Transition of the ability to generate petites in the Saccharomyces/ Kluyveromyces complex

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1 RESEARCH ARTICLE Transition of the ability to generate petites in the Saccharomyces/ Kluyveromyces complex Veronika Fekete 1,Mária Čierna 1, Silvia Poláková 1,2, Jure Piškur 2 & Pavol Sulo 1 1 Comenius University, Faculty of Natural Sciences, Department of Biochemistry, Mlynská Dolina, Bratislava, Slovakia; and 2 Cell and Organism Biology, Lund University, Soelvegatan, Lund, Sweden Correspondence: Pavol Sulo, Comenius University, Faculty of Natural Sciences, Department of Biochemistry, Mlynská Dolina, Bratislava , Slovakia. Tel.: ; fax: ; sulo@fns.uniba.sk Received 8 December 2006; revised 29 May 2007; accepted 29 May First published online 27 July DOI: /j x Editor: Monique Bolotin-Fukuhara Abstract Petite-positivity the ability to tolerate the loss of mtdna was examined after the treatment with ethidium bromide (EB) in over hundred isolates from the Saccharomyces/Kluyveromyces complex. The identity of petite mutants was confirmed by the loss of specific mtdna DAPI staining patterns. Besides unequivocal petite-positive and petite-negative phenotypes, a few species exhibited temperature sensitive petite positive phenotype and petiteness of a few other species could be observed only at the elevated EB concentrations. Several yeast species displayed a mixed moot phenotype, where a major part of the population did not tolerate the loss of mtdna but several cells did. The genera from postwhole-genome duplication lineages (Saccharomyces, Kazachstania, Naumovia, Nakaseomyces) were invariably petite-positive. However, petite-positive traits could also be observed among the prewhole-genome duplication species. Keywords Petite mutation; mtdna; Saccharomyces/ Kluyveromyces complex; ethidium bromide; mitochondria. Introduction In the year 1949, Ephrussi and his coworkers discovered Saccharomyces cerevisiae petite mutants arising spontaneously or after the acriflavine treatment in baker s yeast. The term petite comes from the characteristic reduced colony size in comparison to regular grande large colonies, on solid media with nonfermentable carbon source and low amount of glucose (Ephrussi et al., 1949). Numerous studies later showed that these cytoplasmic petite mutants lacked a functional mitochondrial (r 1 ) genome and exhibited extensive deletion of mtdna (r ) or no mtdna at all (r 0 ) (reviewed in references Dujon, 1981; Piškur, 1994; Chen & Clark-Walker, 1999; Contamine & Picard, 2000). The consequences of r /r 0 petite mutations are (1) impaired mitochondrial protein synthesis, (2) respiratory deficiency, (3) reconfigured metabolism, and (4) the absence of many characteristic protein complexes in mitochondria (such as cytochromes a1a3 and b, oligomycin-sensitive ATPase; Slonimski & Ephrussi, 1949). Later on, it was found that most yeast species do not give rise to petite colonies even after treatment with the intercalating agent ethidium bromide (EB), which massively converts S. cerevisiae r 1 cells into r 0 (reviewed in Chen & Clark-Walker, 1999). To distinguish the ability of yeast to form petite colonies, Bulder (1964a) has introduced the terms petite-positive and petite-negative species. The petite-positive yeasts have been defined as species capable of forming small respiration-deficient colonies after mutagenic treatment. Petite-negative species are not capable of generating small colonies. Instead, they form microcolonies that do die before becoming visible to the naked eye (Bulder, 1964b). Besides the most popular yeast models such as S. cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe, reliable data concerning yeast species are poorly known, as there have not been many comprehensive screenings published after the papers of Bulder (1964a) and de Deken (1966). At that time, yeast taxonomy relied on the phenotypes characterization, but many species have been reclassified since then (Kurtzman & Robnett, 1998, 2003; Kurtzman, 2003). For that reason, the ability to generate petite colonies has not been characterized for many yeast species imbedded in the recent phylogenetic trees.

