The Patterns Produced by Nine Enzymes of Ballistosporogenous Yeasts and Supposedly Related Yeasts Were Studied by Electrophoresis on Poly- Acrylamide Gel

J. Gen. App!. Microbiol., 29, 115-143 (1983)

AN ELECTROPHORETIC COMPARISON OF ENZYMES OF BALLISTOSPOROGENOUS YEASTS

MASASHI YAMAZAKI AND KAZUO KOMAGATA

The Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

(Received February 1, 1983)

The patterns produced by nine enzymes of ballistosporogenous yeasts and supposedly related yeasts were studied by electrophoresis on polyacrylamide gel. Forty-two strains belonged to the genera Sporobolomyces, Sporidiobolus, Aessosporon and Bullera and twenty-four strains to Rhodo- torula, Rhodosporidium, Cryptococcus, unidentified ballistosporogenous yeasts, and Candida edax. Four Sporobolomyces salmonicolor strains, two Sporobolomyces holsaticus strains, two Sporobolomyces odorus strains, five Sporidiobolus salmonicolor strains, two Aessosporon salmonicolor strains, and one strain of Aessosporon dendrophilum produced similar electrophoretic patterns. Moreover, mating was observed between some of these strains. Sporo- bolomyces roseus and Sporobolomyces shibatanus differed from Sporobolo- myces salmonicolor in their glucose-6-phosphate dehydrogenase (EC 1.1.1.49.), but the patterns of the other enzymes were similar; all three species differed clearly from Sporobolomyces singularis, Sporobolomyces gracilis, Sporobolomyces puniceus, and Sporobolomyces antarcticus. Sporobolomyces albo-rubescens showed a peculiar 6-phosphogluconate dehydrogenase (EC 1.1.1.41.) pattern and was similar to two Rhodotorula rubra strains in the pattern of their enzymes. Close relationships were also seen between Sporidiobolus ruinenii and Rhodotorula graminis, and between Bullera alba and a strain in Cryptococcus albidus var. albidus in the electrophoretic patterns of their enzymes. Four unidentified strains which had lost the ability to produce ballistospores had patterns similar to those of Bullera alba, Rhodotorula glutinis, Cryptococcus laurentii var. flavescens, and Cryptococcus macerans. Three colorless strains, putatively derived from a strain of Sporobolomyces roseus, showed the same electrophoretic patterns as the strains from which they originated.

Ballistosporogenous yeasts differ from other yeasts in producing ballistospores

Address reprint requests to: Dr. M. Yamazaki, The Institute of Enology and Viticulture, Yamanashi University, 13-1, Kitashin, 1-chome, Kofu 400, Japan.

115 116 YAMAZAKI and KOMAGATA VOL. 29 that are discharged at maturity by a drop-excretion mechanism. Three genera are described in "The Yeasts" (1): Bullera, Sporobolomyces, and Sporidiobolus. In 1970, VANDER WALT(2) found a homothallic sexual cycle in Sporobolomyces salmonicolor and proposed a new genus Aessosporon for it. FELLand TALLMAN(3) reported matings between strains of Aessosporon salmonicolor and some Sporo- bolomyces species. Thereafter, the sexual species Sporidiobolus salmonicolor and Sporidiobolus pararoseus were described as heterothallic species in the genus Sporidiobolus by FELL and TALLMAN(4). A close relationship between ballistosporogenous yeasts and some species of the genera Rhodotorula and Cryptococcus has been suggested by various biochemical studies (5-14). In previous papers the present authors reported that electrophoretic comparison of enzymes was a useful tool to clarify taxonomical relationships among strains of the genera Rhodotorula and Rhodosporidium (15), among strains of the genus Cryptococcus and related microorganisms (16), and between asporogenous yeasts and their supposed ascosporogenous states (17). The present paper deals with the electrophoretic comparison of nine enzymes of the genera Sporobolomyces, Sporidiobolus, Aessosporon, and Bullera, and discuss- es the relationship between ballistosporogenous yeasts and some strains of species of Rhodotorula, Rhodosporidium, and Cryptococcus.

MATERIALSAND METHODS Yeast strains. The strains used and their sources are listed in Table 1. The identities of all of them were confirmed by the methods of "The Yeasts" (1) and BARNETTet al. (18). Cultivating and harvesting cells, preparation of cellfree extracts, and gel-slab electrophoresis. Methods for cultivating and harvesting cells, preparing cell-free extracts, and gel-slab electrophoresis were essentially the same as those reported previously (15). Staining procedures. The enzymes studied were fructose-l,6-bisphosphate aldolase (FA; EC 4.1.2.13.), 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.41.), malate dehydrogenase (MDH; EC 1.1.1.37.), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49.), glutamate dehydrogenase (GDH; EC 1.4.1.4.), hexokinase (HK; EC 2.7.1.1.), fumarase (Fina; EC 4.2.1.2.), phospho- glucomutase (PGM; EC 2.7.5.1.), and catalase (Cat; EC 1.11.1.6.). The staining procedures used for catalase were described by GREGORYand FRIDOVIcH(19) and for the other enzymes by SIcILIANOand SHAW(20). Figure 1 shows an example of a slab of polyacrylamide gel stained for seven enzymes of some ballistosporogenous yeasts. The relative mobilities (Rm) of the enzyme bands were calculated as the ratio of the distance that the enzyme moved from the origin to the distance that the tracking dye (Bromophenol Blue) moved. Mating tests. Mating tests were made by mixing a loopful of cells of each of a 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 117

