Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis

Adriana Rycovska1, Matus Valach1, Lubomir Tomaska2, Monique Bolotin-Fukuhara3 and Jozef Nosek1,3

1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic
2 Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic
3 Institute of Genetics and Microbiology, University of Paris XI, 91 405 Orsay, France

Correspondence
Jozef Nosek
nosek{at}fns.uniba.sk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The yeast species Candida parapsilosis, an opportunistic pathogen, exhibits genetic and genomic heterogeneity. To assess the polymorphism at the level of mitochondrial DNA (mtDNA), the organization of the mitochondrial genome in strains belonging to the three variant groups of this species was investigated. Although these analyses revealed a group-specific restriction fragment pattern of mtDNA, strains belonging to different groups appear to have similar genes in the same gene order. An extensive survey of C. parapsilosis isolates uncovered surprising alterations in the molecular architecture of their mitochondrial genome. A screening strategy for strains harbouring mtDNA with rearranged architecture showed that nearly all strains from groups I and III possess linear mtDNA molecules terminating with arrays of tandem repeat units, while most of the group II strains have a circular mitochondrial genome. In addition, it was found that linear genophores in mitochondria of strains from different groups differ in the sequence of the mitochondrial telomeric repeat unit. The occurrence of altered forms of mtDNA among C. parapsilosis strains opens up the unique possibility to address questions concerning the evolutionary origin and replication strategy of linear and circular genomes in mitochondria.


Abbreviations: mtDNA, mitochondrial DNA; ITS, internal transcribed spacer

The EMBL/GenBank accession numbers for the sequences reported in this paper are X76196, X76197, X75676, X75681 and AY391843AY391852.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although the yeast Candida parapsilosis is considered to be a benign commensal micro-organism in healthy individuals, it is frequently associated with cases of severe infection in patients with diminished immune function (Weems, 1992; Hazen, 1995; Garber, 2001; Arendrup et al., 2002). Several recent surveys have described genetic and/or genomic heterogeneity among C. parapsilosis isolates (Lott et al., 1993; Branchini et al., 1994; Cassone et al., 1995; Pfaller et al., 1995; De Bernardis et al., 1999). Data from DNA–DNA reassociation, restriction length polymorphism (RFLP) and isoenzyme profiling suggest that the form species C. parapsilosis consists of the three variant groups that may even represent three distinct species (Lin et al., 1995; Roy & Meyer, 1998). This conclusion was further supported by comparison of the DNA sequence within the internal transcribed spacer (ITS) of the rDNA and D1/D2 domain of the gene encoding 26S rRNA (Kurtzman & Robnett, 1998). Although strains from each group have been found in samples from human patients (Lin et al., 1995; Roy & Meyer, 1998; Enger et al., 2001), strains from group I, including the type strain of the species (CBS 604T/ATCC 22019T), seem to predominate among clinical isolates. One of the problems that hampers epidemiological studies is the incorrect identification of group, or even species, which may result from using conventional diagnostic methods (Fenn et al., 1994; Ramani et al., 1998). To solve this problem several approaches employing molecular techniques, such as electrophoretic karyotyping, PCR, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting have been developed for species- and group-specific identification of C. parapsilosis (Carruba et al., 1991; Branchini et al., 1994; Cassone et al., 1995; Pfaller et al., 1995; Pontieri et al., 1996, 2001; Enger et al., 2001). In addition, several studies have reported that molecular markers derived from mitochondrial DNA (mtDNA) are applicable in molecular diagnostics of C. parapsilosis isolates (Camougrand et al., 1988; Su & Meyer, 1989, 1991; Yokoyama et al., 2000; Nosek et al., 2002). Importantly, studies of genetic organization of mtDNA in C. parapsilosis have uncovered that this yeast, in contrast to the related species C. albicans and C. tropicalis, possesses a linear mitochondrial genome terminating with specific structures termed mitochondrial telomeres (Kovac et al., 1984; Nosek et al., 1995). Due to the uniqueness of mitochondrial telomeres in C. parapsilosis compared to related species, these structures may represent a promising diagnostic or therapeutic target (Nosek et al., 1998, 2002).