2 1238 V. Fekete et al. There are two well-separated clades of petite-positive yeasts. Most of them are clustered to the genus Saccharomyces, and a minor part belongs to the Dekkera/Brettanomyces group (Chen & Clark-Walker, 1999). The phylogenetic relationship among yeasts related to Saccharomyces has recently been determined. Multigene sequence analysis placed 75 species of the Saccharomyces/Kluyveromyces complex into 14 well-supported clades (Kurtzman, 2003; Kurtzman & Robnett, 2003). The extensive genome sequencing within this complex revealed that the S. cerevisiae lineage underwent a whole genome duplication event c. 100 million years ago (Wolfe & Shields, 1997; Langkjær et al., 2003; Kellis et al., 2004). It has recently been suggested that the ability to grow anaerobically is associated with this ancient occurrence (Piškur et al., 2006; Merico et al., 2007). The aim of this work was to determine whether the ability to tolerate the loss of mtdna (r 0 tolerance) coincides with the whole duplication event, as well as to establish a reliable methodical background to determine petiteness in yeasts. Our study involved more than one hundred strains covering all species assigned to the Saccharomyces/Kluyveromyces complex (Kurtzman, 2003; Kurtzman & Robnett, 2003). Materials and methods Yeast strains The yeast strains used in this project are listed in Table 1 and they originated from different sources. Dr C. Kurtzman from the Agricultural Research Service Culture Collection (NRRL), US Department of Agriculture, Peoria, IL, USA kindly provided the majority of characterized species (Kurtzman, 2003). To make easier comparison with the older papers we present the designation in CBS collections (CBS abbreviation corresponds to the Culture Collection of Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Center Utrecht, the Netherlands) or in NCYC (for National Collection of Yeast Cultures, Institute of Food Research, Norwich). Superscript T in yeast designations indicates type strains (superscript NT, neotype). The other yeasts employed in this study: Naumovia castellii (former name Saccharomyces castellii) strains CBS 3006, CBS 3007, CBS 4310, CBS 7188, Fr. T5 and Fr. 014; Kazachstania exigua (former name Saccharomyces exiguus) strains CBS 1514, CBS 2141, CBS 134, CBS 4660, CBS 4661, CBS 6440, CBS 8134 and CBS 8135; Kazachstania transvaalensis (former name Saccharomyces transvaalensis) strains CBS 2248, CBS 4906 and Y329; Kazachstania unispora (former name Saccharomyces unisporus) strains CBS 399, CBS 1543, CBS 2420, CBS 2423, CBS 3004 and CBS 4804 were described in Špírek et al. (2003). Saccharomyces cerevisiae strains characterized in this study, CCY (CBS 457), CCY (CBS 4054), CCY (CBS 1426), CCY (CBS 1782), CCY (CBS 436), CCY (CBS 1460), CCY (CBS 400), CCY (CBS 435), CCY (NCYC 410), CCY (CBS 2247), CCY (CBS 2909), CCY (CBS 429), CCY (NCYC 76), CCY (CBS 382), CCY (CBS 1200), CCY (CBS 439) and CCY (CBS 381), were obtained from the Culture Collection of Yeasts (CCY) (former Czechoslovak Collection of Yeasts) located in the Institute of Chemistry, Slovak Academy of Sciences in Bratislava. Also from the CCY collection came hybrid or cybrid species CCY (CBS 308) (nuclear genome from Saccharomyces cariocanus, mitochondrial genome from S. cerevisiae); CCY (CBS 1429) (nuclear genome from S. cerevisiae, mitochondrial genome from Saccharomyces. kudriavzevii); as well as species assigned to Saccharomyces bayanus or pastorianus CCY (CBS 1504), CCY (CBS 425). The proper taxonomic classification of CCY strains has been confirmed by sequencing of the D1/D2 region from large rrna subunit and mitochondrial COX2 gene (Poláková et al., in preparation). Saccharomyces uvarum strain CBS 395 and Saccharomyces pastorianus CBS 1513 were from Jure Piškur collection. Yeast media The yeasts were grown at different temperatures (mostly at 23 1C) in the YPD (1% bactopeptone, 1% yeast extract, 2% glucose), YPGE (1% bactopeptone, 1% yeast extract, 3% glycerol, 2% ethanol) that were solidified for plates with 2% agar. Petite-negativity, petite-positivity The ability to form petite colonies after a treatment with EB was characterized by modification of the procedure described in Špírek et al. (2002). Yeasts cells (or a mixture of spores and mycelia of some Eremothecium species) were cultivated in liquid YPD with different concentrations of EB ( mgml 1 ; mm) (1 2 days) for at least 10 generations (10 12). Cultures were then diluted and 1000 cells as CFU (from EB treated as well as untreated culture) were plated on YPD plates. If some small colonies appeared after 4 7 days of cultivation (in some cases up to 4 weeks, see below), their ability for unlimited growth was examined by replacing on the fresh YPD plates. Respiration deficiency was then determined according to the inability to grow on the plates with the nonfermentable carbon sources such as glycerol and ethanol (YPGE). To exclude the rare event of nuclear petites (pet) or mitochondrial (mit) (Chen & Clark-Walker, 1999; Mller et al., 2001), respectively, the absence of mtdna in selected petite colonies was verified by DAPI staining.

