COENZYME Q SYSTEM IN THE CLASSIFICATION OF APICULATE YEASTS IN THE GENERA NADSONIA, SACCHAROMYCODES, HA NSENIASPORA, KLOECKERA, AND WICKERHAMIA1

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1 J. Gen. App!. Microbiol., 22, (1976) COENZYME Q SYSTEM IN THE CLASSIFICATION OF APICULATE YEASTS IN THE GENERA NADSONIA, SACCHAROMYCODES, HA NSENIASPORA, KLOECKERA, AND WICKERHAMIA1 YUZO YAMADA, MITSUYOSHI ARIMOTO, AND KEIJI KONDO Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University, Shizuoka 422, Japan (Received August 19, 1976) Fourty-three cultures of apiculate yeasts were examined for the Co-Q system, which belong to the genera Nadsonia, Saccharomycodes, Wickerhamia, and Hanseniaspora and its imperfect counterpart of Kloeckera. All the species of these genera were found to have the Q-6 system except for the only species of the genus Wickerhamia (Q-9). The results obtained are discussed in connection with other criteria such as PMR spectra of cell wall polysaccharides, DNA base composition, and serological characteristics. Our method is described as a criterion for grouping yeasts and yeast-like organisms. Since REESS (1) first described apiculate yeasts, a number of species included in the subfamily Nadsonioidae (2) that multiply by bud-fission have been reported. Of the four genera nowadays recognized in the above subfamily, the genus Nadsonia is unrelated to the other three genera, because of having the unusual cell wall that lacks mannan but possesses a high content of chitin (3, 4). MILLER and PHAFF (4,5) also pointed out that the genera Hanseniaspora and Saccharomycodes, together with the imperfect counterpart of the former Kloeckera, had the common cell wall composition which is similar to that found in Saccharomyces cerevisiae. SPENCER and GoRIN (6) showed by their PMR spectrometry of mannose-containing polysaccharides isolated from these apiculate yeasts that only the genus Nadsonia produced galactomannan and the two genera of Hanseniaspora and Kloeckera were divided into 3 groups. A more recent investigation of TSUCHIYA et al. (7) has recognized in the above four genera of Hanseniaspora, Saccharomycodes, 1 This constitutes Part V of a series entitled "Significance of Coenzyme Q System in the Classification of Yeasts and Yeast-like Organisms." This work was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Sendai, April 2, The abbreviations used here for coenzyme Q or ubiquinone are: Co-Q, coenzyme Q; Co-Q, or Q-n with n denoting a specified number of isoprene units in a side chain; e.g., Co-Q6 or Q-6, etc. 293

2 294 YAMADA, ARIMOTO, and KONDO VOL. 22 Wickerhamia, and Kloeckera antigenic structures common to the genus Saccharomyces, and they have set up 2 groups by their slide agglutination technique. NAKASE and KOMAGATA (8) have established 4 subgroups within the genera Hanseniaspora and Kloeckera on the basis of their measurement of DNA base composition. In previous papers (9-12), we reported from a taxonomic point of view the coenzyme Q system of the ascosporogenous yeast and yeast-like genera Hansenula, Pichia, Schizosaccharomyces, and Endomyces, and the asporogenous yeast and yeast-like genera Rhodotorula, Cryptococcus, Sporobolomyces, and Rhodosporidium, and obtained several new informations as to the classification of these organisms. The present paper deals with the apiculate yeasts included in the genera Nadsonia, Saccharomycodes, Hanseniaspora, Kloeckera, and Wickerhamia. MATERIALS AND METHODS Microorganisms. All the strains of apiculate yeasts used in this experiment came from Institute for Fermentation, Osaka, and Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, by courtesy of Drs. I. Banno and T. Hasegawa, and Drs. T. Nakase and T. Tsunoda. These organisms comprise 43 cultures of the ascosporogenous yeast genera Nadsonia, Hanseniaspora, Saccharomycodes, and Wickerhamia, and the asporogenous yeast genus Kloeckera; sources of these cultures are listed in Table 1. Cultivation of microorganisms. Stock cultures were generally maintained on agar slants containing (per liter) glucose 15 g, sucrose 15 g, peptone (Kyokuto) 3 g, and yeast extract (Oriental) 2 g, supplemented with 20% potato extract,2 at 4. For the working cultures, the microorganisms were cultivated as described in a previous paper (9). The growth period was hr at Two species of the genus Nadsonia had shown poorer growth in the liquid medium, so that these organisms were grown on agar plates dispensed in Roux flasks. Extraction and isolation of coenzyme Q. The Co-Q's of the strains to be examined were extracted and partially purified from intact cells, as described previously (9). Determination of coenzyme Q system. For the unequivocal determination, reversed-phase paper chromatography and mass spectrometry were adopted for the Co-Q preparations (9). Reagents and chemicals. Authentic preparations of Co-Q series were prepared as described previously (9-12). Coenzyme Qg was obtained from Sigma Chemical Co., U.S.A. Other reagents and chemicals used in this experiment were commercial preparations (Nakarai). 2 Twenty per cent potato extract was prepared from 200 g of peeled potatoes and filled up to 1,000 ml.