The three groups of C. parapsilosis have been defined without any knowledge regarding structural differences in their mtDNA. In this report, we investigated the variability of mtDNA in the strains from different C. parapsilosis groups and compared their molecular profiles with the closely related species, Lodderomyces elongisporus. The analysis revealed a group-specific pattern at the level of restriction fragments of C. parapsilosis mtDNA. More importantly, the results indicate the striking differences between groups regarding the molecular architecture of the mitochondrial genophore. The presence of variant molecular forms of mtDNA in strains belonging to the same species opens up the unique possibility to analyse the structural differences between linear- and circular-mapping mtDNA, specifically questions concerning the evolutionary origin and replication strategy of linear mitochondrial genomes. Therefore, we developed a strategy for identification of strains with rearrangements in the telomeric region of the mtDNA. This effort uncovered several strains that might be considered as mutants in the mitochondrial telomeres.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strains.
Strains (Table 1) designated CBS were from the Centraalbureau voor Schimmelcultures (Delft, The Netherlands); MCO and PL strains were kindly provided by P. F. Lehmann (Medical College of Ohio, Toledo, USA) and S. A. Meyer (Georgia State University, Atlanta, USA), respectively. Yeast cultures were grown in YPD medium [1 % (w/v) yeast extract (Difco), 2 % (w/v) Bacto Peptone (Difco), 2 % (w/v) glucose] at 28 °C. Biotype profiles of yeast strains were confirmed by using an API20C kit (Biomérieux) according to the manufacturer's instructions.


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Table 1. List of yeast strains and summary of molecular profiling results

 
Electrophoretic karyotyping.
Samples of chromosomal DNA prepared in agarose blocks were separated in a 0·8 % (w/v) agarose gel in 45 mM Tris/borate–1 mM EDTA buffer. Electrophoresis was carried out in a Pulsaphor apparatus (LKB) in a contour-clamped homogeneous electric field (CHEF) configuration with the pulse switching from 60 to 600 s (interpolation) for 72 h at 100 V and 9 °C throughout.

Comparison of ITS rDNA sequences.
Total cellular DNA was isolated from 5 ml yeast cultures as described by Phillippsen et al. (1991). Subsequently, the ITS region of the rDNA was amplified using ITS oligonucleotide primers (Table 2) in a reaction with a 10 : 1 mixture of Taq DNA polymerase (Gibco-BRL) and Vent DNA polymerase (New England Biolabs). The resulting PCR products were cloned into the pDrive cloning vector (Qiagen) and their sequences were determined as indicated below.


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Table 2. Sequences of oligonucleotide primer pairs

 
Analysis of mtDNA.
Screening for linear mtDNA by PFGE was performed as described by Fukuhara et al. (1993). Briefly, whole-cell DNA samples were prepared in agarose blocks and separated in a 0·8–1 % (w/v) agarose gel in 45 mM Tris/borate–1 mM EDTA buffer using a Pulsaphor apparatus (LKB) in CHEF configuration with pulse switching from 5 to 50 s (interpolation) for 24 h at 150 V and 9 °C throughout. Restriction fragment patterns of mtDNAs were examined on small-scale preparations (Defontaine et al., 1991) using the restriction endonucleases HindIII, EcoRV, PvuII or KpnI (New England Biolabs) and separated by agarose gel electrophoresis. For physical mapping and cloning purposes mtDNA was prepared and purified by CsCl gradient centrifugation as described previously (Nosek et al., 1995). The HindIII and EcoRV restriction fragments of mtDNA were cloned into the corresponding sites of the pCR-Script-AmpR-SK(+) plasmid vector (Stratagene) and the sequences at the boundaries of inserts were determined using universal (M13) forward and reverse primers.