3 Petiteness and phylogeny 1239 Table 1. Distribution of petite positivity/negativity in the Saccharomyces/Kluyveromyces complex Species NRRL designation CBS designation Accession number w Former name Petite phenotype Saccharomyces cerevisiae Y NT 1171 AY Saccharomyces cerevisiae 1 Saccharomyces paradoxus Y T 432 U68555 Saccharomyces paradoxus 1 Saccharomyces mikatae Y T 8839 AF Saccharomyces mikatae 1 Saccharomyces cariocanus Y T 7995 AF Saccharomyces cariocanus 1 Saccharomyces kudriavzevii Y T 8840 AF Saccharomyces kudriavzevii 1 Saccharomyces bayanus Y T 380 AY Saccharomyces bayanus 1 Kazachstania servazzii Y T 4311 AY Saccharomyces servazzii 1 Kazachstania unispora Y-1556 T 398 U68554 Saccharomyces unisporus 1 Kazachstania telluris YB-4302 T 2685 U72158 Arxiozyma telluris 1 Kazachstania transvaalensis Y T 2186 U68549 Saccharomyces transvaalensis 1 Kazachstania sinensis Y T 7660 AF Kluyveromyces sinensis 1 Kazachstania africana Y-8276 T 2517 U68550 Kluyveromyces africanus 1/ 1C Kazachstania viticola Y T 6463 AF Kazachstania viticola 1 Kazachstania martiniae Y-409 T 6334 AF Saccharomyces martiniae 1 Kazachstania spencerorum Y T 3019 U84227 Saccharomyces spencerorum 1 Kazachstania rosinii Y T 7127 U84232 Saccharomyces rosinii 1 Kazachstania lodderae Y-8280 T 2757 U68551 Kluyveromyces lodderae 1/ Kazachstania piceae Y T 7738 U84346 Kluyveromyces piceae 1 Kazachstania kunashirensis Y T 7662 AY Saccharomyces kunashirensis 1 1C Kazachstania exigua Y NT 379 U68553 Saccharomyces exiguus 1 Kazachstania turicensis Y T 8665 AF Saccharomyces turicensis 1 Kazachstania bulderi Y T 8638 AF Saccharomyces bulderi 1 Kazachstania barnettii Y T 6946 U84231 Saccharomyces barnettii 1 Candida humilis Y T 5658 U69878 Candida humilis 1 Naumovia castellii Y T 4309 U68557 Saccharomyces castellii 1 Naumovia dairenensis Y T 421 U68556 Saccharomyces dairenensis 1 Candida glabrata Y-65 T 138 U44808 Candida glabrata 1 Nakaseomyces delphensis Y-2379 T 2170 U69576 Kluyveromyces delphensis 1/ 1C Nakaseomyces bacillisporus Y T 7720 U69583 Kluyveromyces bacillisporus 1 Candida castellii Y T 4332 U69876 Candida castellii 1R 1C Tetrapisispora blattae Y T 6284 U69580 Kluyveromyces blattae 1 Tetrapisispora phaffii Y-8282 T 4417 U69578 Kluyveromyces phaffii Tetrapisispora nanseiensis Y T 8763 AF Tetrapisispora nanseiensis 1/ Tetrapisispora arboricola Y T 8765 AF Tetrapisispora arboricola 1/ Tetrapisispora iriomotensis Y T 8762 AF Tetrapisispora iriomotensis Vanderwaltozyma polyspora Y-8283 T 2163 U68548 Kluyveromyces polysporus Vanderwaltozyma yarrowii Y T 8242 AY Kluyveromyces yarrowii 1 1C Zygosaccharomyces rouxii Y-229 T 732 U72163 Zygosaccharomyces rouxii R Zygosaccharomyces mellis Y T 736 U72164 Zygosaccharomyces mellis Zygosaccharomyces baillii Y-2227 T 680 U72161 Zygosaccharomyces baillii Zygosaccharomyces bisporus Y T 702 U72162 Zygosaccharomyces bisporus H Zygosaccharomyces kombuchaensis YB-4811 T 8849 AF Zygosaccharomyces sp. H Zygosaccharomyces lentus Y T 8574 AF Zygosaccharomyces baillii Zygotorulaspora florentinus Y-1560 T 746 U72165 Zygosaccharomyces florentinus 1 1C Zygotorulaspora mrakii Y T 4218 U72159 Zygosaccharomyces mrakii H Torulaspora globosa Y T 764 U72166 Torulaspora globosa 1/ E Torulaspora franciscae Y T 2926 U73604 Torulaspora franciscae Torulaspora pretoriensis Y T 2187 U72157 Torulaspora pretoriensis 1/ Torulaspora delbrueckii Y-866 T 1146 U72156 Torulaspora delbrueckii Torulaspora microellipsoides Y-1549 T 427 U72160 Zygosaccharomyces microellipsoides Lachancea cidri Y T 4575 U84236 Zygosaccharomyces cidri R Lachancea fermentati Y U84239 Zygosaccharomyces fermentati Lachancea thermotolerans Y-8284 T 6340 U69581 Kluyveromyces thermotolerans Lachancea waltii Y-8285 T 6430 U69582 Kluyveromyces waltii H Lachancea kluyveri Y T 3082 U68552 Saccharomyces kluyveri Kluyveromyces aestuarii YB-4510 T 4438 U69579 Kluyveromyces aestuarii H Kluyveromyces nonfermentans Y T 8778 AF Kluyveromyces nonfermentans