3 1976 Significance of Co-Q in Yeast Taxonomy (V) 295 RESULTS AND DISCUSSION As can be seen from the tabulated results (Table 1), the apiculate yeasts examined in the present experiment showed the Co-Q system of Q-6, except for the only species of the genus Wickerhamia (Q-9). The wide-spread system of Q-6 found in these apiculate yeasts was, however, quite identical with those3 of the genera Saccharomyces and Nematospora, which may be indicative of a certain phylogenetic relationship among these ascosporogenous yeasts. Wickerhamia fluorescens is the only species of the genus Wickerhamia, which was first isolated and identified as a new species of the genus Kloeckera but transferred to the newly established genus Wickerhamia on the basis of the shape and number of ascospores (13, 14). The phylogenetic position of this genus is uncertain, but the genus is speculated to be an intermediate which links the Nadsoniae to the Spermophthoraceae (14). It is notable that this species has been found to possess the Q-9 system, which leads us to the assumption that the genus Wickerhamia might occupy a phylogenetically intermediate position that is connected to the above-mentioned Spermophthoraceae, especially to the Q-9 possessors (unpublished data) of this family such as the members of the genus Metschnikowia. MILLER and PHAFF (5) stated that the species of the genus Hanseniaspora and its imperfect counterpart of Kloeckera made a natural group. However, NAKASE and KOMAGATA (8) recognized 4 subgroups within these genera. In view of the only Q-6 system observed in the two genera, it is suggested that the Co-Q system described here and previously would be useful as a criterion for grouping at a genus level. SPENCER and GORIN (6) discriminated the genera Nadsonia, Saccharomycodes, and Hanseniaspora from each other by the PMR spectrometry of isolated mannosecontaining polysaccharides. The spectra of the mannans from Hanseniaspora and Kloeckera species were divided into 3 groups. However, the same Co-Q system found in both the genera indicates only the relation between the perfect and imperfect forms. TSUCHIYA et al. (7) and TsUCHIYA (15) arranged serologically the apiculate yeasts comprising the Hanseniaspora, Kloeckera, Saccharomycodes, and Wickerhamia species into two larger groups (Table 2). Their first group or group I includes H'spora osmophila, H'spora vineae (H'spora osmophila), Kl. africana, Kl. javanica, S'codes ludwigii, etc., as well as several species in the genera Saccharomyces and Candida. Their second group or group II is also composed of H'spora valbyensis, H'spora uvarum, H'spora guilliermondii (H'spora valbyensis), Kl. apiculata, Kl. japonica, and W. fluorescens, together with a number of species in the genera Saccharomyces, Nematospora, Torulopsis, and Candida. It was of interest that these two groups were characterized by the Q-6 and Q-9 systems. Each of these two groups is, therefore, divided into two subgroups by the Co-Q s Unpublished data, which will be published later. The Co-Q systems of the genera Saccharomyces and Debaryomyces are cited in a reference (16).

4 296 YAMADA, ARIM0T0, and KONDO VOL. 22 Table 1. Coenzyme Q system in the apiculate yeasts of the genera Nadsonia, Saccharomycodes, Hanseniaspora, Wickerhamia and Kloeckera.