Fig. 1. Polyacrylamide gel stained for FA, 6PGDH, GDH, MDH, Fina, HK, and PGM. Lane 1 and 2 (FA): 1, Sporidiobolus ruinenii YK 456; 2, Sporobolomyces albo- rubescens YK 421. Lane 3-5 (6PGDH): 3, Sporidiobolus johnsonii YK 457; 4, Spori- diobolus salmonicolor YK 400; 5, Sporidiobolus pararoseus YK 415. Lane 6-8 (GDH) : 6, Bullera alba YK 460; 7, B, alba YK 461; 8, Sporobolomyces albo-rubescens YK 422. Lane 9-11(MDH) : 9, Bullera alba YK 461;10, B. piricola YK 464;11, B, tsugae YK 465. Lane 12-18 (Fma) :12, Sporobolomyces roseus YK 418;13, S. roseus YK 419;14, S. roseus YK 420; 15, S, odorus YK 410; 16, S. odorus YK 411; 17, Sporidiobolus salmonicolor YK 412;18, Sporobolomyces odorus YK 413. Lane 19-21(HK): 19, Sporobolomyces salmonicolor YK 403; 20, Aessosporon salmonicolor YK 451; 21, Sporidiobolus salmonicolor YK 407. Lane 22-25 (PGM) : 22, Bullera alba YK 460; 23, B. alba YK 461; 24, B. alba YK 462; 25, Unidentified YK 482. given pair on corn-meal agar plates. The plates were incubated for 1-2 weeks at 18°, and the growth of the mixed cells was inspected at intervals with the naked eye and microscopically. Teliospore germination followed the standard method (1).

RESULTS Assimilation patterns of carbon compounds and potassium nitrate by strains used Table 2 shows the assimilation of thirty-two carbon compounds and potassium nitrate by the main ballistosporogenous yeasts and the putatively related yeasts. The assimilation patterns of these strains tested agreed well with those given in "The Yeasts" (1) and BARNETTet al. (18). Two strains, YK 416 and YK 417, are identified as Sporobolomyces pararoseus by the system of "The Yeasts" (1), but we called them Sporobolomyces shibatanus because FELL and TALLMAN(4) have changed the name. Four unidentified strains, YK 481, YK 482, YK 484, and YK 485, had lost the ability to produce ballistospores during preservation. The assimilation pattern of strain YK 481 was closest to that of Sporobolomyces roseus and Rhodotorula glutinis. Two strains, YK 482 and YK 484, closely resembled 118 YAMAZAKI and KOMAGATA VOL. 29 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 119 120 YAMAZAKI and KOMAGATA VOL. 29

Table 2. Assimilation of carbon compounds and potassium nitrate

Bullera alba and Cryptococcus laurentii in their assimilation patterns. The strain YK 485 and Cryptococcus macerans had similar assimilation patterns. Three colorless strains, YK 486, YK 487, and YK 488, showed the same assimilation patterns as Sporobolomyces roseus.

Electrophoretic comparison of enzymes in the genus Sporobolomyces The relative electrophoretic mobilities (Rm) of nine enzymes from twenty- three strains of eleven Sporobolomyces species and one strain of Candida edax are 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 121

shown in Table 3. Sporobolomyces holsaticus YK 401 and YK 402 produced identical patterns for nine enzymes. Sporobolomyces salmonicolor YK 403, YK 404, YK 405, and YK 406 exhibited identical patterns in FA, 6PGDH, Fina, HK, and GDH . There were two types of G6PDH : one was detected at Rm 0.23 (YK 403 and YK 404), the other at Rm 0.20 (YK 405 and YK 406). The patterns of MDH and Cat showed slight variations among the four strains. In addition , the PGM of YK 404 differed from the other three strains. The four Sporobolomyces odorus strains 122 YAMAZAKI and KOMAGATA VOL. 29

tested split into two pairs in their electrophoretic patterns. The first pair (YK 410 and YK 411) differed from the second (YK 413 and YK 414) in their 6PGDH, Fina, MDH, HK, PGM, GDH, and Cat patterns. No FA activity was detected in the second pair. Of the three Sporobolomyces roseus strains tested, YK 419 and YK 420 produced identical patterns for all nine enzymes. The other strain YK 418 resembled them with respect to its 6PGDH, Fma, G6PDH, and GDH pattern but not MDH, HK, PGM, and Cat. Sporobolomyces shibatanus YK 416 and YK 417 were identical in the 6PGDH, Fma, MDH (Rm 0.33), G6PDH, HK, GDH, and Cat enzymes but not in PGM ; no FA activity was detected in either of them. Sporobolomyces albo-rubescens YK 421 and YK 422 had identical patterns 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 123

for all nine enzymes. They had a 6PGDH with three bands, at Rm 0.38, 0.40, and 0.42. Sporobolomyces gracilis YK 408 and YK 409 showed identical patterns for 6PGDH, Fina, MDH, G6PDH, HK, PGM, GDH, and Cat: no activity of FA was detected in either of them. Sporobolomyces hispanicus YK 424 had a peculiar 6PGDH which was not detected in other Sporobolomyces species. Sporobolomyces antarcticus YK 425, Sporobolomyces puniceus YK 426, and Sporobolomyces singularis YK 427 each had a peculiar FA which differed from other Sporobolomyces species. In contrast with the species of Rhodotorula (I5) and Cryptococcus (16) each species of Sporobolomyces has, in general, a uniform pattern. 124 YAMAZAKI and KOMAGATA VOL. 29 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 125 126 YAMAZAKI and KOMAGATA VOL. 29

