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Yeast, 2000, 16 1205-1215

MET15  as a visual selection marker for  Candida albicans

Jasmine Viaene, Petra Tiels, Marc Logghe, Sylviane Dewaele, Wim Martinet and Roland Contreras
 

ABSTRACT

To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied. Both MET15 alleles of C. albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene. Growth of this complemented strain on Pb²⁺-containing medium was associated with a colour shift of brown into white colonies. The MET15 alleles of C. albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting. A met15-deficient strain of C. albicans CAI-4 was generated using the ura blaster technique. This strain showed a brown colony colour on Pb²⁺-containing medium, which corresponded with the colony colour of a S. cerevisiae strain lacking the MET15 gene. Unexpectedly, the met15-deficient strain of C. albicans still grew on methionine-depleted medium. However, this growth was severely delayed. In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb²⁺-containing medium. These transformants grew very well on methionine-depleted medium. Colony sectoring was obtained with the replicative plasmid and not with the integrative one. This study demonstrates that the MET15 gene of C. albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability. GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275.

Keywords: MET15; Candida albicans; colony colour; methionine auxotrophy; visual selection marker

INTRODUCTION

The MET15 gene of the yeast Saccharomyces cerevisiae has proved to be an important visual marker for molecular biological and genetical applications in yeast (Cost, 1999). MET15, also named MET17 and MET25, encodes the enzyme O-acetylhomoserine O-acetylserine sulphhydrylase (EC 4.2.99.10), which is a component of the sulphur assimilation pathway. It catalyses both the sulphhydrylation of O-acetylhomoserine to homocysteine and the conversion of O-acetylserine to cysteine. This enzyme is a tetramer with a subunit molecular weight of 50 000 Da (Yamagata, 1976). Met15- deficiency in yeast leads to a nutritional requirement for methionine, cysteine or homocysteine. Met15 mutants accumulate HS- ions, which leak out of the cell. When lead is present in the medium, formation of a dark brown lead sulphide precipitate on the surface of the cell is observed, resulting in a dark brown colony phenotype (Ono et al., 1991; Cost, 1999). For several reasons, Cost and Boeke (1996) described the MET15 gene as one of the most versatile yeast genetic markers. Its size, counter-selectability with methyl-mercury (Singh and Sherman, 1974), the methionine auxotrophy and the easy detection of met15 mutants through the appearance of a colony colour shift on Pb2+- containing medium contribute to the MET15 gene utilization. Since few tools are available in the molecular research of Candida albicans, we have investigated the suitability of its MET15 gene as a visual selection marker. For this purpose, the MET15 gene of C. albicans was isolated and a met15- deficient C. albicans strain was generated.

MATERIALS AND METHODS

Strains

The met15-deficient S. cerevisiae strain BY411 (MATα, trp1Δ63, his3Δ200, ade2Δ, ura3Δ, lys2Δ0, met15Δ0) was obtained from the ATCC collection (ATCC No. 200405). S. cerevisiae INV-Sc1 (MATα, leu2-3,-112, trp1-289, ura3-52, his3Δ1) was purchased from Invitrogen Corp. C. albicans B2630 (ATCC No. 44858) is a clinical isolate obtained from the Janssen Research Foundation. C. albicans CAI-4 (ura3Δ) is described by Fonzi and Irwin (1993). Transformations into Escherichia coli were performed using the K12 MC1061 strain (araΔ139, Δ (ara leu) 7697, Δlac X74, gal U, gal K, r_k- m_k+, Sm^R; Casadaban and Cohen, 1980).

Media

Synthetic dextrose medium (SD), containing 1% glucose and 1.34% Yeast Nitrogen Base without amino acids, was used to study methionine auxotrophy. In the case of ura3-deficiency, this medium was supplemented with 50 µg/ml uridine for C. albicans or 20 µg/ml uracil for S. cerevisiae. Depending on the nutritional requirements, the medium was supplemented with adenine (20 mg/ ml), histidine (20 µg/ml), lysine (30 µg/ml), tryptophan (20 µg/ml) and methionine (20 µg/ml). Lead medium (GLA), containing 0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% ammonium sulphate, 0.016% lead acetate and 0.016% lead nitrate, was used to study the colony colour phenotype. To prepare plates the media were solidified with 2% agar.