5 1976 Significance of Co-Q in Yeast Taxonomy (V) 297 (Table 1. continued) system. The Q-9 possessors were focused on several species of the genera Torulopsis and Candida, and also the only species of the apiculate yeasts (W. fluorescens). Saccharomycodes ludwigii is discriminated from C. albicans by the Co-Q system, though both species have the same antigenic structure. The Co-Q system of W. fluorescens was different from that of Sacch. lactis (Kluyveromyces lactis), while they have very similar antigens. As has been discussed here and previously, it is obvious that our method provides a new information as to the classification of yeasts and yeast-like organisms. The following conclusions can thus be drawn: (a) During the course of these experiments, all kinds of naturally occurring Co-Q have been found in the organisms referred to as yeasts. This suggests the diversity of these organ-

6 298 YAMADA, ARIM0T0, and KONDO VoL. 22 Table 2. Coenzyme Q system in the species of groups I and II of TSUCHIYA et al. (7). isms. (b) Generally, in the respective yeast genera established to date, there occurs a proper or characteristic Co-Q system, e.g., Q-6 in the genus Hanseniaspora and its imperfect counterpart, Kloeckera; Q-7 in the genus Hansenula; Q-8 in the genera Citeromyces and Pachysolen; Q-9 in the genus Debaryomyces3; and Q-10 in the genus Rhodotorula. (c) When a complex distribution in the Co-Q system has been found within a certain genus, e.g., Pichia and Rhodotorula, the genus is inferred to have a heterogeneous nature. The Co-Q system is presented as a potential value in the classification, especially in the designation for grouping at a genus level. (d) Our method consists in employing an organic compound, so that the information obtainable is clear-cut. (e) There have been found only five kinds of natural quinones (Q-6 to Q-10). However, these numbers are too few to classify numerous species of yeasts and yeast-like organisms. Consequently, species which have similar or identical Co-Q system are not necessarily

7 1976 Significance of Co-Q in Yeast Taxonomy (V) 299 related, as pointed out by the following pairs; H. holstii and Rh, infirmo-miniata (Q-8), Wingea robertsii and Rhodosp. toruloides (Q-9), and Schiz. pombe and Rh, rubra (Q-10). In conclusion, it is apparent that the Co-Q system is useful for grouping species when used along with other criteria such as serological characteristics, DNA base composition, DNA-DNA and DNA-RNA hybridizations, PMR spectrometry of cell wall polysaccharides, conventional methods, etc. The authors are indebted to Drs. I. Banno and T. Hasegawa, Institute for Fermentation, Osaka, and to Drs. T. Nakase and T. Tsunoda, Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, for their kind supply of type cultures. Thanks are also due to Dr. K. Komagata, The Institute of Applied Microbiology, University of Tokyo, Tokyo, for his encouragement. REFERENCES 1) M. REESS, "Botanische Untersuchungen uber die Alkoholgarungspilze," Leipzig (1870), cited from Reference 2. 2) J. LODDER, In The Yeasts a Taxonomic Study, 2nd Ed., ed. by J. LODDER, North-Holland Publishing Co., Amsterdam (1970), p.1. 3) D. R. KREGER, Biochim. Biophys. Acta, 13, 1 (1954). 4) M.W. MILLER and H.J. PHAFF, Antonie van Leeuwenhoek J. Microbiol. Serol., 24, 225 (1958). 5) M. W. MILLER and H. J. PHAFF, Mycopathol. Myco!. App!., 10, 113 (1958). 6) J. F. T. SPENCER and P. A. J. GGRIN, J. Bacteriol., 96,180 (1968). 7) T. TsUCHIYA, Y. FUKAZAWA, and S. KAWAKITA, Mycopathol. Mycol. App!., 26, 1 (1965). 8) T. NAKASE and K. KOMAGATA, J. Gen. App!. Microbiol.,16, 241 (1970). 9) Y. YAMADA and K. KONDO, J. Gen. App!. Microbiol.,19, 59 (1973). 10) Y. YAMADA, Y. KANEMATSU, M. OHASHI, and K. KoNDO, Agr. Biol. Chem. (Tokyo), 37, 621 (1973). 11) Y. YAMADA, T. OKADA, 0. UESHIMA, and K. KoNDO, J. Gen. App!. Microbio!., 19, 189 (1973). 12) Y. YAMAI)A, M. ARIMOTO, and K. KoNDO, J. Gen. App!. Mcirobio!.,19, 353 (1973). 13) M. SONEDA, Nagaoa, 34,1(1959). 14) M. SONEDA, Nagaoa, 35, 9 (1960). 15) T. TsUCHIYA, Shinkin to Shinkinsho, 10, 179 (1969). 16) Y. YAMADA and K. KoNDO, Proc. 4th Int. Ferment. Symp., Kyoto, 781(1972).