Electrophoretic comparison of enzymes in the genera Aessosporon and Sporidiobolus The relative electrophoretic mobilities (Rm) of the nine enzymes from strains of two Aessosporon and four Sporidiobolus species are shown in Table 4. Aessosporon salmonicolor YK 451 and YK 452 produced identical patterns for all nine enzymes. Aessosporon dendrophilum YK 454 and YK 455 exhibited different patterns in 6PGDH, MDH, HK, and GDH but identical ones for Fina and G6PDH. A. dendrophilum YK 454 was similar to A, salmonicolor YK 451 and YK 452 with respect to 6PGDH, Fma, MDH, G6PDH, HK, PGM (Rm 0.41-0.42), GDH, and Cat. A. dendrophilum YK 455 and the two A. salmonicolor strains showed different patterns in FA, 6PGDH, MDH, HK, and GDH but similar ones for Fma and G6PDH. Sporidiobolus johnsonii YK 457 and YK 458 produced identical patterns for all nine enzymes. Five Sporidiobolus salmonicolor strains, YK 400, YK 407, YK 412, YK 423, and YK 453, produced similar patterns for FA, 6PGDH, Fma, MDH, HK, GDH, and Cat but differed for PGM. The strain YK 400 and YK

Table 4. Comparison of electrophoretic Rm values of nine enzymes from strains of Aessosporon and Sporidiobolus. 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 127

412 differed from the other three strains in their G6PDH. Sporidiobolus salmonicolor YK 400 and two Sporidiobolus johnsonii strains showed similar patterns except for MDH. Sporidiobolus ruinenii YK 456 and the two Sporidiobolus johnsonii strains showed similar patterns with respect to 6PGDH and Fina and slight variations in their patterns for FA, MDH, HK, PGM, GDH, and Cat, but quite different ones for G6PDH. Moreover, the strain YK 456 differed from five Sporidiobolus salmonicolor strains in its G6PDH and GDH but its pattern for FA, 6PGDH, Fma, PGM, and Cat was similar. Sporidiobolus pararoseus YK 415 differed from the five Sporidiobolus salmonicolor strains in its G6PDH and MDH, rfom Sporidio- bolus ruinenii YK 456 in its G6PDH, HK, and GDH, and from the two Sporidio- bolus johnsonii strains in its FA, MDH, and G6PDH. The five Sporidiobolus salmonicolor strains and the two Aessosporon salmonicolor strains showed similar patterns for all nine enzymes. Thus, with the exception of A. dendrophilum YK 455, there was good agree- ment in the patterns, both within each species of Aessosporon and Sporidiobolus as well as within the genera themselves.

Electrophoretic comparison of enzymes in the genus Bullera The relative electrophoretic mobilities (Rm) of nine enzymes from strains of three Bullera species are shown in Table 5. Bullera alba YK 460. YK 461, YK 462, and YK 463 produced identical patterns for FA, Fma, MDH, G6PDH, GDH, and Cat; no activity of 6PGDH was detected in any of them. The strain YK 463 differed from the other three strains in its PGM. No activity of HK was detected in YK 463; moreover, the HK of YK 462 differed from those of YK 460 and YK 461. No activity of FA, 6PGDH, and Cat was detected in Bullera piricola YK 464, which was similar to the four B. alba strains with respect to its G6PDH and GDH but not in its Fma, MDH, HK, and PGM. Bullera tsugae YK 465 showed a pattern different from all four B.

Table 5. Comparison of electrophoretic Rm values of nine enzymes from strains of the genus Bullera. 128 YAMAZAKI and KOMAGATA VOL. 29 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 129

Fig. 2. Hyphae, clamps, and teliospores formed from a mating between strains showing similar electrophoretic patterns of enzymes. A, Sporobolomyces holsaticus YK 401 and Sporobolomyces salmonicolor YK 403; B, Sporobolomyces holsaticus YK 402 and Sporobolomyces odorus YK 414. Bar =10 ym alha strains in Fina, MDH, G6PDH, HK, and Cat, but not FA, PGM, and GDH. The strain YK 465 and B. piricola YK 464 differed in Fma, MDH, G6PDH, and PGM, but not for HK and GDH. Thus, three Bullera species were distinguishable from one another by the electrophoretic patterns of their enzymes.

Mating tests between strains showing similar electrophoretic patterns of enzymes From the results in the previous study (15), it was expected that ballistosporogenous yeasts would be able to mate with other strains that showed similar electrophoretic patterns of enzymes. Table 6 shows the combinations of strains tested and the results obtained. Conjugations between cells, mycelial development with clamp connections, and formation of spherical teliospores were observed in crosses between five strains {S. holsaticus YK 401 (Fig. 2A), YK 402 (Fig. 2B), S. salmonicolor YK 405, YK 406, and Sporidiobolus salmonicolor YK 412} and seven other strains {S. salmonicolor YK 403 (Fig. 2A), YK 404, S. odorus YK 414 (Fig. 2B), A. salmonicolor YK 451, YK 452, Sporidiobolus salmonicolor YK 423, and YK 130 YAMAZAKI and KOMAGATA VOL. 29

453}. On the other hand, no mating was observed between four strains (S. odorus YK 413, A. dendrophilum YK 454, Sporidiobolus salmonicolor YK 400, and YK 407) and the above twelve strains.