4 1240 V. Fekete et al. Table 1. Continued. Species NRRL designation CBS designation Accession number w Former name Petite phenotype Kluyveromyces wickerhamii Y-8286 T 2745 U69577 Kluyveromyces wickerhamii Kluyveromyces lactis Y-8278 T 2105 U94919 Kluyveromyces lactis Kluyveromyces marxianus Y-8281 T 712 U94924 Kluyveromyces marxianus H Kluyveromyces dobzhanskii Y-1974 T 2104 U69575 Kluyveromyces dobzhanskii Eremothecium gossypii Y-1056 T U43389 Ashbya gossypii H Eremothecium ashbyi Y-1363 U43387 Eremothecium ashbyi H Eremothecium cymbalariae Y U43388 Eremothecium cymbalariae H Eremothecium coryli Y T 2608 U43390 Nematospora coryli Eremothecium sinecaudum Y U43391 Holleya sinecaudum Hanseniaspora valbyensis Y-1626 T 479 U73596 Hanseniaspora valbyensis H Hanseniaspora lindneri Y T 285 U84226 Kloeckera lindneri H Hanseniaspora guilliermondii Y-1625 T 465 U84230 Hanseniaspora guilliermondii H Hanseniaspora uvarum Y-1614 T 314 U84229 Hanseniaspora uvarum Hanseniaspora vineae Y T 2171 U84224 Hanseniaspora vineae Hanseniaspora osmophila Y-1613 T 313 U84228 Hanseniaspora osmophila 1/ E Hanseniaspora occidentalis Y-7946 T 2592 U84225 Hanseniaspora occidentalis H Saccharomycodes ludwigii Y T 821 U73601 Saccharomycodes ludwigii Ability to generate petites was determined after the cultivation with ethidium bromide at 23 1C. 1, petite-positive EB sensitive;, petite-negative EB sensitive; 1/, mixed moot phenotype;, Original collection strain already does not grow on nonfermentable substrate; R, EB resistant (50 mgml 1 EB); 1C, petite-negative phenotype at 28 1C but petite-positive phenotype at 23 1C; H, hypersensitive to EB (unable to grow for 10 generations in the presence of 50 mgml 1 EB); E, extremely slow growth of petites (2 4 weeks to see visible colonies); w, GenBank accession number for D1/D2 domains of 26S rrna gene. Consequently, only the species capable of tolerating full elimination of mtdna (r 0 mutants) were considered as petite-positive. Finally, to rule out the possibility of contamination by some other petite-positive species, the identity of petite colonies was confirmed by sequencing of the amplified D1/D2 region of rrna gene that was compared to published sequences (Kurtzman & Robnett, 1998, 2003). The identity of petite-negative species was assessed in a similar way. Presence of mtdna Presence or absence of mtdna was examined by DAPI staining, according to the specific fluorescence pattern as described previously (Marinoni et al., 1999), with a fluorescent microscope equipped with a DAPI optical filter. Boiling for five minutes in the presence of DAPI is not a universal staining method and does not work in certain species such as Schizosaccharomyces pombe or in yeasts creating small cells (Hanseniaspora clade). Here modifications are required as described by Haffter & Fox (1992) or in Moreno et al. (1991). rrna gene sequencing Genomic DNA was isolated according to Philippsen et al. (1991). The divergent D1/D2 domain (nucleotides for S. cerevisiae) at the 5 0 end