Complementation of the met15-deficient S. cerevisiae strain and characterization of the clones

Complementation of the met15-deficient S. cerevisiae strain BY411 was achieved by transformation of either a genomic DNA library or a cDNA library according to the LiAc method (Ito et al., 1983). The genomic DNA library was generated by subcloning DNA fragments of 6000-10 000 bp into the BamHI site of plasmid YCp50 (Johnston and Davis, 1984). Those fragments were obtained by a partial digestion of genomic DNA of C. albicans B2630 with Sau3A. The cDNA library was prepared in the plasmid pSCGAL10-SN (Goldman et al., 1992), using mRNA isolated from C. albicans B2630, grown on SD medium. The cDNA was synthesized with flanking SfiI-NotI restriction sites to allow cloning behind the gal10-cytochrome c hybrid promoter in sense orientation. After transformation, the resulting transformants were pooled and plated onto methionine-free medium to select transformants containing the MET15 gene. In the case of the cDNA library, 1% galactose was used instead of dextrose to induce the hybrid promoter. Plasmid DNA was prepared from the positive clones using the method of Elder et al. (1983), followed by electroporation of the DNA into E. coli MC1061. Plasmid DNA was isolated from these transformants and analysed by restriction digest to determine the length of the insert (1600 bp for the cDNA insert, corresponding with the expected size of polyadenylated MET15 cDNA, and 6000-7800 bp for the genomic DNA insert). Based on the sequence information of the first 349 bp of the C. albicans MET15 open reading frame (forward sequence of sample 265223H07 of the Candida Sequencing Project at Stanford's DNA Sequencing and Technology Center; http://candida.stanford.edu/bin/ gbrowse2?265223-265223H07), the downstream primer 315823 (5'-CTACAATGCCTTCTCAC-3'), ranking from nucleotides x5 to +12, was synthesized. A PCR was performed using plasmid DNA (50 ng) of the cDNA transformants, primer 315823 and an upstream primer (5'-GGATCATAAGCTTGTGAATTCG- 3k) located on the plasmid pSCGAL10-SN, approximately 50 bp behind the cDNA insert. Brie¯y, the hot start PCR was carried out in a PTC-200 Thermal Cycler (MJ Research) according to the following program, with a first cycle of denaturation for 10 min at 95°C followed by a touch down started at 59°C for 2 min minus 1 degree per cycle (nine cycles). Then 25 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 1 min and elongation at 72°C for 2 min were performed. The PCR was carried out with 2.5 U Taq DNA polymerase (Boehringer-Mannheim), 200 mM dXTPs and the buffer supplied by the manufacturer, supplemented with MgCl2 (final concentration of 3 mM). Most of the clones gave rise to a PCR fragment of t1700 bp, suggesting the presence of the MET15 cDNA. The 5k end of the cDNA insert of four positive clones was sequenced using the primer 5k-CGCAAACACAAATACACACAC- 3k located at the end of the gal10-cytochrome c hybrid promoter. Sequencing was performed following an ABI Taq DyeDeoxy Terminator Cycle Sequencing protocol (ABI). The resulting sequences were compared with the one obtained from the Stanford Candida data bank (as described above), showing that the cDNA of MET15 was isolated. Using the walking primer strategy, the entire C. albicans MET15 cDNA was sequenced. To determine the presence of the MET15 sequence on the genomic DNA clones (obtained after complementation) sequence analyses were performed with the downstream primer 315823 (described above) and the upstream primer 5k-CAACTGGTTGACCAGCATG- 3k located at position +31. This primer was selected based on the MET15 cDNA sequences. Comparison of the obtained sequences with those of the MET15 cDNA and the Stanford data bank, revealed the presence of the genomic MET15 sequence. The position and orientation of the MET15 gene on the genomic DNA insert was identified by PCR using different combinations of the primers described above and two additional primers located on the plasmid YCp50 (one downstream primer at the left site of the genomic DNA insert (5'-TCCTGCTCGCTTCGCTACTTG-3') and one upstream primer at the right site (5'- CGGTGATGTCGGCGATATAGG-3'). The PCR was performed following standard conditions. A restriction endonuclease map of the genomic DNA insert was constructed. Based on this map and on the position and orientation of the MET15 gene an easy strategy was found to shorten the genomic DNA insert. Digestion with EcoRI, followed by religation of the largest fragment, a modified YCp50 plasmid (377 bp of YCp50 were eliminated) was obtained containing a genomic DNA insert of -2200 bp. This plasmid was called YCpCaMET15. The shortened genomic DNA insert was sequenced completely using the walking primer strategy. Beside the open reading frame of 1320 bp, 542 bp upstream and 314 bp downstream sequence were present.

Complementation experiments in the S. cerevisiae BY411 strain were confirmed with both the shortened genomic DNA clone and the cDNA clone.

