in response to A factor (heavy arrows) and the cell-recognizing factor (light arrows). LITERATURE CITED 1. Ashworth, J. M., and J. E. Smith. 1973. The Society for General Microbiology, symposium 23. Microbial differentiation. Cambridge University Press, New York. 2. Austin, D. J., J. D. Bu'Lock, and G. W. Gooday. 1969. Trisporic acids: sexual hormones from Mucor mucedo and Blakeslea trispora. Nature (London) 223:1178-1179. 3. Banno, I. 1967. Studies on sexuality of Rhodotorula. J. Gen. Appl. Microbiol. 13:167-196. 4. Barksdale, A. W. 1969. Sexual hormones of Achlya and other fungi. Science 116:831-837. 5. Bucking-Throme, E., W. Duntze, L. H. Hartwell, and T.
R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 6. Crandall, M., L. H. Lawrence, and R. T. Saunders. 1974. Molecular complimentality of yeast glycoprotein mating factors. Proc. Natl. Acad. Sci. U.S.A. 71:26-29. 7. Duntze, W., D. Stotzler, E. Bucking-Throme, and S. Kalbitzer. 1973. Purification and partial characterization of a-factor, a mating-type specific inhibitor of cell reproduction from Saccharomyces cerevisiae. Eur. J. Biochem. 35:357-365. 8. Nutting, W. H., H. Rapoport, and L. Machlis. 1968. The structure of sirenin. J. Am. Chem. Soc. 90:6434-6438.