DISCUSSION

Relationships among ballistosporogenous yeasts in the electrophoretic patterns of their enzymes S, salmonicolor, S. holsaticus, and S. hispanicus are nitrate-positive and hyphae- forming species. S. hispanicus differs from S. salmonicolor in its inability to assimilate sucrose and raffinose, and S. holsaticus from S. salmonicolor in its ability to assimilate maltose, melezitose, and a-methyl-D-glucoside, though their mor- phological and other physiological properties are similar (1). VANDER WALT(2) described a homothallic sexual cycle in S. salmonicolor in which a diploid teliospores was formed by fusion of haploid mother and daughter cells, and he proposed a new genus Aessosporon for it. BANDONIet al. (21) found mating reactions between the type strains of S. hispanicus, S. odorus, and S. salmonicolor. Conjugation between compatible strains was followed by formation of a mycelium with clamp connections and chlamydospores, but the teliospores did not germinate. Therefore, they concluded that these species were conspecific and that the oldest name, S. salmonicolor, should be accepted. FELL and TALLMAN(3) reported mating between strains of A. salmonicolor, S. odorus, S. holsaticus, S. salmonicolor var. fischerii, and S. hispanicus, though no germination of the teliospore was observed. Moreover, FELLand TALLMAN(3) stated that A. salmonicolor belongs to the same heterothallic sexual species as S. salmonicolor. Thereafter, they described the heterothallic sexual species Sporidiobolus salmonicolor and Sporidiobolus pararoseus (4). In this study, two strains of S. holsaticus, four strains of S. salmonicolor, two strains of S. odorus group 2, two strains of A. salmonicolor, A. dendrophilum YK 454, and five strains of Sporidiobolus salmonicolor produced similar electrophoretic enzyme patterns. Sporidiobolus salmonicolor YK 400, YK 407, YK 412, YK 423, and YK 453 were once classified as Sporobolomyces holsaticus, S. salmonicolor var. fischerii, S. odorus, S. hispanicus, and A. salmonicolor, respectively, and they were described as compatible mating strains of Sporidiobolus salmonicolor by FELL and TALLMAN(4). The enzymes of Sporidiobolus salmonicolor YK 423 and Sporobolo- myces hispanicus YK 424 clearly differed, though the assimilation of carbon compounds and potassium nitrate were identical (Table 2). S. hispanicus YK 424, therefore, is a species distinct from Sporidiobolus salmonicolor YK 423. Twelve of the above sixteen strains mated as shown in Table 6. The shape of teliospores was the same as described by FELL and TALLMAN(4). Germination of these teliospores is still under investigation. However, these results indicate that five strains (S. salmonicolor YK 403, YK 404, S. odorus YK 414, A. salmonicolor 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 131