PCR analysis.
The analysis was performed as described previously (Nosek et al., 2002) with modifications in the primer pairs (Table 2). Briefly, amplification reactions (20–50 µl) were performed using Taq DNA polymerase (Gibco-BRL) on cell lysates or isolated total cellular DNA (see above). Reactions were done in a thermal cycler (Biometra) using a standard three-step programme: 3 min at 95 °C, followed by 25–30 cycles of 45 s at 94 °C, 1 min at 43–49 °C and 1 min at 72 °C, with a final step of 3 min at 72 °C. Samples were then separated by agarose gel electrophoresis (1·0–1·5 %, w/v, agarose containing 0·5 µg ethidium bromide ml–1) at 5–10 V cm–1 for 45–60 min in 45 mM Tris/borate–1 mM EDTA buffer.

Miscellaneous.
Enzymic DNA manipulations, Southern blot analysis and cloning procedures were performed as described by Sambrook & Russell (2001). The DNA sequence was determined using the BigDye terminator sequencing kit and an ABI 310 automatic analyser (Applied Biosystems). Sequence assembly and analysis was performed using the Vector NTI package (Informax) and BLAST (http://www.ncbi.nlm.nih.gov/blast/). The sequence alignments and tree calculation were done using the AlignX program (Informax).

Reproducibility of results.
All PCR and PFGE analyses were repeated at least twice with the same results.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains of C. parapsilosis display group-specific molecular profiles of mtDNA
The variability of strains (Table 1) belonging to genetically distinct groups of C. parapsilosis was examined by PFGE and by restriction enzyme analysis of mtDNA. The results of both approaches indicate that electrophoretic karyotypes as well as patterns of mtDNA restriction fragments are group-specific features (Figs 1 and 2). Only a minor polymorphism in chromosome length was found among strains of the same group (data not shown). However, mtDNA-derived profiles appear to be highly conserved within groups I and III, while some heterogeneity was observed among strains in group II (see below). Strain CBS 5301, whose original classification as C. parapsilosis was questioned recently (Nosek et al., 2002), displays a profile unrelated to any of the three groups (lane 4 in Figs 1 and 2). To examine its relationship to distinct groups of C. parapsilosis and the closest related species, L. elongisporus, the sequences of the ITS region of the rDNAs were determined and compared. As described by Lin et al. (1995) and Enger et al. (2001), the strains from distinct C. parapsilosis groups form three adjacent branches on the phylogenetic tree. However, the ITS sequence of CBS 5301 clearly clusters with the sequences of two strains of L. elongisporus (CBS 2605T and CBS 2606; Fig. 3). Together with the electrophoretic karyotype and restriction enzyme analysis of mtDNA, the results strongly suggest that CBS 5301 represents an anamorphic strain of L. elongisporus.



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Fig. 1. PFGE analysis. DNA samples were prepared and separated by PFGE using a programme for chromosomal (a) and mtDNA separation (b) as described in Methods. Note that circular-mapping mitochondrial genomes are represented mainly by polydisperse linear mtDNA molecules (Williamson, 2002) and, in contrast to linear-mapping mtDNAs, do not exhibit a distinct band in PFGE (Fukuhara et al., 1993). Lanes: 1, C. parapsilosis CBS 604T (group I); 2, MCO 456 (II); 3, MCO 448 (III); 4, CBS 5301; 5, L. elongisporus CBS 2605T; 6, CBS 2606; 7, CBS 6120.

 


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Fig. 2. Comparison of C. parapsilosis and L. elongisporus strains. Samples of mtDNA were digested with EcoRV (a) and PvuII (b) and separated by agarose gel electrophoresis (see Methods). Lanes: 1, C. parapsilosis CBS 604T (group I); 2, MCO 456 (II); 3, MCO 448 (III); 4, CBS 5301; 5, L. elongisporus CBS 2605T; 6, CBS 2606; 7, CBS 6120; M, molecular mass marker ({lambda} DNA digested with PstI).

