A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application

Gene 292 (2002) 141–149 www.elsevier.com/locate/gene

A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application Debra Aker Willins, George H. Shimer Jr 1, Guillaume Cottarel* Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02453, USA Received 16 July 2001; received in revised form 4 February 2002; accepted 22 April 2002 Received by B. Dujon

Abstract We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also contructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Fungus; Infection; Expression; Vector; Essential; Allele replacement

1. Introduction Candida species are the major cause of life-threatening fungal infections and represent the fourth most common isolates from blood cultures in American hospitals (Georgopapadakou, 1998). Candida albicans is an important opportunistic pathogen of immunocompromised patients, causing superficial mucocutaneous infections as well as serious systemic infections, which cause almost 30% Abbreviations: Ade, adenine; ADE2, gene encoding aminoimidazole ribonucleotide carboxylase; ARS, autonomously-replicating sequence; bp, base pairs; C., Candida; CEN, centromere; His, histidine; HIS3, gene encoding imidazoleglycerol-phosphate dehydratase; Leu, leucine; LEU2, gene encoding isopropylmalate dehydrogenase; MT-1, metallothionein I; NEO, gene encoding neomycin/kanamycin resistance; ORF, open reading frame; p, plasmid; S., Saccharomyces; Ura, uracil; URA3, gene encoding orotidine-5 0 -phosphate decarboxylase * Corresponding author. Tel.: 11-781-398-2487; fax: 11-781-398-2476. E-mail address: [email protected] (G. Cottarel). 1 Present address: Cubist Pharmaceuticals, Inc., 24 Emily Street, Cambridge, MA 02139, USA.

mortality in these patients (Gale et al., 1998). The frequency of systemic fungal infections has increased in the last several decades, largely due to an increase in the number of immunosuppressed patients and patients treated with long-term antibiotic therapy, chemotherapy, and invasive procedures or devices (Georgopapadakou, 1998). The major responsible pathogen is C. albicans, normally a commensal organism, but the relative fraction of infections caused by non-albicans Candida species is increasing (Fidel et al., 1999). There is a strong need for antifungal agents with improved spectrum, efficacy, and safety and therefore a need to develop better molecular genetics tools to identify genes important for fungal growth and virulence. Although C. albicans is the most commonly-isolated pathogenic Candida species, it is difficult to perform genetics experiments with this organism because it lacks a haploid state and does not undergo sexual crosses. We therefore focussed our attention on the haploid fungus C. glabrata, a pathogen which can cause infection with the same mortality rate as C. albicans (Fidel et al., 1999) and is

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00648-0

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responsible for 10–15% of Candida infections (Cormack and Falkow, 1999). C. glabrata is the fourth most common Candida species in blood isolates and, at some sites of infection, it represents the second or third most common cause of Candida infection after C. albicans (Fidel et al., 1999). The frequency of C. glabrata infections is increasing, a cause for concern because they are difficult to treat and frequently resistant to azole compounds (Fidel et al., 1999). C. glabrata shows promise as a useful model organism because it is amenable to molecular genetics. However, compared with other Candida species, C. glabrata is relatively poorly studied. It undergoes efficient homologous recombination (Cormack and Falkow, 1999) and auxotrophic mutants are available (Cormack and Falkow, 1999; Kitada et al., 1995; Zhou et al., 1994) but there are few autonomously-replicating vectors. In order to provide additional molecular genetics tools for working with this organism, we developed a simple method for deletion of C. glabrata genes that is amenable to high-throughput application and constructed two vectors for complementation and other expression experiments. Combined, these tools would be particularly useful in anti-fungal drug discovery to identify genes essential for growth or virulence of this organism and to screen anti-fungal compounds. 2. Materials and methods 2.1. Strains and media C. glabrata Dhis3, Dtrp1, Dura3 strain ATCC (American Type Culture Collection) 200989 (Kitada et al., 1995) and C. glabrata clinical isolate ATCC 38326 were used as sources of genomic DNA for polymerase chain reaction (PCR) and ATCC 200989 was also used as a host for DNA-mediated transformation. C. glabrata strains were grown at 308C on YEPD (Yeast Extract Peptone Dextrose) or CSM (Complete Supplement Mixture) minimal media using components from Q-Biogene (formerly Bio101). In plasmid expression experiments pictured in Figs. 3 and 4, media for uninduced conditions was prepared from copper and zinc-free YNB (Yeast Nitrogen Base) (Q-Biogene) with the addition of zinc sulfate to 0.4 mg per liter. Media for induced conditions was prepared by the addition of cupric sulfate to 0.2 mM and zinc sulfate to 0.4 mg per liter (both chemicals were from Sigma Chemical Co.). Geneticin was from Gibco-BRL Life Technologies. C. glabrata was transformed with DNA using the Frozen EZ Yeast Transformation Kit (Zymo Research) and genomic DNA was prepared from transformants using the YeaStar Genomic DNA Kit (Zymo Research). 2.2. Construction of deletion strains Each deletion fragment was generated by PCR from three pieces: (1) a fragment containing 400 bp of sequence derived from the region upstream of the gene of interest, with a short 40