YK 451, and YK 452) are the Al mating type of Sporidiobolus salmonicolor, and four strains (S. holsaticus YK 401, YK 402, S. salmonicolor YK 405, and YK 406) are the A2 mating type. These results also clearly reveal the inadequacy of delimit- ing species by the assimilation of carbon compounds only. Hereafter in this study, we designate S. salmonicolor, S. holsaticus, and S. odorus group 2 as the S. salmonicolor group. Thus, nine haploid mating strains of Sporidiobolus salmonicolor were found among the strains of S. salmonicolor, S. holsaticus, S. odorus, and A. salmonicolor, as a result of mating tests suggested by electrophoretic comparison of their enzymes. However, four strains (S. odorus YK 413, A. dendrophilum YK 454, Sporidiobolus salmonicolor YK 400, and YK 407) failed to mate with any strains with similar electrophoretic enzyme patterns. Moreover, four strains (A. salmonicolor YK 451, YK 452, Sporidiobolus salmonicolor YK 412, and YK 453) also failed to mate with most of the strains tested. Such failures might be ascribed to an un- satisfactory medium or other cultural conditions. In addition to that, it is likely that the mating capacity of these strains has degenerated. S. odorus group 1 and group 2 differed in their enzymes patterns, though their patterns of assimilation of carbon compounds and potassium nitrate were similar (Table 2). As shown above, the electrophoretic patterns of S. odorus group 2 were similar to those of S. salmonicolor and S. holsaticus, and in fact, the strain YK 414 mated with them (Table 6). Two strains of S. odorus group 2, YK 413 and YK 414, were found to be compatible mating strains by BANDONIet al. (22). More- over, the enzymes of Sporidiobolus salmonicolor YK 412, which was once classified as Sporobolomyces odorus, were similar to those of the two strains of S. odorus YK 413 and YK 414. Therefore, S. odorus group 1 and group 2 are thought to represent two distinct species. S. roseus differed from the S. salmonicolor group in its G6PDH, MDH, and GDH pattern, but the patterns of the other enzymes were similar. According to STORCKet al. (9), the GEC contents of the DNAs of S. roseus and S. salmonicolor differed by about 10 mol %, however, the strains used in this study were not the same as those used by them. S. roseus also differed from S. shibatanus in its MDH, G6PDH, GDH, and Cat pattern, and from S. albo-rubescens in its FA, 6PGDH, GDH, and Cat pattern. Thus, S. roseus is a species distinct from the S. salmonicolor group, S. shibatanus, and S. albo-rubescens according to the electrophoretic patterns of their enzymes. FELLand TALLMAN(4) corrected the name of the species known under the il- legitimate epithet "pararoseus" to Sporobolomyces shibatanus. S. shibatanus differed from the S. salmonicolor group in its G6PDH, but the patterns of their other enzymes were similar. According to STORCKet al. (9), the GEC contents of the DNAs of S. pararoseus ranged from 51.5 to 60 mol %. Moreover, S. pararoseus differed from S. salmonicolor and S. odorus in its antigens (S). As far as this study is concerned, all the strains of S. shibatanus including Sporidiobolus pararoseus were similar, in addition, they seem to have a close relationship with the S. 132 YAMAZAKI and KOMAGATA VOL. 29 salmonicolor group. The DNA of S. albo-rubescens has the same GEC content as those of S. holsaticus and S. salmonicolor (9). S. albo-rubescens had a peculiar 6PGDH, in addition, it certainly differed from S. salmonicolor group in the pattern of G6PDH and GDH. Consequently, S. albo-rubescens seems to differ phylogenetically from the S. salmonicolor group. S. gracilis differed from the S. salmonicolor group in its pattern for 6PGDH, Fina, MDH, G6PDH, HK, PGM, and GDH, and in addition, it differed from other Sporobolomyces species in its pattern for most enzymes. The ballistospores of S. gracilis are described as nearly symmetrical, though the genus Sporobolomyces is defined as forming assymmetrical ballistospores (1). According to TSUCHIYA et al. (5), S. gracilis differed from S. salmonicolor, S. odorus, and S. pararoseus (=S. shibatanus) antigenetically. From these results, S. gracilis is thought to differ phylogenetically from the S. salmonicolor group and other Sporobolomyces species. S. singularis is included in the genus Sporobolomyces because of the formation of asymmetrical ballistospores, though it is not pigmented (1). S. singularis YK 427 differed from other Sporobolomyces species except S. antarcticus and S. puniceus in the assimilation of lactose, 2-keto-gluconate and 5-keto-gluconate (Table 2). These characters were similar to those encountered in the genus Bullera. S. singularis YK 427 differed from the S. salmonicolor group in its pattern for FA, 6PGDH, G6PDH, HK, PGM, and GDH; and, in addition, it differed from other Sporobolo- myces species in most of its enzyme patterns. S. puniceus was isolated by NAKASE and KoMAGATA(23) and described by them as Candida punicea. The formation of ballistospores was reported by AHEARNet al. (24) and YARROW(25), therefore, it was reclassified in the genus Sporobolomyces (26). STADELMANN(27) speculated that S. puniceus and S. singularis should be transferred from the genus Sporobolo- myces to the genus Bullera considering their assimilation patterns of carbon compounds. Moreover, FIASON(28) stated that the pale pink color of S. puniceus was not due to carotenoids and suggested that this species might be included in the genus Bullera. In this study, S. puniceus YK 426 differed from the S. salmonicolor group in the pattern of FA, Fma, G6PDH, PGM, and GDH, in addition, it differed from other Sporobolomyces species in the pattern of most enzymes. As described above, S. singularis and S. puniceus occupy a peculiar position in the genus Sporobolomyces. It is obvious that further investigations are required to determine whether they be- long to the genus Sporobolomyces or the genus Bullera. S. antarcticus was isolated from sediment of Lake Vanda in Antarctica and described as a new species of the genus Sporobolomyces by GoTO et al. (29). S. antarcticus YK 425 produced a peculiar pattern for FA and 6PGDH which was dis- similar to those of other Sporobolomyces species. According to STADELMANN(27), this species is probably identical with Candida edax. In this study, no activity of FA, 6PGDH, and Cat was detected in C. edax YK 513, and these two strains differed in the pattern of Fma, MDH, G6PDH, HK, and PGM. Therefore, we be- 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 133 lieve that S. antarcticus is a species distinct from C. edax. S. hispanicus YK 424 was similar to A. dendrophilum YK 455 with respect to its 6PGDH, Fina, MDH, G6PDH, and GDH pattern. However, mating was not observed between YK 424 and YK 455. A. dendrophilum was isolated by VAN DER WALT and SCOTT(30) and described as a new species of the genus Bullera, namely, B. dendrophila. Thereafter, its perfect stage was assigned to the genus Aessosporon because of its sexual characteristics by VANDER WALT(31). Of the two A. dendrophilum strains tested, YK 454 which was derived from the type strain was similar to the S. salmonicolor group as shown above. However, no mating was observed between YK 454 and strains having similar electrophoretic patterns. A. dendrophilum YK 454 and YK 455 showed identical assimilation patterns for carbon compounds and potassium nitrate, and they clearly differed from S. hispanicus YK 424 and the S. salmonicolor in their assimilation patterns (Table 2). Con- sequently, further investigations will be required to clarify the relationships between S. hispanicus YK 424 and A. dendrophilum YK 455, and between A. dendrophilum YK 454 and the S, salmonicolor group. Sporidiobolus yeasts differ from Sporobolomyces yeasts only by the presence of the dikaryotic mycelium with clamps and teliospores. PANDONIet al. (22) reported that the dikaryotic phase and chlamydospore germination of S. odorus (=group 2) resembled those of Sporidiobolus johnsonii. The enzymes of these two species have similar electrophoretic patterns except for MDH and G6PDH, and their assimilation patterns of carbon compounds and potassium nitrate are also similar. Moreover, Sporidiobolus johnsonii YK 457 was similar to the S. salmonicolor group with respect to its FA, 6PGDH, Fma, G6PDH, HK, GDH, and Cat pattern Sporidiobolusjohnsonii and the S. salmonicolor group exhibited similar G+C contents of DNAs (9, 32). As far as this study is concerned, Sporidio- bolus johnsonii seems to be closely related to the S. salmonicolor group. Sporidiobolus ruinenii YK 456 and Sporidiobolus johnsonii YK 457 showed slight variations in the patterns of their enzymes. According to HoLZSCHUet al. (32), these two species differed by 1.2 mol % G+C in the base composition of their nuclear DNAs and shared only about 8.5 % of their nuclear DNA base sequences. Therefore, Sporidiobolus ruinenii seems to be a species distinct from Sporidiobolus johnsonii. Sporidiobolus pararoseus YK 415 was once classified as Sporobolomycespararo- seus, but it was found to be a haploid heterothallic sexual strain and was described as a mating type A 1 of Sporidiobolus pararoseus by FELL and TALLMAN(4). This strain YK 415 was similar to Sporobolomyces shibatanus YK 416 and YK 417 with respect to its pattern for 6PGDH, Fma, MDH, G6PDH, PGM, GDH, and Cat. However, no mating between YK 415 and two strains YK 416 and YK 417 was observed. A mating test between strains of mating type A2 and strains YK 416 and YK 417 is required. The genus Bullera is considered to be closely related to the genus Sporobolo- 134 YAMAZAKI and KOMAGATA VOL. 29 myces but is distinguished from it by the formation of symmetrical ballistospores. However, STADELMANN(27) described a new species Bullera piricola forming asymmetrical as well as symmetrical ballistospores. The Bullera species differed from the Sporobolomyces species in the monosaccharide components of their hydrolyzed whole cells (14). The three Bullera species were not similar to any of the Sporobolo- myces yeasts tested in the electrophoretic patterns of their enzymes. As far as this study is concerned, the three Bullera species seem to have little relationship to the Sporobolomyces species.