Pulsed-field gel electrophoresis and Southern blot

A BioRad contour-clamped homogeneous electric field CHEF DR III system was used for pulsed-field gel electrophoresis. Chromosomal DNA was prepared in agarose plugs following the instructions of the manufacturer. A 0.8% agarose gel in 1rTAE buffer (6.2% acetic acid, 50 mM EDTA, pH 8, 0.24% Tris) was used to separate the chromosomes. The gel was electrophoresed with a 106° angle at 14°C at 3 V/cm for 40 h, with a linear ramping of the switch interval from 120-480 s. After separation, the chromosomes were visualized with ethidium bromide and the MET15 gene was mapped by Southern analysis. Therefore, the gel was incubated with 0.25 M HCl at 30°C for 5 min and neutralized in 0.5 M Tris, pH 7, at room temperature for 30 min. Capillary alkali transfer of the DNA was carried out onto a Hybond N+ membrane (Amersham). The MET15 probe was prepared by digesting plasmid pUCCaMET15 (see below) with XbaI and NcoI, followed by random labelling of the 1228 bp MET15 fragment with α ³²P dCTP, using the High Prime kit of Boehringer-Mannheim.

Construction of a met15 C. albicans knock-out

The disruption cassette used to create a met15 knock-out of C. albicans was constructed in the following way: nearly the entire MET15 sequence (2170 bp) was isolated from YCpCaMET15 by a Sau3A-EcoRI digest. After blunting the sticky ends with T4 DNA polymerase, the MET15 fragment was subcloned into the HincII site (dephosphorylated with calf intestinal phosphatase) of plasmid pUC18 (Vieira and Messing, 1982), creating plasmid pUCCaMET15. A 567 bp fragment of the coding MET15 sequence (encoding for the amino acids 181-369; Figure 1), ¯anked by HincII sites, was removed from pUCCaMET15 by HincII digestion and replaced with the hisG-URA3-hisG cassette, isolated from pMB7 (Fonzi and Irwin, 1993) using an Ecl136II-StuI digest, resulting into plasmid pUCCaMET15D::URAHIS (Figure 3). The MET15/hisG-URA3-hisG disruption cassette (5645 bp) was isolated from pUCCaMET15D::URAHIS by AvaI-HindIII digestion. The resulting fragment was used to create a double knockout of MET15 in C. albicans CAI-4, using the `ura blaster' technique of Fonzi and Irwin (1993). For transformation of the disruption cassette, a modified procedure (Logghe et al., in preparation) of the spheroplasting protocol described by Herreros et al. (1992) was used.

The transformants were further analysed by Southern blot. Genomic DNA was prepared using the Nucleon extraction and purification kit (Amersham) and digested with AccI. The MET15 probe used in the Southern blot was prepared by PCR using the PCR DIG Probe Synthesis Kit (Boehringer-Mannheim). The PCR was performed using plasmid pUCCaMET15Δ::URAHIS (500 pg) as a template, together with the lower primer 5k- CCATACAAGTAGGATGTACTG-3k (located in the first part, in front of the hisG-URA3-hisG cassette, of the coding MET15 sequence) and the upper primer 5k-GTTGTCCCTCAATTCACTCC- 3k (located in the MET15 promoter sequence). Standard PCR conditions were used. Southern blot hybridization and detection were performed following the DIG Easy Hyb procedure and the Detection Starter Kit II of Boehringer-Mannheim, respectively.

To determine the exact integration of the disruption cassette in the MET15 locus, the transformants have also been analysed by PCR (data not shown). Therefore, an upstream primer (5'-GTGAGGCATGAGTTTCTGC- 3k) located in the URA3 gene and a downstream primer (5'-CAGCATGTATCAA CAGTG-3') located in the 5k untranslated region of MET15 (upstream of the MET15 sequence of the disruption cassette) were used. The hot start PCR was performed on genomic DNA following standard PCR conditions.

Growth curves

For growth curves, yeast cells were grown for 24 h in SD medium supplemented with uridine and methionine. These cultures were diluted to an OD600 of 0.08 into fresh SD medium supplemented with uridine or uridine and methionine. Growth was monitored in microtitre plates using the Bioscreen C system (Labsystems).

Construction of the integrative and replicative plasmids

To construct the integrative plasmids pScMET15 and pCaMET15 containing the MET15 gene of S. cerevisiae or C. albicans, respectively, the pGAL1PNiST-1 vector (Logghe et al., in preparation) was used. This vector was opened with BamHI and BglII, after which the ends were flushed with T4 DNA polymerase. The MET15 gene of S. cerevisiae was isolated from plasmid pGC3 (Cost and Boeke, 1996) with a SpeI digest and the C. albicans MET15 gene was obtained by Sau3A/ EcoRI digestion of YCpCaMET15 (see above). The overhanging ends of both MET15 genes were blunted. The gene fragments were ligated into the pGAL1PNiST-1 vector, producing plasmid pScMET15 and pCaMET15.