of the large ribosomal subunit of rrna gene was amplified by PCR and sequenced as described in Kurtzman & Robnett (1998). Sequences were compared with the NCBI database by the simple standard nucleotide nucleotide BLAST program. Results Petite phenotypes In the available literature, there is a severe discrepancy among the published methods on petite generation in various yeasts. We attempted to upgrade methodology that would be useful for rapid, simple and reliable screening of the petite phenotype. After the cultivation with EB (50 mgml 1 ), we observed a few variations of the basic petite phenotype. Petite-positive (EB sensitive) The typical features were as follows. (1) Cultivation with EB induced petite colonies at high levels (several tens of percents) and the overall number of petite and grande colonies corresponded to the number of the plated cells. (2) Petite mutants formed regular-shape colonies, unable to grow on the medium with a nonfermentable carbon source, but capable of growing infinitely on glucose. (3) Petite colonies occasionally arose spontaneously. (4) Microcolonies typical for petite-negative species were absent. (5) DAPI staining often revealed petite colonies consisting of cells

5 Petiteness and phylogeny 1241 completely lacking any mtdna signal (Fig. 1a and d). A minor variation was observed for Candida castellii, which was resistant to commonly used concentration of EB (25 mgml 1 ) and unable to form spontaneously petite colonies. However, if the culture was treated with elevated concentrations of EB (up to 200 mgml 1 ), it behaved like regular petite-positive yeasts, although the efficiency of petite formation was reduced up to 50%. Petite-negative (EB sensitive) After the treatment with EB, only big grande respiring colonies were visible on the plates and their number was significantly lower than the number of plated cells. In addition, a vast number of petite-negative species formed small, irregular-shaped microcolonies, comprising a few to several thousand cells, visible only under the microscope (Fig. 1b). Several yeast species and most of the Eremothecium species were not capable of growing in the presence of EB for ten generations (Table 1). However, when these hypersensitive species were plated on YPD after the exposure to EB, they formed microcolonies like other petitenegative species. Two species Lachancea cidri and Zygosaccharomyces rouxii (formerly Zygosaccharomyces) were resistant to commonly used concentrations of EB. The petite phenotype was easily distinguishable at the elevated concentrations of EB (up to 200 mgml 1 ), according to the reduced number of grande colonies and the formation of small microcolonies. Mixed moot phenotype In this case, EB (50 mgml 1 ) induced all three types of colonies that appeared on YPD plates after cultivation of 2 4 weeks. Besides respiring grande colonies and some smaller petite colonies, the majority of the plated cells ( %) generated microcolonies. In contrast to microcolonies Fig. 1. Petite phenotypes. (a) Petite-positive phenotype sensitive to ethidium bromide (Candida glabrata). (b) Petite-negative phenotype sensitive to ethidium bromide (Tetrapisispora phaffii). (c) Mixed moot petite phenotype (Kazachstania lodderae). Plating on YPD after the cultivation with EB (left); microscopic detail (middle); plating on YPD after the cultivation without EB (right). (d) DAPI staining grande (r 1 ) colony (left); petite (r 0 ) colony (right) (Candida glabrata).