Electrophoretic comparison of enzymes between strains of Sporobolomyces roseus and their supposed colorless variants The relative electrophoretic mobilities (Rm) of eight enzymes from three S. roseus strains and their supposed colorless variants are shown in Table 7. When the three strains YK 418, YK 419 and YK 420 which were identified as S. roseus were purified on plates, colorless colonies, which we numbered YK 486, YK 487, and YK 488, appeared spontaneously on each plate. These three strains (YK 486, YK 487, and YK 488) formed asymmetrical ballistospores, and were also similar to S. roseus YK 418, YK 419, and YK 420, respectively, in their assimilation of carbon compounds and potassium nitrate (Table 2). The unidentified YK 486 and S. roseus YK 418 produced identical patterns for 6PGDH, Fina, MDH (Rm 0.34), G6PDH, PGM, and GDH, and they showed a slight variation for HK. No activity of MDH (Rm 0.27) or Cat was detected in YK 486. The unidentified YK 487 and S. roseus YK 419 exhibited identical patterns in 6PGDH, Fma, MDH (Rm 0.40), G6PDH, HK, PGM, and GDH. No activity of MDH (Rm 0.31) or Cat was detected in YK 487. The unidentified YK 488 showed the same electrophoretic pattern as S. roseus YK 420 in 6PGDH, Fma, MDH, G6PDH, HK, PGM, and GDH. No activity of Cat was detected in

Table 7. Comparison of electrophoretic Rm values of eight enzymes between Sporobolomyces roseus strains and their colorless putative variants. 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 135

YK 488. Thus, no activity of Cat was detected in any of the three colorless strains, but the patterns of the other enzymes were very like those of the respective S. roseus strains. From these results, we assume that YK 486, YK 487, and YK 488 are colorless variants each of which evolved from S. roseus YK 418, YK 419, and YK 420, respectively. As shown above, electrophoretic comparison of enzymes is also thought to be one of the useful tools for identifying color variants.

Relationships between ballistosporogenous yeasts and supposedly related yeasts in the electrophoretic patterns of their enzymes A close relationship between ballistosporogenous yeasts and the genera Rhodotorula and Cryptococcus has been suggested by their antigenic structures (5), cell-wall structures (6,12), enzyme associations in tryptophan synthesis (7), G+C contents of DNAs (8, 9), proton-magnetic-resonance spectra of cell-wall mannans and structures of their polysaccharides (10), a formation of rhodotorulic acid (11), coenzyme Q systems (13), and monosaccharide patterns of hydrolyzed whole cells (14). Therefore, the genera Rhodotorula and Cryptococcus have been suspected of containing ballistosporogenous yeasts that had lost the ability to produce ballistospores. The relative electrophoretic mobilities (Rm) of nine enzymes from four unidentified strains, YK 481, YK 482, YK 484, and YK 485, some ballistosporogenous yeasts and supposedly related yeasts are shown in Table 8. These four strains had lost the ability to produce ballistospores after prolonged maintenance in culture.

Sporobolomvicesand supposedly related yeasts According to Pxusso and WELLS(6), the structure of the cell wall and the mode of budding in S. roseus are the same as those in Rhodotorula glutinis and R. rubra. The unidentified YK 481, S. roseus YK 419; and R. glutinis YK 106 showed similar assimilation patterns for carbon compounds and potassium nitrate (Table 2). The strain YK 481 and S. roseus YK 419 produced similar patterns for 6PGDH, G6PDH, Fina, HK (Rm 0.44-0.45), and Cat but not for FA, MDH, PGM, and GDH. On the other hand, the strain YK 481 and R. glutinis YK 106 exhibited similar patterns in FA, 6PGDH, G6PDH, HK (Rm 0.45-0.46), MDH (Rm 0.42), and GDH, but not in PGM. Thus, the strain YK 481 is related to R. glutinis YK 106 rather than S. roseus YK 419 according to the electrophoretic patterns of their enzymes. However, the strain YK 481 did not mate with R. glutinis YK 106. HuTTERand DE Moss (7) indicated that S. salmonicolor was similar to R. glutinis with respect to their enzyme associations in tryptophan synthesis. S. salmonicolor YK 404 and R. glutinis var. dairenensis YK 116 produced similar patterns for FA, 6PGDH, MDH, and HK (Rm 0.45), but not for G6PDH, PGM, and GDH. S. salmonicolor YK 404 differed from R. glutinis YK 116 in its inability to assimilate 136 YAMAZAKI and KOMAGATA VOL. 29

Table 8. Relationships between ballistosporogenous yeasts and their supposedly related yeasts on the basis of their electrophoretic patterns of nine enzymes. 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 137

Table 8. Continued.

