AN ELECTROPHORETIC COMPARISON OF ENZYMES IN STRAINS OF SPECIES IN THE FISSION YEAST GENERA SCHJZOSACCHAROMYCES, 0CTOSPOROMYCES, AND HA SEGA WAEA1

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1 J. Gen. Appl. Microbiol., 33, (1987) AN ELECTROPHORETIC COMPARISON OF ENZYMES IN STRAINS OF SPECIES IN THE FISSION YEAST GENERA SCHJZOSACCHAROMYCES, 0CTOSPOROMYCES, AND HA SEGA WAEA1 YUZO YAMADA, KANA AIZAWA, AKIKO MATSUMOTO, YASUYOSHI NAKAGAWA, AND ISAO BANNO* Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University, Shizuoka 422, Japan * Institute for Fermentation, Osaka, Jusohon-machi, Yodogawa-ku, Osaka 532, Japan (Received June 10, 1987) A taxonomic study below the generic or at the specific level was made of the electrophoretic patterns of five enzymes in fourteen strains of Schizosaccharomyces, Octosporomyces, and Hasegawaea species. The five enzymes were glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphoglucomutase, and fumarase. All of the six strains of Schizosaccharomyces pombe examined were linked to each other with a similarity value of 40 c or more. The type strain of Schizosaccharomyces malidevorans was closely related to that of S, pombe with a similarity value of 60%. The similarity values of three strains of Octosporomyces octosporus were 80 c or more. All of the three strains of Hasegawaea japonica var. japonica examined had a uniform electrophoretic enzyme pattern. The similarity value between the strains of H. japonica var. japonica and H. japonica var. versatilis was 60%. The three species, S. pombe, 0. octosporus, and H. japonica had quite different electrophoretic enzyme patterns. Their similarity values were all 000. The Co-Q systems of the strains were reinvestigated. These data are discussed from the taxonomic point of view. The genus Schizosaccharomyces was established by LINDNER (1) with a single species, Schizosaccharomyces pombe Lindner (1893) for classifying a fission yeast strain isolated from African beer. KLOCKER (2) placed only the genus Schizosaccharomyces Lindner in the family Schizosaccharomycetaceae. Since then, 1 This constitutes Part XXII of a series entitled "Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms." For Part XXI, see ref. 6. Address reprint requests to: Dr. Y. Yamada, Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University, 836 Ohya, Shizuoka 422, Japan. 363

2 364 YAMADA, AIZAWA, MATSUMOTO, NAKAGAWA, and BANNO VOL. 33 four species have been accepted in the fission yeast genus classified presently in the subfamily Schizosaccharomycetoideae of the family Saccharomycetaceae (3-5): Schizosaccharomyces pombe (type species), Schizosaccharomyces octosporus Beijerinck (1894), Schizosaccharomyces japonicus Yukawa et Maki (1931) var. japonicus, Schizosaccharomyces japonicus var. versatilis Wickerham et Duprat ex Slooff (1970), and Schizosaccharomyces malidevorans Rankine et Fornachon (1964). In a previous paper (6), two of the authors (Y. Yamada and I. Banno) divided the fission yeast genus into three groups and distinguished them at the generic level on the basis of the differences in ascospore morphology (7) correlated with those of chemotaxonomic characteristics, namely, the Co-Q system (8) and the presence or absence of linoleic acid in cellular fatty acid composition (9): (i) the genus Schizosaccharomyces Lindner, which includes S, pombe and S. malidevorans, characterized by the presence of four warty ascospores per ascus, the Q-10 system, and the absence of linoleic acid; (ii) the genus Octosporomyces Kudriavzev emend. Yamada et Banno, which includes Octosporomyces octosporus (Beijerinck) Kudriavzev (1960) (- S. octosporus), characterized by the presence of eight smooth ascospores with papillae per ascus, the Q-9 system, and the absence of linoleic acid; and (iii) the genus Hasegawaea Yamada et Banno, which includes Hasegawaea japonica (Yukawa et Maki) Yamada et Banno (1987) var. japonica (- S. japonicus var. japonicus) and H, japonica var. versatilis (- S. japonicus var. versatilis), characterized by the presence of six to eight smooth ascospores without papillae per ascus, the absence of Co-Q, and the presence of linoleic acid. This paper is concerned with electrophoretic enzyme patterns in strains assigned to the fission yeast genera to confirm the classification of the four species mentioned above below the generic level, along with reinvestigation of the Co-Q system. MATERIALS AND METHODS Yeast strains. Fourteen strains of the four species in the fission yeast genera Schizosaccharomyces, Octosporomyces, and Hasegawaea were cultured aerobically in 100 ml of medium containing 100 glucose, 0.5 c peptone, 0.3 c yeast extract, and 0.3% malt extract (ph 6.0) at 25 C for 24 hr. The inoculum size was one twelfth for final cultures. Preparation of Co-Q and identification of the Co-Q system. The preparation of Co-Q and the identification of the Co-Q system were carried out as described previously (8). Preparation of enzymes. The intact cells harvested by centrifugation were suspended in 50 mm Tris-HCl (ph 7.8) containing 0.57 mm L-ascorbic acid, 0.64 mm L-cysteine, and 400 mm sucrose, and disrupted by sonication (10 khz) at 180W for 18 min. The enzyme preparation was obtained by centrifugation at 12,500 rpm for 60 min, and the resulting precipitates were discarded. Electrophoresis and staining of enzymes. Five enzymes were examined:

3 1987 Enzyme Patterns in Fission Yeast Genera 365 glucose-6-phosphate dehydrogenase (NADP-dependent, EC ), 6-phosphogluconate dehydrogenase (NADP-dependent, EC ), hexokinase (EC ), phosphoglucomutase (EC ), and fumarase (EC ). Isocitrate dehydrogenase (NADP-dependent, EC ) and malate dehydrogenase (NADdependent, EC ) were too weak to be examined by gel electrophoresis. The electrophoresis and staining procedures of the enzymes were as described by YAMAZAKI (1 D). A 7.5,, polyacrylamide gel slab was used (130 x 135 x 2 mm, the socalled 8.3 gel). The enzyme solutions ( ,ig of protein) were applied (1], 12). The electrophoretic mobilities (Rm's) of the enzymes were obtained relative to bromphenol blue. The similarity values in the electrophoretic patterns of enzymes were calculated by the following formula: s (%) = NS/(NS + Nd) x 100 (s, similarity value; NS, the number of enzymes with identical relative mobility; Nd, the number of enzymes with different mobility). The dendrogram based on the calculated similarity values was drawn by the complete linkage method (13). Chemicals. Acrylamide (monomer, EPI-7) was obtained from Nakarai Chemicals, Ltd., Kyoto, Japan. N,N,N',N'-Tetramethylethylenediamine ( ) was from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Glucose-6- phosphate, glucose- l -phosphate, and 6-phosphogluconate were products of Oriental Yeast Co., Ltd., Tokyo, Japan. RESULTS AND DISCUSSION Table 1 shows the Co-Q system of strains of species in the genera Schizosaccharomyces, Octosporomyces, and Hasegawaea. The data confirmed the results obtained previously (8). The genus Octosporomyces was distinguished from the genus Schizosaccharomyces by the Q-9 system. The genus Hasegawaea was characterized by the absence of Co-Q. One of the authors (Y. Yamada) reported that S. japonicus SHF 1 (= IFO 0341) had the Q-9 system (8). However, the strain IFO has been reidentified as 0. octosporus (I. Banno, unpublished data). The strain was probably mislabeled. The Rm's of five enzymes are shown in Table 2. In the examined fourteen strains of Schizosaccharomyces, Octosporomyces, and Hasegawaea species, the Rm's of glucose-6-phosphate dehydrogenase varied from 0.22 to The lower Rm of 0.22 appeared to be characteristic of the 0. octosporus strains. The Rm's of 6- phosphogluconate dehydrogenase were from 0.25 to The strains of S. pombe and S. malidevorans had a lower Rm at 0.25, though one of the strains showed no enzyme band. The type strain of 0. octosporus (CBS 371) had a high Rm (0.72). The Rm of hexokinase was higher in the 0. octosporus strains (0.53). Others were from 0.34 to Phosphoglucomutase revealed two or three isozyme bands (Rm's, 0.41 to 0.48). However, the 0. octosporus strains had a single band (Rm, 0.30). The bands Z The Rm's were: 0.22 in glucose-6-phosphate dehydrogenase, 0.39 in 6-phosphogluconate dehydrogenase, 0.53 in hexokinase, 0.30 in phosphoglucomutase, and 0.32 and 0.34 in fumarase. The calculated similarity value was 80% between the strain and 0. octosporus CBS 371 (type strain).

4 366 YAMADA, AIZAWA, MATSUMOTO, NAKAGAWA, and BANNO VOL. 33 Table 1. The strains examined of species in the genera Schizosaccharomyces, and Hasegawaea and the Co-Q system of the strains. Octosporomyces, of fumarase appeared from Rm 0.29 to H. japonica, including the two varieties, showed higher Rm's at 0.46 to These data are summarized in Fig. 1 as a triangle matrix designed to emphasize similarity based on the electrophoretic patterns of the five enzymes. The dendrogram was drawn based on the calculated similarity values (Fig. 2). Two strains of 0. octosporus had a uniform electrophoretic enzyme pattern (s=100%). The type strain (CBS 371=IF ) was linked to the two strains with a similarity value of 80%. All strains of S. pombe examined were linked to each other with a similarity value of 40 c or more. Two of the six strains had a uniform electrophoretic enzyme

5 1987 Enzyme Patterns in Fission Yeast Genera 367 Table 2. The electrophoretic mobilities of five enzymes in strains of Schizosaccharomyces, Octosporomyces, and Hasegawaea species. Fig. 1. The triangle matrix based on the calculated similarity values of five enzymes in strains of Schizosaccharomyces, Octosporomyces, and Hasegawaea species. The three species, S. pombe, 0. octosporus, and H. japonica constitute their own clusters separately. Schizosaccharomyces malidevorans is synonymous with S. pombe. * Type strain.