 


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Fig. 3. The phylogenetic relationship among C. parapsilosis and L. elongisporus strains. The tree was constructed from the sequences of the ITS region (ITS1–5·8S rRNA–ITS2) from the indicated yeast strains using the AlignX program (Informax).

 
Physical mapping of mtDNA in different groups of C. parapsilosis
Previous investigations demonstrated that the mitochondrial genome in C. parapsilosis CBS 7157 (SR23) consists of a population of linear DNA molecules that terminate with a variable number of the 738 bp unit repeated in tandem (Nosek et al., 1995). Southern hybridization and subsequent DNA sequence analysis uncovered a standard set of mitochondrial genes starting with nad3 near the left telomere and terminating with atp6 close to the right telomere (Fig. 4) (Kovac et al., 1984; Nosek & Fukuhara, 1994a, b; Nosek et al., 1995; J. Nosek, M. Novotna, Z. Hlavatovicova, D. W. Ussery, J. Fajkus & L. Tomaska, unpublished). Results of the restriction enzyme analysis of mtDNA by HindIII, EcoRV, PvuII and KpnI indicated that the restriction fragment patterns seem to be constant among group I strains. This is in agreement with results described by Camougrand et al. (1988) and by Su & Meyer (1989, 1991).



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Fig. 4. Genetic organization of mtDNA in C. parapsilosis groups. Gene contents were deduced by sequencing the termini of EcoRV and HindIII restriction fragments [MCO 456 (group II) and MCO 448 (group III)]. In the case of the group I strain CBS 7157 (SR23), the map is derived from previous studies (Kovac et al., 1984; Nosek & Fukuhara, 1994a, b; Nosek et al., 1995; J. Nosek, unpublished). While mtDNA of strains from groups I and III displays a linear map terminating with telomeric tandem arrays (shown as black arrows), the group II strain possesses a circular-mapping mtDNA.

 
Since strains MCO 456 and MCO 448 from groups II and III, respectively, exhibit restriction fragment patterns different from the type strain CBS 604T and other group I strains, it was of interest to find whether these alterations represent an intraspecies polymorphism or reflect profound differences in genetic organization of the mtDNA. Restriction enzyme mapping of mtDNA by a combination of single- and double-digestions with restriction endonucleases indicate that strains CBS 2916 and MCO 448 (group III) possess linear-mapping mitochondrial genomes, while MCO 456 (group II) displays a circular-mapping mtDNA (Fig. 4). These results are in agreement with PFGE analysis that revealed a distinct band corresponding to the linear mitochondrial genome in the group I and III strains, but not in the group II strain (Fig. 1b).

To study the genetic organization of mtDNA in strains from groups II and III, the plasmid libraries of EcoRV and HindIII fragments of mtDNA from strains MCO 456 and MCO 448 were constructed. Next, the gene contents in both mtDNAs were inferred by sequencing the termini of the restriction fragments inserted within the plasmid vector. The data were then compared with the GenBank database as well as with the complete sequence of mtDNA of the group I strain CBS 7157 (Nosek & Fukuhara, 1994a, b; Nosek et al., 1995; J. Nosek, M. Novotna, Z. Hlavatovicova, D. W. Ussery, J. Fajkus & L. Tomaska, unpublished). This approach led to the identification of nad1-5, cytb, atp6 and rrnL genes in MCO 456 and nad1-5, cytb, cox1, cox3, atp6 and rrnL in MCO 448. Subsequently, these sequences were localized on the restriction enzyme maps of both mtDNAs. The results indicate that mitochondrial genomes in group II and III strains have the same gene order as found in group I strains (Fig. 4). In several cases the coding regions were fully sequenced and the data show a significant degree of homology when compared with their counterparts from group I (Table 3).