6 1242 V. Fekete et al. arising from true petite-negative species, these were larger, multi-layered, and usually they had more than thousands of cells, and were therefore clearly visible under the microscope (Fig. 1c). These colonies, like in petite-negative species, exhibited only a limited growing ability and did not grow at all after a transfer to the fresh medium. Less frequent small colonies, visible by naked eye (petite-like), possessed infinite growing capacity, did not grow on a nonfermentable carbon source and consisted of cells lacking any mtdna (r 0 strains). Mixed phenotype was characteristic for Kazachstania lodderae forming up to 10% of respiration deficient colonies with unlimited growth, Kazachstania africana (1%), Nakaseomyces delphensis (0.1%), Tetrapisispora nanseiensis (1%), Tetrapisispora arboricola (1%), Torulaspora globosa (50%), Torulaspora pretoriensis (50%) and Hanseniaspora osmophila (up to 10%). Interestingly, the moot phenotype was found also in Saccharomyces carlsbergensis CBS 1513, which is an interspecific hybrid. Strain CBS 1513 is the first lager brewing yeast that was pure-cultured and probably most lager brewing yeasts used today are closely related to this strain (reviewed in Kodama et al., 2005). Saccharomyces strains isolated from different habitats over the world exhibited uniform petite-positive EB-sensitive phenotype (strains are listed in the section Materials and methods ). Distribution of petite-negativity/positivity in the Saccharomyces/Kluyveromyces complex The ability to generate petites and a list of the examined species is summarized in Table 1. Apparently, petiteness is a species-specific feature, because a number of different wellcharacterized isolates assigned to Kazachstania exigua, Kazachstania unispora, Kazachstania transvaalensis and N. castellii isolates (strains listed in the section Materials and methods ; Špírek et al., 2003) were capable of tolerating the loss of mtdna. When Kurtzman & Robnett (2003), (Kurtzman, 2003) analysed the family Saccharomycetaceae they reassigned several species to the currently accepted genera, and proposed five new genera (Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma, Zygotorulaspora). We present the examined yeast species under their former as well as their recent names to avoid confusion (Table 1, Fig. 2). The ability to tolerate the elimination of mtdna was found within the clades closely related to S. cerevisiae (Saccharomyces, Kazachstania, Naumovia, Nakaseomyces, and partially Tetrapisispora). With the increasing phylogenic distance from S. cerevisiae, the occurrence of petite-positivity drops, albeit a few, species from less-related clades (Zygotorulaspora florentinus, Torulaspora globosa, Torulaspora pretoriensis and H. osmophila) were capable to generate petites (Table 1, Fig. 2). Discussion Pitfalls in determination of petiteness Our approach eliminated several drawbacks in the characterization of the petite forming ability. First, it is important to spot the symptoms of mtdna elimination, such as the formation of petites or microcolonies introduced by Bulder (1964b). Some yeasts reported as resistant to the commonly used concentrations of EB were already classified as petitenegative (Šubík et al., 1974). However, cultivation with an elevated concentration of EB (200 mgml 1 ) allows to determine petiteness in any characterized species. Obvious EB doses used were 25 mgml 1 (Fox et al., 1991; Dunn et al., 2006) or 5 50 mgml 1 (Piškur et al., 1998; Mller et al., 2001; Schneider-Berlin et al., 2005). However, Maleszka (1994) reported a concentration of 2.5 mm for C. parapsilosis (equals 1000 mgml 1 ), which is five times higher than the maximal concentration used in our work. Besides the potential to grow infinitely, it is important to determine the inability of petites to grow on nonfermentable substrates. The complete elimination of mtdna could be determined simply by DAPI staining (Fig. 1d). Because of the nuclear petite mutants that are also pleiotropically deficient in cytochrome oxidase (Tzagoloff & Dieckmann, 1990), the DAPI approach is much more reliable than the traditional monitoring of cytochromes a1a3 (Bulder 1964a). Staining is also more convenient for rapid screening procedure than Southern blot hybridization (Fox et al., 1991) or purification of mtdna by CsCl bisbenzimid gradient (Mller et al., 2001). Our screening procedure also allows us to determine the petite phenotype if the original strains (such as Kazachstania transvaalensis) do not grow, or grow extremely slowly, on the media with a nonfermentable carbon source (such as the Hanseniaspora clade). In spite of this inability, r 0 mutants can be distinguished according to smaller colony size and the DAPI staining analysis. Although the oldest data are difficult to trace due to the missing CBS numbers (Bulder, 1964a; de Deken, 1966), the identity of many species can be deduced according to their former designation in the CBS database. In general, the results from different sources correspond with our observations (Bulder, 1964a; de Deken, 1966; Piškur et al., 1998; Middelhoven & Kurtzman, 2003; Merico et al., 2007). A few minor variations may result from employment of different strains, minor contaminations, different cultivation conditions (especially temperature) or be due to the variable sensitivity to the intercalating agents. Temperature sensitive petite phenotype Initial experiments were carried out at 28 1C, which is optimal for the growth of Saccharomyces. However, later on we tested species that were hardly capable of growing on

7 Petiteness and phylogeny 1243 Fig. 2. Distribution of petite-positive/negative species in phylogenetic tree. Petiteness of examined species from Saccharomyces/Kluyveromyces complex is emphasized by different colors: petite-positive, green; petite-negative, red; mixed phenotype, green/red; not determined, black. The phylogenetic tree was adapted from Kurtzman & Robnett (2003).