The replicative plasmids pRM2ScMET15 and pRM2CaMET15 were constructed by ligating both MET15 genes into the SmaI restriction site of pRM2 (Logghe et al., in preparation).

Results

Isolation of the MET15 gene of C. albicans

Both C. albicans MET15 alleles (containing the promoter) were isolated by complementation of the met15-deficient S. cerevisiae strain BY411 (Cost and Boeke, 1996), using a genomic DNA library of C. albicans B2630 (clinical isolate). Subcloning of a 2.2 kb genomic DNA fragment was sufficient to complement the methionine auxotrophy and to evoke a white colony colour phenotype on Pb2+- containing medium. Sequence analysis revealed an open reading frame of 1320 nucleotides (440 amino acids). Fortunately, bearing on the unusual CUG codon usage in C. albicans (Santos and Tuite, 1995), MET15 contains no CTG codons, which facilitated heterologous expression. In addition, we also isolated the MET15 cDNA by complementation with a cDNA library, which was controlled by the gal10-cytochrome c hybrid promoter of S. cerevisiae. Comparison of the genomic and cDNA MET15 sequence showed no introns. Six nucleotide substitutions were found between both MET15 alleles. Five of them were silent, whereas only one (at position 745 of the coding sequence) corresponded with a conservative amino acid substitution (isoleucine to valine). The MET15 sequence is shown in Figure 1.

Chromosomal localization of MET15

Chromosomal localization of the MET15 gene was determined by pulsed-field gel electrophoresis using the C. albicans CAI-4 strain (Fonzi and Irwin, 1993), followed by Southern analysis using a MET15 probe comprising 545 bp of the 5k untranslated region and 683 bp of the MET15 ORF. A clear signal was obtained with chromosome 4 (Figure 2).

Generation of a met15-deficient strain of C. albicans

To evaluate the MET15 gene as a marker for C. albicans, a met15-deficient strain of C. albicans CAI-4 was generated using the `ura blaster' technique of Fonzi and Irwin (1993) (Figure 3). Both MET15 alleles were knocked out by replacing 567 bp of the coding sequence (amino acids 181-369) with a 1.1 kb hisG fragment of Salmonella typhimurium (Figure 4). The resulting met15-defi- cient strain shows a brown colony colour on Pb2+- containing medium. The lead concentration used for C. albicans was lower than that described for S. cerevisiae (Ono et al., 1991), as concentrations above 0.07% have been found to be toxic for C. albicans. To obtain the brown colony colour with the met15 knockout and the white colony colour with the wild-type, a mixture of lead nitrate and lead acetate, both in a concentration of 0.016%, was found to be optimal. Unexpectedly, the met15 knockout strain can still grow in the absence of methionine; however, the growth is strongly delayed compared with the wild-type CAI-4. Growth was monitored on solid (Figure 5A) as well as in liquid medium. Growth curves are represented in Figure 5B.

MET15 complementation

Complementation experiments were carried out with the met15-deficient C. albicans strain, using the MET15 gene of either S. cerevisiae or C. albicans. In both cases the gene specific promoters were used. The MET15 genes were cloned into a replicative and an integrative C. albicans vector (Figure 6). The double knockout met15 strain was transformed with the resulting constructs, using the spheroplast transformation method of Logghe et al. (in preparation). The empty vectors served as a negative control. Transformants were selected on uridine-free medium and further studied on minimal medium (SD) without methionine and on Pb2+- containing medium. Complementation could not be obtained with the control vectors and with the S. cerevisiae MET15-containing plasmids. In contrast, complementation with the MET15 gene of C. albicans resulted in a wild-type growth pattern on methionine-free medium and white colonies on Pb2+-containing medium (Figure 7). Colony sectoring appeared with the replicative plasmid but not with the integrative one, indicating a decreased stability of the replicative plasmid on non-selective growth media (Figure 7).

Discussion

C. albicans is certainly not a model organism to work with. Its diploid genome and the absence of a sexual cycle, and the lack of good molecular genetic tools, both hinder molecular and genetic research. In this work, we have evaluated the MET15 gene of C. albicans as a visual selection marker. Previously, the MET15 gene of S. cerevisiae has been described as one of the most versatile yeast genetic markers (Cost and Boeke, 1996).