base segment of homology to the 5 0 end of the marker gene encoding imidazoleglycerol-phosphate dehydratase (HIS3); (2) the C. glabrata HIS3 marker (sequences from 223 bp before the ATG translation start codon to 171 bp after the translation stop codon); and (3) a fragment containing 400 bp of downstream sequence from the gene of interest, with a short 40 base segment of homology to the 3 0 end of the HIS3 marker. These three pieces were generated by PCR with C. glabrata genomic DNA from strain ATCC 200989 (fragments from the genes encoding aminoimidazole ribonucleotide carboxylase, ADE2 and gene encoding isopropylmalate dehydrogenase, LEU2) or ATCC 38326 (HIS3 fragment) and primers listed in Table 1 as shown in Fig. 1A. PCR amplification was performed in a 50 ml reaction with 0.3 mM of each primer and approximately 100 ng of genomic DNA template using PCR SuperMix High Fidelity enzyme (Gibco-BRL Life Technologies). PCR conditions were as follows: (a) 928C for 2 min; (b) nine cycles of 928C for 30 s, 508C for 30 s, and 688C for 4 min; (c) 29 cycles of 928C for 30 s, 508C for 30 s, 688C for 4 min plus an additional 20 s per cycle; and (d) 688C for 7 min. To generate a fragment containing the HIS3 marker gene flanked by 400 bp segments upstream and downstream of the gene of interest, the three fragments were purified on a Qia-quick column (Qiagen) and used as templates in a final PCR reaction with only the two outermost primers (left and right) with the same conditions described above. As numbered relative to the ATG translation startsite, the ADE2 deletion fragment lacked sequences from 1240 to 11497 bp (215 bp before the STOP codon) or about 74% of the open reading frame (ORF) and the LEU2 deletion fragment lacked sequences from 220 to 1852 bp (245 bp before the STOP codon) or about 78% of the ORF. The DNA was precipitated with ethanol and approximately 30 mg was used to transform C. glabrata strain ATCC 200989 (Dhis3, Dtrp1, Dura3). His 1 transformants were selected, patched onto CSM medium lacking histidine, and tested for deletion of the ADE2 or LEU2 genes by their growth phenotype after replica-plating onto selective media. 2.3. Construction of pFPG1, pFPG2, and expression test plasmids To construct the expression plasmid pFPG1, the promoter and terminator regions of the C. glabrata metallothionein I gene (MT-1) (GenBank accession number J05133) (Mehra et al., 1989) were cloned into the Saccharomyces cerevisiae vector pRS416 (GenBank accession number U03450) (Sikorski and Hieter, 1989). The promoter region of MT-1 (bases 2308 to 2 1 relative to the ATG translation start) was amplified by PCR with hybrid primers containing the SacII restriction enzyme site (P CgMT-1 up and P CgMT-1 down) using genomic DNA from strain ATCC 38326 and cloned into the SacII site of pRS416. In a similar manner, the terminator region of MT-1 (bases 11 to 1 253 relative to the STOP codon) was amplified with hybrid primers containing the KpnI restriction site (T CgMT-1 up and