8 1244 V. Fekete et al. YPD at this temperature (among them Kazachstania piceae, Kazachstania barnettii and others). To exclude the synergistic effect of the temperature on the petiteness we examined the ability to tolerate the loss of mtdna after the cultivation with EB at lower temperatures (13, 18, 23 1C). Indeed, Kazachstania africana, Kazachstania kunashirensis, N. delphensis, C. castellii, Vanderwaltozyma yarrowii and Zygotorulaspora florentinus, which were unequivocally petite negative at 28 1C, generated petite colonies at temperatures of 23 1C or lower. Species Kazachstania africana and C. castellii were capable of forming visible sectored colonies containing much more than thousands of cells at 28 1C, but their proliferating ability is limited as they could not grow after the placement on the fresh YPD plates. With the exception of Zygotorulaspora florentinus (formerly Zygosaccharomyces florentinus), petite colonies from the aforementioned species grew at higher temperatures (28 1C), implicating the role of temperature during the process of petite formation. Transition of the ability to generate petites We characterized petiteness of all species assigned to the Saccharomyces/Kluyveromyces complex (Fig. 2). Interestingly, the genera from postduplication lineages (Saccharomyces, Kazachstania, Naumovia, Nakaseomyces) are invariably petite-positive. Watson et al. (1980) reported petite-positivity of thermophilic enteric yeasts Tetrapisispora pintolopesii and Candida slooffii. These species were recently reclassified to the genus Kazachstania by Kurtzman et al. (2005), indicating that this clade consists entirely of species that tolerate the loss of mtdna. The lineages branching out just prior to and after the whole genome duplication are a mixture of petite-negative and petite-positive species (Fig. 2). Apparently, a clear ability to generate petites has been fixed in the lineage, which underwent the whole genome duplication but not in all Tetrapisispora species. However, the petite positive trait emerges sporadically also in the species from the preduplication lineages (Zygotorulaspora florentinus, Torulaspora globosa, Torulaspora pretoriensis, H. osmophila). In pre- and postduplication yeasts the partially incapacitated ability to generate petites can sporadically be found, and they exhibit mixed moot phenotype. Herein, besides respiring grande colonies and some petite colonies, the majority of the plated cells ( %) generate microcolonies. Mixed phenotype most often occurs in the Tetrapisispora clade (Fig. 2). To ascertain whether a particular yeast species is a mixture of petite-negative and petite-positive variants, we examined petite phenotype in single cell cultures of Kazachstania lodderae, N. delphensis, Tetrapisispora nanseiensis and Kazachstania africana. Ten different cultures arising from distinct single colonies exhibited unchanged mixed petite phenotype, with the similar ratio of true petites and microcolonies indicating that this is a typical feature of the entire yeast population. Evidently, the characteristic of this phenotype is that the main part of the population does not tolerate the loss of mtdna, but a significant portion, 1 50%, does. Therefore, it could be considered as a transition step from petite-negativity to petite-positivity, where cells are still not perfectly tuned for the life without the mitochondrial functions. We assume that a similar effect was already spotted by Bulder (1964a), who also noticed that petite colonies occurred rather infrequently in Schizosaccharomyces pombe, Brettanomyces lambicus (currently refers to Dekkera anomala) and Saccharomyces florentinus (currently likely refers to Zygotorulaspora florentinus). Petite-positivity can be easily converted to petite-negativity through single mutation and vice versa (reviewed in Chen & Clark-Walker, 1999; Contamine & Picard, 2000; Dunn et al., 2006). Even though a single mutation can switch over petite phenotype, mutations that allow the transition of just a minor part of the yeast population (likewise in yeasts with moot phenotype) have not been reported yet. The partial ability and unusual high frequency of petite formation distinguishes this class of yeasts from petite susceptible species such as Schizosaccharomyces pombe and Kluyveromyces lactis. These yeasts