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Table 3. Percentage identity of DNA sequences and deduced protein products of mitochondrial genes among strains from the three different C. parapsilosis groups and C. albicans (GenBank accession no. AF285261)

Numbers in parentheses indicate the homology when conservative amino acid substitutions are included. NA, Complete sequence is not available.

 
The overall length of the mitochondrial genome was estimated to be approximately 23 kbp in both MCO 456 (group II) and MCO 448 (group III) strains. Since mtDNA of CBS 7157 (30·9 kbp) contains two and three introns within the cytb and cox1 genes, respectively (J. Nosek, M. Novotna, Z. Hlavatovicova, D. W. Ussery, J. Fajkus & L. Tomaska, unpublished), shorter mtDNAs in group II and III strains may reflect the absence of some of these sequences. Our preliminary results support this idea, since a partial DNA sequence analysis of the cytb gene indicated that strain MCO 448 lacks the bi2 intron (data not shown).

Mitochondrial telomeres differ between strains belonging to the variant groups
DNA sequence analysis of terminal regions uncovered that linear mitochondrial genomes from strains in groups I (CBS 7157) and III (MCO 448) differ in the sequence of their telomeric regions. While the mitochondrial telomeres in the strain belonging to group I consist of arrays of tandem repeats of a 738 bp unit (Nosek et al., 1995), the telomeric repeat in the group III strain is remarkably shorter (620 bp). However, the two sequences possess highly conserved regions and overall identity is 72 % (for details see the GenBank/EMBL database entries AY391851, AY391852, X76196 and X76197).

Survey of C. parapsilosis strains
To analyse the frequency of linear- and circular-mapping mtDNA in this species, we employed the PFGE method for screening of linear mitochondrial genomes (Fukuhara et al., 1993). This approach revealed a distinct band corresponding to linear mtDNA molecules in almost all strains from groups I and III, but only in one group II strain (MCO 471; Table 1).

Mitochondria of strain CBS 7157 harbour extragenomic minicircular DNA molecules derived solely from the mitochondrial telomere repeat motif that seem to play a key role in the dynamics of the linear mtDNA terminal structures (Tomaska et al., 2000; Nosek & Tomaska, 2002). Therefore, we examined their occurrence in strains from the three groups by probing Southern blots of electrophoretically separated undigested total cellular DNA with an [{alpha}32P]dCTP-labelled telomeric probe. The results show that the presence of the minicircles (Fig. 5) correlate with the linear form of mtDNA (Table 1), supporting the hypothesis that minicircles are involved in mitochondrial telomere maintenance.



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Fig. 5. Screening for the presence of mitochondrial telomeric minicircles by Southern blot analysis. Samples of undigested total cellular DNA were electrophoretically separated in a 1 % agarose gel and transferred onto a nylon membrane. Blots were then hybridized with a radioactively labelled probe derived from the 620 bp EcoRV telomeric fragment from mtDNA of strain MCO 448 (group III). The scale pattern indicates the presence of multimers of the telomeric minicircular DNAs. Lanes: 1, CBS 604T (group I); 2, CBS 1954 (I); 3, CBS 2152 (I); 4, CBS 2193 (I); 5, CBS 2194 (I); 6, CBS 2195 (I); 7, CBS 2197 (I); 8, CBS 2211 (I); 9, CBS 2215 (I); 10, CBS 2916 (III); 11, CBS 6318 (I); 12, CBS 7157 (I); 13, CBS 8050 (I); 14, CBS 8181 (I); 15, CBS 5301; 16, MCO 433 (I); 17, MCO 441 (I); 18, MCO 448 (III); 19, MCO 456 (II); 20, MCO 457 (II); 21, MCO 462 (II); 22, MCO 471 (II); 23, MCO 478 (I); 24, PL 429 (III); 25, PL 448 (III); 26, PL 452 (II). All strains except CBS 5301 (lane 15) are C. parapsilosis.