D.A. Willins et al. / Gene 292 (2002) 141–149 Table 1 Oligonucleotide primers used a

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Table 1 (continued) Primer

Sequence

ACT GTG TGT CAA GTT CCA ATT GAG

CgCEN down

GTC AAA ATG ATC AAG CTT CCG TTG ACG CGT AAC TAA TGA TGC AAT TTT TG

CgADE2-CgHIS inner left

GTC TTT TCT GAG GAA TTT CAA GTT TCT GAG TCA TTT TCC TCG CCA ATG CAT TAA CAT CCA CAT GCT CGA

CgARS up

GCA TCA TTA GTT ACG CGT CAA CGG AAG CTT GAT CAT TTT GAC CCC ATC

CgADE2-CgHIS inner right

TGT GCC ATT CAT AAA CGT GAT CAC TTT ACG TAG CAG GCA ACC CCA CTA TTG TTC AAA TGC CAA GAG GTG

CgARS down

AGG TCC GCC GGC GGA CTT ACG CTC TAT CCG GTA ACG TTA G

CgADE2-ATG CgADE2-right

CTA CGT GTT ACA CTG GAA TGA AGG

GAA CCG CTC GAG CGG ATG GAC TCT AGA ACT GTC GGT ATT

CgLEU2-left

CCC TAT CTT GAA ACT GGT TAT GGT

CgADE2-STOP

GAA CCG CTC GAG CGG CTA TTT GGA CTC TAG GTA CTT TTC

CgLEU2-Cg HIS inner left

GTC TTT TCT GAG GAA TTT CAA GTT TCT GAG TCA TTT TCC TCG CCA GTG TGT GTA TAG TGT ATC CTC TTC

CgLEU2-ATG

CAA CGC GGA TCC GCG ATG GCT GTG ACC AAG ACA ATT GTA

CgLEU2-CgHIS inner right

TGT GCC ATT CAT AAA CGT GAT CAC TTT ACG TAG CAG GCA ACC CCA TAT GAG CCA TGT CAT GGC TCT GCT

CgLEU2-STOP

CAA CGC GGA TCC GCG CTA AGC TAA TAG TTC CCT GAC AGC

neo-ATG CgLEU2-right

CAG ATG ATT CAC CGG TTT GAT AGT

CAA CGC GGA TCC GCG ATG AGC CAT ATT CAA CGG GAA ACG

Cg HIS3-left

TGG CGA GGA AAA TGA CTC AGA AAC TTG

neo-STOP

CAA CGC GGA TCC GCG TTA GAA AAA CTC ATC GAG CAT CAA ATG AAA CTG C

Cg HIS3-right

TGG GGT TGC CTG CTA CGT AAA GTG

Primer

Sequence

CgADE2-left

Primer sequences are given from the 5 0 end (left) toward the 3 0 end (right). a

Cg HIS3-B

TGC TCT CCC GTA CAG AAA CAG ACC

Cg HIS3-C

CCA TCG CCA TCA GAG AGG CAA GAA

CgADE2-A

TGA AAG ATA CTT TTC TGC CAC TCC

CgADE2-D

GAT GAC TAT CAG AAA TTT GGC GTT GAT

CgADE2-E

GTA GGG TAG ATT TCC AAT TTG GGG

CgADE2-F

GAG TAC CTG TCA AGG GCT CAT TCT

CgLEU2-A

CCA ATT CTG TGT TTC CCG GAA ATG

CgLEU2-D

GTT CGT TTG CCG ATA CAT GCG AAT

CgLEU2-E

TCA CCT GGT GGA ACT ACA ATT GTC

CgLEU2-F

TAC TTC CAT CTG CCT CCT TGG CAT

P CgMT-1 up

AGG TCC CCG CGG GGA CGC CCT TCA TAC ACA TCC TAC ACT

P CgMT-1 down

AGG TCC CCG CGG GGA TGT GTT TGT TTT TGT ATG TGT TTG TTG

T CgMT-1 up

CAA CGG GGT ACC CCG TTG CAT TAA CAA CTA AAG CAA ACT ACT

T CgMT-1 down

CAA CGG GGT ACC CCG TTT CGT CGT GGA AGC GTG GAT CGT

CgCEN up

AGG TCC GCC GGC GGA TCT AGA AAA TAC ATA GTG AAT CT

T CgMT-1 down) and cloned into the KpnI site of pRS416, at the opposite end of the multiple cloning site. Proper amplification and construction were verified by DNA sequencing across both inserts. This vector was designated pFPG1. To generate pFPG2, the C. glabrata centromere 8 (CEN8) and autonomously-replicating sequence 10 (ARS10) regions (GenBank accession numbers U43926 and U43925) (Kitada et al., 1996) were cloned into pFPG1, as follows. The CENARS region was generated by PCR from two