are capable to convert to r 0 variants due to single nuclear mutations. However, the frequency is extremely low (one to a few colonies per experiment) and requires long-term exposure (14 17 days) to EB (Haffter & Fox, 1992; Chen & Clark-Walker, 1995, 1999). Mutations converting Kluyveromyces lactis to petitepositive have been already identified in three largest subunits of the mitochondrial F1-ATPase, suggesting the essential nature of the functional ATPase in mitochondria, especially in the cells that have lost mtdna. Apparently, two key features are important for the ability to survive without mtdna. It is the capacity to provide enough energy from glycolysis, when the key energy source is disabled (Merico et al., 2007), and the capability of supporting mitochondrial biogenesis (Chen & Clark-Walker, 1999; Clark-Walker, 2003; Smith & Thorsness, 2005). Functional protein import machinery is essential for the mitochondrial biogenesis and consequently for the viability of cells. Two energy supplies, membrane potential and ATP, are needed for mediating protein translocation across and insertion into the inner membrane (for reviews Mokranjac & Neupert, 2005; de Marcos-Lousa et al., 2006). Consequently, survival and growth of yeasts depends on the generation of a voltage gradient, across the mitochondrial inner membrane (St-Pierre et al., 2000; Clark-Walker, 2003; Schnaufer et al., 2005). However, in cells lacking mtdna it can be generated only as a result of the electrogenic nature of

9 Petiteness and phylogeny 1245 an ADP/ATP translocator, an inner membrane protein exchanging the cytosolic ATP 4 for mitochondrial ADP 3 (for reviews see Pebay-Peyroula & Brandolin, 2004; Nury et al., 2006). Keeping a constant level of ADP inside the mitochondria by ATP hydrolysis is essential for the maintenance of membrane potential. Therefore, the activity of the F 1 -ATPase subunit is indispensable and can be tuned up by the mutations in F 1 subunits (Chen & Clark-Walker, 1995; Clark-Walker, 2003; Smith & Thorsness, 2005) or by increasing the overall mitochondrial ATPase activity (Kominsky & Thorsness, 2000). Unfortunately, the majority of the examined species are wild-type diploids and most of the moot species are very likely homothalic (Butler et al., 2004; Piškur et al., 2006). This is the major setback for genetic experiments that can distinguish whether moot phenotype is due to the mutation. Alternatively, it could be just a selection of a part of the population with tuned up maintenance of mitochondrial membrane potential (with elevated mitochondrial ATPase activity or increased capacity to transport cytosolic ATP into the mitochondria). Apparently, the whole genome duplication is directly and indirectly connected to the petiteness. However, the whole genome duplication is not involved in the petite positive phenotype in some of the Brettanomyces/Dekkera species (Bulder, 1964a; Šubík et al., 1974; Hoeben et al., 1993; Woolfit et al., 2007). It is very likely that petite-positive species are interspread also among other unrelated yeasts, such as Candida albicans (Gyurko et al., 2000), but they are not known in filamentous fungi, algae or plants. On the other hand, tolerance to the loss of mtdna has been reported in some protozoa (Schnaufer et al., 2002) and animal cell lines (Inoue et al., 1997). The remarkable feature is that more evolved cells from higher eukaryotes (mammals, avians) are petite-positive, if they grow in the cell culture, even though the organisms as a whole do not tolerate the elimination of mtdna (Desjardins et al., 1986; King & Attardi, 1989). The distribution of petite-positivity appears to be thought-provoking paradox in evolution, as it is hard to understand why the majority of lower eukaryotes cannot tolerate mtdna elimination, while human cells do. The mosaic distribution pattern of petite-positivity is mysterious because it is difficult to understand the advantage of petite positivity or negativity. Perhaps it could have been just an outcome of tinkering in evolution (Jacob, 1977) associated with the fermentative life-style and therefore it emerges randomly but infrequently. Acknowledgements We thank C. Kurtzman and E. Sláviková for providing yeast strains employed in this study. We also thank I. 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