 
Recently, we have demonstrated that mitochondrial telomeres represent specific molecular markers with potential in molecular diagnostics of C. parapsilosis (Nosek et al., 2002). Based on the differences in the mitochondrial telomeres of CBS 7157 and MCO 448 and in the corresponding region (nad3atp6) of the circular-mapping mtDNA of MCO 456, we designed a panel of oligonucleotide primers derived from telomeric and subtelomeric regions (Table 2). Subsequent PCR analysis (Fig. 6, Table 1) showed that these primers allow the identification of strains with alterations within the telomeric regions of their mitochondrial genomes. To confirm the results, restriction fragment maps of mtDNA were constructed and compared (Fig. 7).



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Fig. 6. Screening for strains with an altered molecular architecture. PCR reactions were performed as described in Methods with the indicated primer pairs (Table 2) and products were electrophoretically separated on a 1·5 % agarose gel. Lanes: 1, CBS 604T (group I); 2, MCO 456 (II); 3, MCO 448 (III); 4, CBS 2194 (I); 5, MCO 457 (II); 6, MCO 462 (II); 7, MCO 471 (II); 8, PL 448 (III); 9, PL 452 (II).

 


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Fig. 7. Restriction enzyme mapping of mtDNA in group II and III strains. (a) Samples of mtDNA from C. parapsilosis strains were digested with EcoRV and HindIII, respectively, and electrophoretically separated. Lanes: 1, MCO 456 (group II); 2, MCO 457 (II); 3, MCO 462 (II); 4, MCO 471 (II); 5, PL 452 (II). (b) Restriction fragment maps of group II strains. Except for strain MCO 471, all group II strains contain circular-mapping mtDNAs. The asterisk indicates the position of faint bands with lengths of about 0·9, 1·0, 1·2 and 1·3 kbp, respectively. (c) Comparison of linear- and circular-mapping strains of group III strains. Note that mtDNA of CBS 2916 exhibits an identical restriction map to strains MCO 448 and PL 429.

 
Polymorphisms in mtDNA
In contrast to the conserved restriction fragment profiles of mtDNA in strains from the groups I and III, we observed a polymorphism within group II (Fig. 7a). The variability is within the sequence between the genes rrnL and nad4 and is present on the 5·0 kbp EcoRV fragment in strain MCO 456. In strains MCO 457, MCO 462, MCO 471 and PL 452, the length of the corresponding restriction fragment is approximately 6·5 kbp. The mtDNA of strain PL 452 also contains an additional 0·6 kbp EcoRV fragment, the position of which was not precisely determined. Since mtDNAs of strains from groups I and III contain the cox1 gene within this region, it is possible that the differences are attributed to the presence of intronic sequence(s) that might be absent in strain MCO 456. The polymorphism also affects the largest EcoRV fragment. In strains MCO 456, MCO 457, MCO 462 and PL 452 the length of this fragment varies from 13·0 to 13·5 kbp while in strain MCO 471 this same region is divided into two separate fragments of approximately 6·8 kbp split exactly between the atp6 and nad3 genes. This corresponds with the results of PFGE analysis that indicate the presence of a linear mitochondrial genome in MCO 471 (Table 1) and suggests that this strain has the molecular architecture of mtDNA typical of group I and III strains.