pieces: (1) a fragment containing CgCEN8, with an NgoMIV restriction enzyme site at the 5 0 end and a short 18 base segment of homology to CgARS10 at the 3 0 end; and (2) a fragment containing CgARS10, with a short 18 base segment of homology to CgCEN8 at the 5 0 end and an NgoMIV restriction enzyme site at the 3 0 end. These two pieces were generated by PCR amplification with C. glabrata genomic DNA from strain ATCC 38326 using primers CgCEN up and CgCEN down or CgARS up and CgARS down, respectively (Table 1). To generate a fragment containing the entire CEN–ARS region, the two fragments were used as templates in a final PCR reaction with only the two outermost primers (CgCEN up and CgARS down). This assembled CEN–ARS region was cloned into the NgoMIV site of pFPG1 to generate pFPG2. Proper amplification and construction were verified by DNA sequencing across the insert.

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Fig. 1. Construction of the deletion fragments. (A) Position of primers used to construct the deletion fragment. The black box depicts the C. glabrata HIS3 gene including its upstream and downstream flanking sequences. The unshaded and gray boxes represent sequences flanking and within the coding sequence of your favorite gene (YFG), the gene to be deleted. The inner-left and inner-right primers are hybrid primers with some sequence derived from HIS3 and some from YFG, as depicted by the shading. (B) The three individual PCR fragments generated in the initial PCR reaction. (C) The final PCR product (used to generate the C. glabrata deletion strains) results from a PCR reaction containing the left and right primers with the left flank, HIS3, and right flank PCR products. (D) Position of primers used in a PCR test to determine whether the deletion fragment integrated at the desired locus. PCR reactions were performed with A/D, A/ B/E, and C/D/F primer sets.

To evaluate expression and complementation, we cloned the coding sequence of the C. glabrata ADE2 and LEU2 genes, and a neomycin/kanamycin resistance gene, into pFPG1. We amplified the entire coding sequence of the ADE2 gene using genomic DNA from C. glabrata strain ATCC 38326 and hybrid primers containing the XhoI restriction site (CgADE2-ATG and CgADE2-STOP), then cloned the ORF into the XhoI site of pFPG1. DNA sequencing showed that the pFPG1-ADE2 plasmid carries a GTG valine codon instead of the expected GTA valine codon at amino acid position 231 (additional changes as compared to the GenBank sequence, accession no. AF030388, were identical between two independent clones and were probably due to strain differences or sequence errors). An ADE2 version of pFPG2 was constructed by cloning the C. glabrata CEN–ARS NgoMIV fragment from pFPG2 into the pFPG1-ADE2 plasmid, selecting a clone with the CEN– ARS fragment in the same orientation as in pFPG2. Similarly, the entire LEU2 ORF (amplified with CgLEU2-

