0026-2617/05/7404- © 2005 Pleiades Publishing, Inc. 0459 Microbiology, Vol. 74, No. 4, 2005, pp. 459–462. Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 533–536. Original Russian Text Copyright © 2005 by Yurkov. The Saccharomyces sensu stricto complex, which, it was recently proposed, should be restricted to the genus Saccharomyces [1], contains at least six biological sib- ling species: Saccharomyces cerevisiae Meyen ex Hansen, S. paradoxus Bachinskaya, S. bayanus Sac- cardo, S. cariocanus Naumov et al., S. kudriavzevii Naumov et al., and S. mikatae Naumov et al. Isolation of the strains of this complex is almost completely lim- ited to industrial fermentation processes. Not much is known about their distribution in nature. Representa- tives of Saccharomyces paradoxus have been most fre- quently found in natural habitats. This species has a vast natural habitat and is represented by a number of divergent populations: European [2–4], Far Eastern [5], Hawaiian [6], and North American [7]. However, the Saccharomyces cerevisiae habitat is thought of as inter- rupted, since representatives of this species have never been isolated in Siberia, where only representatives of the close species Saccharomyces cerevisiae have been found. During our investigations of yeast communities on plant substrates in Novosibirsk oblast [8], we isolated, from oak exudates, two strains that were identified as S. paradoxus according to the results of biochemical tests. Molecular biological investigations confirmed the affiliation of these isolates with the species S. para- doxus. Both strains demonstrated intense sporulation. The isolation of these yeast strains in western Siberia is of considerable interest. Earlier, only representatives of S. cerevisiae [9] had been isolated in this region. The joint occurrence of different Saccharomyces species was previously noted in Far East [5]. The strains were deposited with the Collection of the Soil Biology Department, Faculty of Soil Science, Moscow State University, and given the strain designa- tions KBP-3828 and KBP-3829. MATERIALS AND METHODS Samples of pedunculate oak (Quercus robur) exu- date were obtained in August 2003 from the oak plant- ing territory of the Central Siberian Botanical Garden, Siberian Division, Russian Academy of Sciences (Novosibirsk). In total, we analyzed 12 oak exudates from 8 trees. The samples of oak exudates were taken together with bark pieces. Two strains were isolated from two exudate flows in different parts of one tree trunk. The isolation of yeasts was carried out in accor- dance with the enrichment culture method. The bark pieces were incubated in a liquid malt extract medium at room temperature for 2–4 days until the appearance of signs of intensive fermentation. Then, the culture was plated onto malt extract agar that had been acidi- fied to pH 4–4.5 with 40% lactic acid so as to inhibit bacterial growth. The plates were incubated at room temperature for a week. The grown yeast colonies were isolated in pure cultures. Identification of the isolates was performed, based on morphological and physiological properties, accord- ing to determination manuals [10, 11]. Isolation and amplification of DNA was performed using a standard method. The amplification of 5.8S rDNA and the internal transcriptional spacers ITS1 and ITS2 (5.8S-ITS fragment) was accomplished with the use of the primers pITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and pITS4 (5'-CCTCCGCTTATTGATATGC-3'). A PCR reaction was run in 30 μl of a reaction mixture that con- tained a PCR buffer, 20 mM (NH 4 ) 2 SO 4 , 3 mM MgCl 2 , 0.25 mM dNTP, 0.30 μmol of each primer, 1.25 units of Tag polymerase (Sintol, Russia), and 20 ng of the ana- lyzed genomic DNA. A Tertsik thermal cycler (DNK- tekhnologiya, Russia) was operated on the following regime: initial heat denaturation of native DNA at 94°C EXPERIMENTAL ARTICLES First Isolation of the Yeast Saccharomyces paradoxus in Western Siberia A. M. Yurkov Soil Science Faculty, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia Received September 6, 2004; in final form, December 16, 2004 Abstract—Two ascomycetous yeast strains have been isolated near Novosibirsk from oak exudate. The strains have been identified as Saccharomyces paradoxus Bachinskaya based on the results of biochemical tests. The conspecificity of the isolates with S. paradoxus was confirmed by electrophoretic karyotyping and restriction analysis of the ITS region of its rDNA. This first isolation of S. paradoxus in Siberia provides evidence for the continuity of its natural habitats. Key words: Saccharomyces paradoxus, exudate, western Siberia, karyotyping, restriction analysis.