6 368 YAMADA, AIZAWA, MATSUMOTO, NAKAGAWA, and BANNO VOL. 33 Fig. 2. The dendrogram based on the calculated similarity values of five enzymes in strains of Schizosaccharomyces, Octosporomyces, and Hasegawaea species. The dendrogram is drawn by the complete linkage method (13). Considering the present data in connection with the previous ones (11, 12), it can be concluded that a couple of strains showing a similarity value of 40% or more are accommodated in the same species. * Type strain. pattern. It is noted that the type strain of S. malidevorans (IFO 1608 =CBS 5557) was closely related to that of S. pombe (IFO 1628=CBS 356). The similarity value between the two type strains was 60%. According to The Yeasts, a Taxonomic Study, 3rd ed. (5), S. malidevorans is discriminated from S. pombe by inability to ferment maltose. This indicates that S, malidevorans is synonymous with S. pombe. All the strains of H. japonica var. japonica examined gave a similarity value of Between the two varieties, H. japonica var. japonica and H, japonica var. versatilis, the similarity value was 60%. The only differences are phenotypically in the shape and iodine reaction of the mature ascospores (5). The fission yeast strains examined comprise three separate large clusters: the genus Schizosaccharomyces including those of S. pombe, the genus Octosporomyces including those of 0, octosporus, and the genus Hasegawaea including those of H. japonica var. japonica and H, japonica var. versatilis. The similarity values were all 0% among the three large clusters (Fig. 2). In this study, differing from the previous studies (11, 12), the authors examined only five enzymes, all of which are situated on the main pathways for carbohydrate catabolism. The results obtained above confirmed the usefulness of the electrophoretic enzyme patterns for classifying microorganisms below the generic level, especially at the specific level (Fig. 2). The authors thank Dr. D. Yarrow, Yeast Division, Centraalbureau voor Schimmelcultures, Delft, The Netherlands for sending them a yeast culture. This work was supported by a Grant-in-Aid for Cooperative Research from the Ministry of Education, Science and Culture of Japan (No ).

7 1987 Enzyme Patterns in Fission Yeast Genera 369 REFERENCES 1) P. LINDNER, Wochenschr. Brau., 10, 1298 (1893). 2) A. KLOCKER, In Handbuch der Technischen Mykologie, ed. by F. LAFAR, Fischer, Jena (1907), p ) W. CH SLOOFF, In The Yeasts: a Taxonomic Study, 2nd ed., ed. by J. LODDER, The North Holland Publishing Co., Amsterdam (1970), p ) N. J. W. KREGER-VAN RIJ, In The Yeasts: a Taxonomic Study, 3rd ed., ed. by N. J. W. KREGER-VAN Ru, Elsevier Publishers, Amsterdam (1984), p. 2. 5) D. YARROW, In The Yeasts: a Taxonomic Study, 3rd ed., ed. by N. J. W. KREGER-VAN RIJ, Elsevier Publishers, Amsterdam (1984), p. 414, 6) Y. YAMAHA and I. BANNO, J. Gen. App!. Microbio!., 33, 295 (1987). 7) M. MIKATA and I. BANNO, IFO Res. Commun., 13, 45 (1987). 8) Y. YAMAHA, M. ARIMGTO, and K. KONDO, J. Gen. App!. Microbio!., 19, 353 (1973). 9) J. L. F. KoCK and J. P. VAN DER WALT, Syst. App!. Microbiol., 8, 163 (1986). 10) M. YAMAZAKI, In Biseibutsu no Kagakubunrui Jikkenho, ed. by K. KOMAGATA, Gakkai Shuppan Center, Tokyo (1982), p ) Y. YAMAHA, M. WATANABE, M. AKITA, and I. BANNO, J. Gen. App!. Microbio!., 32, 157 (1986). 12) Y. YAMAHA, K. AIZAWA, and I. BANNO, J. Gen. App!. Microbio!., 32, 367 (1986). 13) T. KANEKO, In Biseibutsu no Kagakubunrui Jikkenho, ed. by K. KOMAGATA, Gakkai Shuppan Center, Tokyo (1982), p. 309.