ATG and CgLEU2-STOP) was cloned into the BamHI site of pFPG1. The pFPG1-LEU2 plasmid carries no mutations compared to the expected sequence (again, additional changes as compared to the GenBank sequence, accession no. U90626, were identical between four independent clones and were probably due to strain differences or sequence errors). A LEU2 version of pFPG2 was constructed by cloning the C. glabrata CEN–ARS NgoMIV fragment from pFPG2 into the pFPG1-LEU2 plasmid, selecting a clone with the same CEN–ARS orientation as in pFPG2. Finally, we amplified the entire Tn903 neomycin/kanamycin resistance ORF with plasmid pNK2887 DNA as template using hybrid primers containing the BamHI restriction site (neo-ATG and neo-STOP) and cloned the ORF into the BamHI site of pFPG1. The pFPG1 plasmid with the gene encoding neomycin/kanamycin resistance (NEO) carries a leucine (Leu) codon instead of an isoleucine codon at position 4 compared to the expected sequence reported in GenBank accession no. X06402. A NEO version of pFPG2

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was constructed by cloning the C. glabrata CEN–ARS NgoMIV fragment from pFPG2 into the pFPG1-NEO plasmid, selecting a clone with the same CEN–ARS orientation as in pFPG2. 3. Results 3.1. A method for deletion or modification of Candida glabrata genes The PCR-based technique, splicing by overlap extension (SOEing) or recombinant PCR, has been used to generate hybrid DNA fragments for gene disruption and allele replacement (Horton et al., 1989; Higuchi, 1990; Amberg et al., 1995). For this technique, two individual PCR products are generated with chimeric primers so that a segment at the 3 0 end of the left fragment is identical to a segment at the 5 0 end of the right fragment. In a second PCR reaction, the two PCR products are mixed and denatured. They anneal at their common segment and are extended to make a long final product, which is amplified by primers hybridizing to its ends. We have previously used a variation of this technique with three PCR products to generate hybrid DNA fragments replacing S. cerevisiae genes of interest with a marker gene and successfully used such fragments to generate S. cerevisiae deletion mutants in a high-throughput manner with a large number of genes (Willins et al., 2002). Here, we applied a similar variation of SOEing to modify C. glabrata genes in a manner amenable to high-throughput application. To demonstrate the utility of this method, we generated hybrid DNA fragments to delete most of the chromosomal copy of the C. glabrata ADE2 and LEU2 genes and replace them with the C. glabrata HIS3 gene (Fig. 1). These DNA fragments were used to transform C. glabrata strain ATCC 200989 (Dhis3, Dtrp1, Dura3), selecting for the HIS3 marker. After replica-plating onto selective media, we found that on average 20% of the transformants had the adenine 2 (Ade 2) or Leu 2 growth phenotype expected for a deletion of the chromosomal locus (Table 2). Genomic DNA was prepared from five transformants of each phenotype (Ade 2, Ade 1, Leu 2, Leu 1) and used in a PCR test with Table 2 Results of the deletion experiment a Gene deleted

Transformants that fail to grow on selective media (%)

ADE2

(1) 30 (2) 11

LEU2

(1) 28 (2) 15

a

C. glabrata was transformed with DNA fragments to replace the ADE2 and LEU2 genes. Independent transformants were replica-plated onto selective media (-Ade -His and -Leu -His media) and non-selective media. Results presented are from two independent experiments with about 100 transformants each.