Restriction enzyme mapping using EcoRV and HindIII localized the polymorphic region between genes atp6 and nad3 (Fig. 7b). The 1·1 kbp HindIII fragment of MCO 456 varies in size from 0·45 to 1·3 kbp in strains MCO 457, MCO 462 and PL 452. Interestingly, four non-stoichiometric bands produced by HindIII (0·9, 1·0, 1·2 and 1·3 kbp) that map in this region were observed in strain MCO 462, indicating either instability of this region or a heteroplasmic state of the cells (Fig. 7a, lane 3). Furthermore, the circular-mapping mtDNA of strain PL 448 lacks telomeres and possesses a restriction fragment corresponding to the fusion of the left and right termini (Fig. 7c). This indicates that, similar to group II strains, mitochondrial genomes identified in group III strains have identical genetic organization and differ only in their linear/circular state.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Taxonomic remarks
Differences among C. parapsilosis isolates led to the suggestion that distinct groups may represent separate species (Lin et al., 1995). A relatively low degree of DNA relatedness among the groups argues for this possibility (Roy & Meyer, 1998). Kurtzman & Robnett (1997, 1998) found that strain NRRL Y-17456 belonging to group II differs from the type strain (CBS 604T/ATCC 22019T) by 6 nt in the D1/D2 domain of rRNA and concluded that this strain represents a new species of Candida. Although comparisons of rRNA sequences are successfully employed in the molecular taxonomy of yeasts, in an evolutionary perspective, a species does not necessarily need to be homogeneous in molecular terms and therefore biological criteria for the species need to be examined (Naumov, 1987). However, crossing strains from different groups followed by an analysis of fertility in the progeny is not possible in yeasts where the sexual state is unknown. Regardless of this taxonomical problem, the data presented in this work clearly show that genetic organization as well as coding sequences in mtDNA are highly conserved among the three groups of C. parapsilosis, pointing to their close phylogenetic relationship. Moreover, the results indicate that mtDNA profiles enable discrimination between the groups, exemplifying that investigations of mtDNA polymorphism represent a powerful tool to distinguish subspecies variants or close, recently separated species.

Evolutionary implications
The existence of linear genophores in mitochondria of diverse phylogenetic taxa evokes questions concerning their evolutionary origin and relatedness to circular-mapping mtDNAs. In several cases it has been observed that the form of mitochondrial genome may differ among closely related species (Bridge et al., 1992; Fukuhara et al., 1993; Martin, 1995; Laflamme & Lee, 2003). Investigations of mtDNA-binding proteins indicate that similar machineries are responsible for the maintenance of both forms (Miyakawa et al., 1995, 1996; Nosek et al., 1999; Kaufman et al., 2000; Tomaska et al., 2001). The occurrence of linear- and circular-mapping genomes in different strains of the same species indicates that both types of mitochondrial genome do not differ radically in their life styles (Fukuhara et al., 1993). One such example has been reported in two strains of the saturn-spored yeast, Williopsis suaveolens. While mitochondria of strain CBS 1670 with a linear-mapping genome harbour mtDNA molecules terminating with inverted repeats closed by a single-stranded hairpin loop, strain CBS 255 possesses a mitochondrial genome with similar genetic organization except that its map is circular (Fukuhara et al., 1993; Drissi et al., 1994). Here we report another example of intraspecies variability in mtDNA architecture in several strains of C. parapsilosis. The structure of the linear mitochondrial genome found in this species substantially differs in the type of mitochondrial telomere from that of W. suaveolens (Nosek et al., 1995). While nearly all strains in C. parapsilosis groups I and III possess a linear mtDNA that terminates with arrays of tandem repeats, only one group II isolate (MCO 471) has a linear genome in its mitochondria. However, most of the strains in group II and PL 448 from group III display a circular map of their mtDNAs. Moreover, the linear mtDNAs in groups I and III differ in the sequences of their telomeric motifs.

The genetic organization and homology of the coding regions appear to be highly conserved among linear- and circular-mapping mitochondrial genomes found in C. parapsilosis strains, indicating that both forms originated from a common ancestor via a relatively simple mechanism. At present it is not possible to make any definitive conclusion as to whether a circular-mapping mtDNA represents either an ancestral form or a rearranged derivative (mutant) that lost the mitochondrial telomeres and solved the ‘end-replication problem’ (Olovnikov, 1971,1973; Watson, 1972) by circularization of the genophore, similar to bacteriophage {lambda} (reviewed by Taylor & Wegrzyn, 1995) or nuclear chromosomes in the telomerase-deficient mutant of the fission yeast Schizosaccharomyces pombe (Nakamura et al., 1998).