maltose, melezitose, a-methyl-D-glucoside, salicin, and DL-lactate (Table 2). S. odorus group 1 (YK 410 and YK 411) was similar to R. glutinis YK 103 with respect to its FA, 6PGDH, HK, PGM, and GDH pattern but not to its MDH and G6PDH pattern. Moreover, S. odorus group 1 was similar to Rhodosporidium diobovatum YK 223 with respect to its FA, 6PGDH, MDH (Rm 0.29-0:30), G6PDH, and HK (Rm 0.46) but not to its PGM and GDH pattern. The physiological properties of S. shibatanus (=S. pararoseus) are somewhat similar to those of R. rubra. Sporidiobolus pararoseus YK 415 was similar to R, rubra YK 152 with respect to its FA, MDH, G6PDH, and HK pattern but not to its 6PGDH, PGM, and GDH pattern. It was also similar to R. rubra YK 144 forr its FA, MDH, 138 YAMAZAKI and KOMAGATA VOL. 29

G6PDH, HK, and GDH pattern but not PGM and 6PGDH. On the other hand, Sporidiobolus pararoseus YK 415 was similar to R. glutinis YK 114 for its FA, 6PGDH, Fina, MDH (Rm 0.33-0.34), G6PDH, and HK (Rm 0.45) but not for its PGM and GDH. According to STORCKet al. (9), the G+ C contents of DNAs of S. pararoseus (=S. shibatanus) ranged from 51.5 to 60.0 mol %. Moreover, NAKASEand KOMAGATA(33) reported that R. rubra showed G+C contents of 60.0- 60.2 mol %. Thus, Sporobolomyces shibatanus (=Sporobolomyces pararoseus= Sporidiobolus pararoseus) is similar to certain strains of R. rubra in physiological and biochemical properties. In the previous paper (15), R. glutinis and its varieties were classified into six groups by the electrophoretic patterns of their enzymes, and three groups were found to be anamorphs of the Rhodosporidium species, but no relationship could be discerned between the other three groups and any species of Rhodotorula and Rho- dosporidium. Therefore, as shown above, R. glutinis and R. rubra appear to include strains having lost the ability to form and discharge ballistospores, though no mating was observed among strains tested in this study. S. albo-rubescens is similar to R. rubra in its physiological properties, and the latter is supposed to be the asporogenous state of S. albo-rubescens (1). Of the sixteen R. rubra strains tested in the previous paper (15), YK 155 and YK 156 had a 6PGDH with three bands, and their Rm values were similar to those of S. albo- rubescens YK 421 and YK 422. In addition, they were similar to R. rubra YK 155 with respect to their FA, Fma, MDH, G6PDH (Rm 0.31), HK, PGM, GDH, and Cat patterns, and they were also similar to R. rubra YK 156 in their FA, Fma, MDH, G6PDH, HK, PGM (Rm 0.48-0.49), and GDH patterns. These strains also showed similar assimilation of carbon compounds and potassium nitrate (Table 2). According to NAKASEand KOMAGATA(33), R. rubra YK 155 exhibited a G+C content of 60.2 mol %. STORCKet al. (9) suggested that S. albo-rubescens YK 422 showed a G+ C content of 63 mol %. The values of the G+ C contents of DNAs reported by STORCKet al. (9) were generally about 2-3 mol % higher than those of NAKASEand KOMAGATA(33). Therefore, these strains are thought to have similar G+ C contents of DNAs. From these results, we assume that R. rubra includes S. albo-rubescens strains which have lost the ability to produce ballistospores, and that YK 155 and YK 156, having three bands for 6PGDH, correspond to them.

Sporidiobolus ruinenii and Rhodotorula graminis Rhodotorula graminis is considered to represent yeast-like strains of Sporidio- bolus ruinenii which have lost the ability to form ballistospores, chlamydospores, and true hyphae (1). In the previous paper (15), R. graminis strains were classified into two groups by the electrophoretic patterns of their enzymes. Two strains, YK 119 and YK 120, were included in group 1, and YK 121 derived from the type strains was included in group 2. FELL (34) stated that R. graminis might 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 139 consist of two distinct species, namely, Rhodosporidium malvinellum and Sporidio- bolus ruinenii. However, the electrophoretic patterns of the R. graminis strains differed from the Rhodosporidium malvinellum strains (15). The enzymatic pattern of R. graminis YK 121 was similar to those of the Rhodosporidium diobovatum strains, but those of YK 119 and YK 120 differed from those of other Rhodotorula and Rhodosporidium species (15). R. graminis YK 119 and YK 120 were once classified as Rhodotorula rosa by GoTo and YoKOTSUKA(35) and as Sporobolo- myces coprophilus by SUGIYAMAand GoTo (36); later, these two strains were re- identified as R, graminis (1). The electrophoretic patterns of the enzymes of YK 119 and YK 120 and of Sporidiobolus ruinenii YK 456 showed close similarities. Therefore, R. graminis YK 119 and YK 120 were thought to be strains of Sporidio- bolus ruinenii which had lost the ability to form ballistospores, chlamydospores, and true hyphae.