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primers as shown in Fig. 1D to assess whether the deletion fragments had integrated at the homologous locus in the chromosome as expected, replacing the wildtype locus by double crossover recombination. In each case, all transformants expected to have the deletion by their growth phenotype also showed correct integration by the PCR test and none of the transformants with a wildtype phenotype did (data not shown). One Dade2 strain (FSG015) and one Dleu2 strain (FSG020) were chosen for further study. This method can easily be adapted for high-throughput deletion of C. glabrata genes. Any non-essential gene can be deleted directly. For essential genes, it is necessary to complement the deletion-associated growth defect by expressing the wildtype gene before introducing the deletion. Toward that end, we constructed a plasmid that would express a wildtype copy of C. glabrata genes. 3.2. Construction of Candida glabrata expression plasmids We constructed a C. glabrata expression plasmid, pFPG1, by cloning a regulatable promoter and a terminator into the pRS416 plasmid (Sikorski and Hieter, 1989), which carries the S. cerevisiae gene encoding orotidine-5 0 -phosphate decarboxylase (URA3) and the S. cerevisiae CEN-ARS region. Transcription from the C. glabrata MT-1 metallothionein gene, one of the mostly tightly-regulated genes described for the organism, is induced about 10-fold in media containing 0.1 mM copper sulfate, with very little transcription in media lacking copper (Mehra et al., 1989). The pFPG1 expression plasmid carries the C. glabrata MT-1 promoter and terminator at different restriction enzyme sites flanking the multiple cloning site of pRS416, leaving several internal restriction enzyme sites available for cloning genes to be expressed (Fig. 2A). We also constructed a second version of the expression plasmid, pFPG2, which carries the C. glabrata CEN8 and ARS10 regions in addition to the S. cerevisiae CEN-ARS region (Fig. 2B). These regions confer plasmid stability in C. glabrata (Kitada et al., 1996). 3.3. Expression of test genes To evaluate the expression vectors, we cloned the entire ORFs of the C. glabrata ADE2 and LEU2 genes into pFPG1 and pFPG2 to test the function of the plasmids in expression and complementation of the ADE2 and LEU2 deletions. We also expressed the entire ORF of the neomycin/kanamycin resistance gene. The first set of experiments were performed with pFPG1, which lacks the C. glabrata CEN-ARS region. The pFPG1ADE2 plasmid and empty pFPG1 as a control were transformed into strain FSG015, the C. glabrata Dade2 strain we constructed, and tested for their ability to complement the adenine-dependent growth of that strain (Fig. 3A). As expected, strain FSG015 carrying the empty plasmid failed to grow under either uninduced or induced conditions. Strain FSG015 carrying the pFPG1-ADE2 plasmid failed

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Fig. 2. Map of the pFPG1 and pFPG2 expression plasmids. P and T CgMT-1 represent the promoter and terminator, respectively, from the C. glabrata metallothionein I gene. Restriction sites marked are unique to the plasmid. Sc represents S. cerevisiae–derived sequences, Cg represents C. glabrata-derived sequences. (A) The pFPG1 vector. (B) The pFPG2 vector.

to grow under uninduced conditions but grew well in induced conditions. This indicated that ADE2 expressed from pFPG1 was sufficient to complement the ADE2 deletion and that induction of the MT-1 promoter was necessary for adequate expression. In a similar manner, the pFPG1-LEU2 plasmid and empty pFPG1 as a control were transformed into strain FSG020, the C. glabrata Dleu2 strain we constructed, and tested for their ability to complement the leucine-dependent growth of that strain (Fig. 3B). As expected, strain FSG020 carrying the empty plasmid failed to grow under either uninduced or induced conditions. Strain FSG020 carrying the pFPG1-

LEU2 plasmid grew moderately well under uninduced conditions and well under induced conditions. This indicated that LEU2 expressed from pFPG1 complemented the LEU2 deletion. However, in this case, induction of the MT-1 promoter was not absolutely necessary for expression. Presumably in pFPG1 there is a low level of transcription from the metallothionein promoter under uninduced conditions and the resulting expression is sufficient to complement the leucine deficiency of strain FSG020. The neomycin/kanamycin resistance gene from Tn903 confers resistance of C. glabrata to the antibiotic G418, geneticin (Cormack and Falkow, 1999). The pFPG1-NEO