Mitochondrial telomeres play essentially the same biological role(s) as their nuclear counterparts, i.e. they have to (i) ensure the complete replication of the linear genophore, (ii) mask the ends from DNA repair machinery and (iii) protect them from exonucleolytic degradation and/or end-to-end fusions. Identification of a specific mitochondrial telomere-binding protein (mtTBP) (Tomaska et al., 1997; Nosek et al., 1999) and mitochondrial telomeric loop (t-loop) structures (Tomaska et al., 2002) similar to the t-loops present at the ends of mammalian chromosomes (Griffith et al., 1999) substantiates the analogy. Observation of extragenomic telomeric minicircles in mitochondria of several yeast species (Tomaska et al., 2000) uncovered yet another general theme in telomere biology. Recently, we have demonstrated that the minicircles replicate via a rolling-circle mechanism which generates tandem arrays of the telomeric sequence that may recombine with linear molecules of mtDNA to lengthen the telomeres (L. Tomaska, A. M. Makhov, J. D. Griffith & J. Nosek, unpublished). This may parallel alternative mechanisms of nuclear telomere maintenance involving telomeric small polydisperse circular DNAs detected in several telomerase-negative tumour cell lines (Regev et al., 1998) and Xenopus oocytes (Cohen & Mechali, 2002), or an elongation of telomeres by recombination with DNA circles as demonstrated in the yeast Kluyveromyces lactis (Natarajan & McEachern, 2002).

Recently, we proposed a hypothesis that mitochondrial telomeres might have evolved from mobile genetic elements (e.g. transposons, plasmids, telomeric minicircles) that invaded mitochondria, integrated into a circular-mapping mtDNA and eventually resulted in the formation of linear mtDNA molecules of defined length, terminating with specific telomeric structures (Nosek & Tomaska, 2002, 2003). Insertions and excisions of such elements may represent a simple mechanism for switching between the two forms of the mitochondrial genophore. Some support for this idea comes from the correlation between the occurrence of the linear-/circular-mapping genome and the presence/absence of extragenomic telomeric minicircles (Fig. 5, Table 1). One possibility, although speculative, is that some drugs might interfere with the replication of mitochondrial telomeres and/or telomeric minicircles thus selecting for the formation of a circular-mapping genome without any apparent changes in mitochondrial physiology and/or maintenance of the mtDNA. Hence, the variability in the telomeric region might be generated by antifungal treatment of patients infected with C. parapsilosis. The identification of a collection of strains with altered mtDNA architecture among C. parapsilosis isolates opens the unique possibility of uncovering molecular mechanism(s) that trigger the alteration of the mtDNA form and may be instrumental in experimental testing of the above hypotheses.


   ACKNOWLEDGEMENTS
 
We thank L. Kovac (Comenius University, Bratislava, Slovakia) for continuous support, helpful discussions and comments; H. Fukuhara (Institut Curie, Orsay, France) for critical reading of the manuscript and the gift of the yeast strains, P. F. Lehmann (Medical College of Ohio, Toledo), S. A. Meyer (Georgia State University, Atlanta) and A. Vaughan-Martini (University of Perugia, Perugia, Italy) for providing the yeast strains, and G. Minarik (Comenius University, Bratislava, Slovakia) and Y. Hugodot (University of Paris XI, Orsay, France) for excellent technical assistance with automatic DNA sequencing. A. J. Cesare (University of North Carolina, Chapel Hill, NC, USA) is acknowledged for valuable editorial advice. This work was supported in part by grants from the Howard Hughes Medical Institute (55000327), the Slovak grant agencies VEGA (1/9153/02 and 1/0006/03) and APVT (20-003902), and the Fogarty International Research Collaboration Award (1-R03-TW05654-01). J. N. received an EMBO short-term fellowship.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 12 December 2003; revised 14 January 2004; accepted 15 January 2004.