Both MET15 alleles of C. albicans, as their cDNA, were isolated by complementation of a met15-deficient S. cerevisiae strain. Sequence analysis revealed an open reading frame of 1320 nucleotides devoid of CTG codons and introns. Six differences were detected between the two alleles, resulting into only one amino acid substitution: isoleucine into valine. The other five mutations were silent. However, both alleles were functional in the met15-deficient strain of S. cerevisiae. Not only was methionine prototrophy obtained, but also a white colony colour phenotype on Pb2+-containing medium. Remarkably, the white colony colour did not appear when the met15-deficient strain was complemented with the MET15 cDNA of C. albicans, although methionine prototrophy was obtained. The genomic and cDNA clones differ in both the promoter and terminator sequences and the copy number of the plasmids. The cDNA was cloned behind the strong hybrid gal10-cytochrome c promoter, while the genomic clone contained a 550 bp upstream fragment, most likely containing the entire MET15 promoter. The genomic DNA library was constructed in a centromeric plasmid and the cDNA in a multicopy plasmid. The absence of colony colour shift when MET15 cDNA was used is difficult to explain, but differences in the expression level of MET15 could possibly be involved.

Both alleles of the MET15 gene of C. albicans CAI-4 are localized on chromosome 4, as we conclude from the results obtained after pulsed- field gel electrophoresis and Southern blot. Thus, large chromosomal translocations or further aneuploidy, concerning this gene, do not seem to be present.

A met15-deficient strain of C. albicans was generated using the ura blaster technique to investigate the suitability of the isolated MET15 gene as a visual selection marker. Growth of this met15 double knock-out strain was characterized by the formation of brown colonies on Pb2+-containing medium. Complementation of the knock-out with an integrative plasmid, containing the MET15 gene of C. albicans, gave rise to white colonies. Using a replicative plasmid, not only were white colonies obtained, but also colony sectoring was visible at high frequency, indicating the instability of the replicative plasmid. Based on these results, we conclude that the MET15 gene of C. albicans can be used to study plasmid stability and to screen for stability-enhancing elements. This had already been demonstrated for S. cerevisiae by Cost and Boeke (1996).

The met15-deficiency of the C. albicans met15 double knockout strain could not be restored by the MET15 gene of S. cerevisiae. Neither methionine prototrophy nor a white colony colour phenotype on Pb2+-containing medium was obtained. Nevertheless, the MET15 gene of S. cerevisiae contains no CTG codons and shows an amino acid homology of 69% with the C. albicans MET15. Probably the MET15 promoter of S. cerevisiae is not functional in C. albicans, although the MET15 promoter of C. albicans seems to work well in S. cerevisiae.

Unfortunately, the met15 double knock-out of C. albicans is still able to grow on methionine-free medium, although the growth is strongly delayed compared to the wild-type. Complementation with the C. albicans MET15 gene results in a normal growth pattern. This incomplete methionine auxotrophy of the double knockout strain could be explained by the presence of an additional monofunctional O-acetylserine sulphhydrylase. This enzyme synthesizes cysteine from O-acetylserine, after which cysteine is converted to homocysteine via the cystathionine pathway. Homocysteine can further be converted to methionine (for review, see Yamagata, 1989). Brzywczy and Paszewski (1993) mentioned the absence of this enzyme in S. cerevisiae, which explains the observed methionine auxotrophy in the case of met15-deficiency in S. cerevisiae. Other met15-deficient yeast strains, such as Schizosaccharomyces pombe, are not methionine-auxotrophic, due to the presence of this additional sulphhydrylase. A met15 knockout of Kluyveromyces lactis showed the same growth pattern as our C. albicans knockout strain (Brzywczy and Paszewski, 1993). This indicates that MET15 is important but not essential for growth on methionine-free medium. We tried to optimize our knockout strain to obtain complete methionine auxotrophy by using inhibitors of the methionine pathway. Ethionine was tested in concentrations between 0.5 and 25 mM, supplemented to the medium (data not shown). Surprisingly, the wild-type C. albicans CAI-4, as well as the C. albicans strain B2630, showed no growth inhibition. This is in contrast with the S. cerevisiae INVSc1 strain, which already showed full growth inhibition at the lowest ethionine concentration tested. The C. albicans met15 knockout grew even better when ethionine was present. A nearly wild-type growth pattern was obtained, suggesting that C. albicans possess the ability to convert ethionine.

In general, we conclude that MET15 can be used as a visual selection marker, for example to identify transformants and study plasmid stability. However, further optimization of the met15 double knockout strain will be necessary to use MET15 as a significant selection marker in C. albicans.

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