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plasmid and empty pFPG1 as a control were transformed into C. glabrata strain ATCC 200989 and tested for geneticin resistance under uninduced and induced conditions (Fig. 3C). We found that this strain was relatively resistant to geneticin, requiring about 1 mg/ml geneticin to suppress growth with an empty pFPG1 plasmid. In the presence of 1 mg/ml geneticin, strain ATCC 200989 carrying the empty plasmid failed to grow under either uninduced or induced conditions, whereas the same strain carrying the pFPG1NEO plasmid grew moderately well under uninduced conditions and well under induced conditions. This indicated that neomycin/kanamycin resistance was expressed from pFPG1. In this case, induction of the MT-1 promoter in pFPG1 was not absolutely necessary to obtain adequate expression of the NEO gene. The second set of experiments were performed in a similar fashion with the pFPG2 version of the vector, which carries the C. glabrata CEN-ARS region in addition to the S. cerevisiae CEN-ARS region. It has been shown previously

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that plasmids containing the C. glabrata CEN-ARS region are maintained in C. glabrata at about one copy per cell (Kitada et al., 1996). By contrast, the S. cerevisiae centromere does not function in C. glabrata and plasmids carrying the S. cerevisiae CEN-ARS region are maintained at 10–30 copies in C. glabrata (Zhou et al., 1994). Therefore, one would expect our pFPG2 vector to be stably maintained in a lower copy number than pFPG1, which carries only the S. cerevisiae CEN-ARS region. Strain FSG015 (C. glabrata Dade2) carrying pFPG2ADE2 failed to grow under either uninduced or induced conditions (Fig. 4A), indicating that the level of expression from the pFPG2 plasmid is not sufficient to complement the ADE2 deletion. By contrast, strain FSG020 (C. glabrata Dleu2) carrying pFPG2-LEU2 failed to grow under uninduced conditions and grew moderately well under induced conditions (Fig. 4B). This indicated that LEU2 expression from pFPG2 was sufficient to complement the LEU2 deletion and that induction of the MT-1 promoter was necessary

Fig. 3. Growth phenotypes of C. glabrata strains carrying the expression plasmid pFPG1. To evaluate the expression vector, growth of strains carrying the expression plasmid pFPG1 with or without a wildtype test gene were compared on selective media lacking or containing copper sulfate to induce the MT-1 promoter. All strains were grown for 3 days. (A) C. glabrata Dade2 strain with expression plasmid pFPG1 lacking or carrying the wildtype ADE2 gene, on -His -Ura -Ade media. (B) C. glabrata Dleu2 strain with expression plasmid pFPG1 lacking or carrying the wildtype LEU2 gene, on -His -Ura -Leu media. (C) C. glabrata strain with expression plasmid pFPG1 lacking or carrying the wildtype neomycin/kanamycin resistance gene (which confers resistance to the antibiotic geneticin), on -Ura media supplemented with 1 mg/ml geneticin.

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Fig. 4. Growth phenotypes of C. glabrata strains carrying the C. glabrata CEN-ARS expression plasmid pFPG2. To evaluate the expression vector, growth of strains carrying the expression plasmid pFPG2 with or without a wildtype test gene were compared on selective media lacking or containing copper sulfate to induce the MT-1 promoter. All strains were grown for 3 days. (A) C. glabrata Dade2 strain with expression plasmid pFPG2 lacking or carrying the wildtype ADE2 gene, on -His -Ura -Ade media. (B) C. glabrata Dleu2 strain with expression plasmid pFPG2 lacking or carrying the wildtype LEU2 gene, on -His -Ura Leu media. (C) C. glabrata strain with expression plasmid pFPG2 lacking or carrying the wildtype neomycin/kanamycin resistance gene (which confers resistance to the antibiotic geneticin), on -Ura media supplemented with 1 mg/ml geneticin.