Bullera and supposedly related yeasts B. alba strains which have lost the ability to form and discharge ballistospores would be classified as one of three varieties of Cryptococcus laurentii due to similarities of physiological properties. The strain YK 482 and B. alba YK 461 produced identical patterns for FA, Fina, MDH (Rm 0.29), G6PDH, PGM, GDH, and Cat but not HK. The strain YK 484 and B. alba YK 461 exhibited similar patterns in MDH (Rm 0.29-0.30), G6PDH, PGM, GDH, and Cat but not in FA, Fma, and HK. On the other hand, the strain YK 484 and C. laurentii var. flavescens YK 325 produced similar patterns for FA, 6PGDH, Fma (Rm 0.21), MDH, G6PDH, HK, PGM, and GDH. In the previous paper (16), C. laurentii and its varieties were heterogeneous as judged by the electrophoretic patterns of their enzymes. Therefore, YK 482 was thought to be a strain of B. alba which had lost the ability to form ballistospores. Moreover, a strain which had lost the ability to produce ballistospores also seems to be included in the strains of C. laurentii. The strain YK 484 might be an asporogenous state of an unknown Bullera species since the electrophoretic pattern of its enzymes were different from those of known Bullera species. B. alba YK 461 and Cryptococcus albidus var. albidus YK 304 also produced similar patterns for FA, MDH, Fma, G6PDH, PGM, and GDH but not HK. This strain YK 304 was isolated by GoTo (37) and described as an ascospore-forming yeast for which he proposed the name Naganishia globosus. This strain YK 304 differed from the strain YK 300 derived from the type strain of C. albidus var. albidus in their respective enzymes (16). On the other hand, B. alba YK 461 was similar to C. laurentii var. flavescens YK 325 with respect to its MDH, G6PDH, PGM, and GDH pattern but not to its FA, Fma, and HK pattern. B. alba YK 461 differed from C. albidus var. albidus YK 304 in the assimilation of galactose, meli- biose, ribitol, 2-keto-gluconate, 5-keto-gluconate, and potassium nitrate (Table 2). Thus, the B. alba strains tested seem to be related to certain strains in C. albidus 140 YAMAZAKI and KOMAGATA VOL. 29 rather than to the C. laurentii strains according to the electrophoretic patterns of their enzymes. Therefore, C. albidus also seems to include strains that have lost the ability to discharge ballistospores. The strain YK 485 and Cryptococcus macerans YK 331 produced identical patterns for FA, 6PGDH, Fina, MDH, and GDH and slight variations for HK and PGM. These two strains also exhibited similar assimilation patterns of carbon compounds and potassium nitrate (Table 2). C. macerans was thought to be a heterogeneous species on the basis of the electrophoretic patterns of its enzymes (16). Therefore, C. macerans seems to include strains which have lost the ability to discharge ballistospores, though no mating was observed between YK 485 and YK 331.

A diagrammatic illustration of relationships among ballistosporogenous yeasts, anamorphic and teleomorphic species Figure 3 is a diagrammatic representation, based on the electrophoretic enzyme patterns, summarizing this and previous studies (15,16) of the relationships among ballistosporogenous yeasts, anamorphic states belonging to the genera Rhodotorula and Cryptococcus, and their teleomorphic states, and yeast-like fungi of the genera Rhodosporidium, Filobasidium, Filobasidiella, and Tremella. Some Rhodotorula species were thought to be composed of anamorphic states of the genus Rhodosporidium and strains of the genera Sporobolomyces and Spori- diobolus which had lost the ability to produce ballistospores. However, teleomorphic states and the relationship with other genera have not yet been detected in Rhodotorula lactosa, R. marina, R. minuta, and R. aurantiaca. Some Cryptococcus species were suspected of being composed of anamorphic states of Filobasidium, Filobasidiella, and Tremella and strains of the genus Bullera which had lost the ability to produce ballistospores. However, relationships between the majority of Cryptococcus species and other yeasts and yeast-like genera are not yet clear. Therefore, further studies are required. A relationship between R. rubra and C. flavus was assumed because of their enzymatic patterns. However., contrary to our expectation, Rhodotorula species seem to have little relationship to Cryptococcus species. Sporobolomyces species also seem to have little relationship to Bullera species. Sporobolomyces, Aessosporon, and Sporidiobolus have a closer relationship to Rhodotorula than Bullera. However, relaitonships in S. gracilis, S. antarcticus, S. puniceus, and S. singularis are still not clear. B, alba had a close relationship to Cryptococcus, though relationships between the two species B. tsugae and B. piricola and other yeasts are still unclear. In the future, electrophoretic comparison of the enzymes of various yeasts and yeast-like organisms is needed to clarify the relationships in these yeasts.

Usefulness of an electrophoretic comparison of enzymes in yeast taxonomy As shown above, ballistosporogenous yeasts and some Rhodotorula and 1983 Enzymatic Patterns of Ballistosporogenous Yeasts 141 142 YAMAZAKI and KOMAGATA VOL. 29

Cryptococcus species were thought to be closely related. The strains which had lost the ability to discharge ballistospores would be classified in the genus Rho- dotorula or the genus Cryptococcus if they were studied without knowing that ballistospores were formerly formed. However, when their enzymes were compared electrophoretically, their relationships become clearer. Possible haploid mating partners of Rhodosporidium and Sporidiobolus were detected by comparing the electrophoretic patterns of the enzymes of various strains and such strains did indeed cross with the known haploid mating type strains or strains showing similar electrophoretic patterns. Therefore, the electrophoretic comparison of enzymes is useful in studying the relationship of an asporogenous yeasts to a sporogenous one. It allows the relationship between the teleomorphic and anamorphic states to be more certainly established. Moreover, this technique is useful in identifying the strains which have lost characteristics important in defining a genus or a species, e. g., formation of ballistospores, production of pigment, assimilation of potassium nitrate, etc.

The authors thank Prof. S. Goto, Yamanashi University, Kofu, Japan and Dr. D. Yarrow, Centraalbureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands for their encour- agement and invaluable suggestions. They also thank Dr. T. Iijima, Institute for Fermentation, Osaka, Japan, Dr. K. Yamada, Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan, Dr. A. Kockova-Kratochvilova, Institute of Chemistry of Slovak Academy of Sciences, Bratislava, Czechoslovakia, and Dr. R. J. Bandoni, University of British Columbia, Vancouver, Canada for supplying cultures.

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