for adequate expression. Furthermore, C. glabrata strain ATCC 200989 carrying pFPG2-NEO failed to grow under uninduced conditions and grew well under induced conditions (Fig. 4C). This indicated that neomycin/kanamycin resistance was expressed from pFPG2 as well and that induction of the MT-1 promoter was necessary for adequate expression. In summary, pFPG2 apparently exhibits lower expression levels (probably because of a lower plasmid copy number), which allows regulated expression for the LEU2 and NEO genes.

4. Discussion Current PCR-based strategies to delete S. cerevisiae genes make use of DNA fragments containing about 50 bp of homology with the chromosome (Winzeler et al., 1999; Niedenthal et al., 1999). By contrast, the frequency

of homologous recombination into the C. glabrata chromosome has been found to be very low with 50 bp and even 100 bp of homology but high with 200 bp of homology or more (Cormack and Falkow, 1999). Because we did not expect standard PCR-based techniques to be effective for C. glabrata, we developed a technique based on deletion fragments with longer homology regions. Here we describe an effective cloning-free method for deletion of C. glabrata genes using PCR to produce homology regions of several hundred base pairs or longer. Given its similarity to a high throughput method we have used for deletion of large numbers of S. cerevisiae genes (Willins et al., 2002), we expect that it will be amenable to high throughput. Although we applied this technique to genes expected to have a deletion phenotype, any C. glabrata gene could potentially be deleted in this way, as long as sufficient DNA sequence is available to design primers for the deletion fragment and for a PCR test to confirm proper integration. Furthermore, we have

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successfully applied a similar strategy to delete several C. albicans genes (D.A. Willins, A. Santos, S. Hogan, and G. Cottarel, unpublished results) and we anticipate that the procedure will be applicable to other fungi as well. Finally, the same technique is also broadly applicable to modifying genes and replacing alleles in other ways, such as introducing mutations or generating fusions to reporter genes or alternative promoters. We have also constructed two C. glabrata expression vectors which allow the expression of a cloned gene under the control of the C. glabrata MT-1 copper-regulated promoter. Both pFPG1 and pFPG2 vectors carry the S. cerevisiae URA3 marker gene and an S. cerevisiae CEN-ARS region. In addition, the pFPG2 vector carries the C. glabrata CEN–ARS region for stable plasmid maintenance in C. glabrata. We have successfully expressed the C. glabrata ADE2 gene in the pFPG1 vector and the C. glabrata LEU2 gene in both vectors and used them to complement the auxotrophies of the ADE2 and LEU2 deletion strains, repectively. We have also expressed the neomycin/kanamycin resistance gene in both vectors, conferring resistance to the antibiotic geneticin. Furthermore, we found that expression of the ADE2 gene from pFPG1 and expression of the LEU2 and NEO genes from pFGP2 is regulated by copper induction of transcription from the MT-1 promoter. These vectors could be used for a variety of expression and complementation applications, including regulatable expression. Potentially, they could be useful for heterologous expression as well, although none of the three test genes were expressed from pFGP2 in S. cerevisiae in the presence of 0.2 or 0.02 mM cupric sulfate (not shown). In C. glabrata, expression from the MT-1 promoter in the pFGP1 vector is high enough to produce the expected growth phenotypes for all three test genes, indicating that it is appropriate for expression and complementation applications. If regulated expression is critical, one can evaluate expression from the pFPG1 vector and also the pFPG2 vector, which contains the C. glabrata CEN-ARS region. The pFPG2 vector apparently exhibits a lower expression level, probably due to a lower plasmid copy number. We obtained copper-regulated expression for each of the three test genes using one of these two vectors, indicating the vectors would be valuable in a variety of expression applications.

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