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Eukaryotic Cell, July 2007, p. 1239-1247, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00414-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Highly Efficient Gene Targeting System Induced by Transient Repression of YKU80 Expression in Candida glabrata{triangledown} ,{dagger}

Keigo Ueno,1 Jun Uno,1 Hironobu Nakayama,2 Kaname Sasamoto,1 Yuzuru Mikami,1 and Hiroji Chibana1*

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan,1 Suzuka National College of Technology, Shiroko, Suzuka, Mie 510-0294, Japan2

Received 30 December 2006/ Accepted 4 May 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C. glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, H2O2, UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of opportunistic fungal infection has been increasing, particularly in patients who are immunocompromised by human immunodeficiency virus infection or leukemia or who are receiving immunosuppressive therapy for organ transplantation or chemotherapy for cancer. Systemic fungal infections in such patients are often life threatening. Candida albicans and Candida glabrata are among the opportunistic pathogenic fungi that cause candidiasis (7). C. albicans remains the most commonly encountered species in clinical practice but other species (non-C. albicans species), such as C. glabrata, now cause significant levels of disease (35). In one study, C. glabrata was the second most commonly documented species, isolated from up to 20% of all candidiasis patients (31). It is known that C. glabrata has a low susceptibility to fluconazole, which is the first drug administered in clinical situations (12); thus, candidiasis caused by C. glabrata is associated with high mortality (17). Despite this increase in incidence, little is known about the molecular basis of C. glabrata virulence in contrast to that of C. albicans.

C. glabrata offers some advantages for the study of fungal pathogenesis. It is more closely related to Saccharomyces cerevisiae than to C. albicans (2, 12), suggesting that more of the wealth of information accumulated by decades of study of S. cerevisiae might be transferable to C. glabrata. C. glabrata is a haploid fungus, whereas C. albicans is diploid, making gene targeting in C. albicans at least twice as difficult as that in C. glabrata (4). The whole genome sequence of C. glabrata has been completed, thus allowing the comprehensive identification of all genes (10). Taking these attributes together, C. glabrata has now come of age as a feasible model organism for the genome-wide study of pathogenicity in fungi and for the identification of antifungal drug targets.

Gene targeting is one of the most important approaches for analysis of gene function. Genome-wide functional analyses are facilitated by efficient gene targeting using constructs flanked at each end with short homologous sequences, the lengths of which are typically 40 to 60 bp. These short homologous sequences can be easily produced by single-step PCR using a pair of oligonucleotide DNA primers containing homologous flanking sequences. However, targeted integration with short homologous sequences is relatively inefficient in C. glabrata, a characteristic it shares with higher eukaryotes and most fungi except S. cerevisiae (8, 22, 28, 40). S. cerevisiae has highly efficient gene targeting, with exogenous DNA fragments correctly integrated into the targeted locus with an efficiency close to 100% and requiring only short target sequences of 40 bp in length (3, 40). As a consequence, a complete set of gene knockout strains of S. cerevisiae has been successfully constructed (13).

The targeted integration of a DNA fragment requires homologous recombination, which is one of the repair mechanisms for DNA double-strand breaks (DSB). Nonhomologous end joining (NHEJ) is another mechanism of DSB repair, but it does not require such homologous DNA sequences (9, 30). We observed that in C. glabrata, NHEJ seems to predominate at integration events involving DNA fragments with flanking short homologous sequences. Therefore, we reasoned that if the activity of NHEJ could be suppressed, then the frequency of homologous recombination should increase. Several genes, such as KU70, KU80, and LIG4 and their homologues, have been identified as key genes for NHEJ in eukaryotes, including mammals, plants, and fungi. Recently, mutants have been constructed in which the genes homologous to human KU70 or KU80 were disrupted in Neurospora crassa (28); Kluyveromyces lactis (22); Aspergillus fumigatus (8); koji molds, including Aspergillus niger (25), Aspergillus sojae, and Aspergillus oryzae (38); Cryptococcus neoformans (16); and Sordaria macrospora (32). Drastically improved efficiency of gene targeting was found in these Ku mutants. It has also been shown that when NHEJ was suppressed, it led to sensitization to some DNA-damaging agents, including methyl methane sulfonate (MMS) (5, 16, 37). These findings have enabled important advances in meeting the objective of high-throughput construction of targeted recombinant fungal strains. After construction of the desired recombinant strain, the NHEJ genes are usually returned to the cell via mating. Although the sexual cycle has not been identified in C. glabrata, here we have developed a new system that delivers efficient gene targeting and restores the expression of YKU80 with a single transformation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and media. All strains used in this study are described in Table 1.


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  		TABLE 1. Strains used in this study

 
C. glabrata strains were grown routinely at 37°C on YPD (1% [wt/vol] yeast extract, 2% [wt/vol] Bacto peptone, 2% [wt/vol] dextrose) and SD (0.67% [wt/vol] yeast nitrogen base without amino acids, 2% [wt/vol] dextrose) media. Various growth supplements were added to SD medium as described in a standard experimental manual (6). For the screening of sulfometuron methyl (SM) resistance as was performed for HET80, the cells were plated onto SD plates plus 20 µg/ml histidine with 20 µg/ml SM (Wako, Inc.; dissolved in dimethyl sulfoxide to prepare 2 mg/ml stock solution). For the selection of nourseothricin (NSTC)-resistant strains, cells were plated onto YPD plates containing 100 µg/ml NSTC (HKI Jena, Germany; 10 mg/ml in water as stock solution). In order to test for the recovery of live C. glabrata from infected mice, homogenized kidney was spread on agar plates consisting of 1.5% (wt/vol) agar, 0.5% (wt/vol) yeast extract, 1% (wt/vol) polypeptone, 1% (wt/vol) dextrose, 1,000 units/ml penicillin G, and 100 µg/ml streptomycin sulfate (Wako Chemicals, Japan). For the repression of genes of interest, inactivation of the tetracycline regulatable promoter (Tet-P) was carried out in appropriate medium with 20 µg/ml doxycycline ([ICN Biomedicals Inc.] dissolved in 50% ethyl alcohol to prepare 2 mg/ml stock solution).

Escherichia coli DH5{alpha} was used for DNA manipulation. General recombinant DNA procedures were performed as described previously (15, 36).

Primers. For a list of the primers used in this study, see Table S1 in the supplemental material.

Plasmid construction. The plasmids used in this study are listed in Table 2. All plasmids were based on pBluescriptII SK(+) (Stratagene).


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  		TABLE 2. Plasmids used in this study

 
pKUILV2-1. A 6-kb DNA fragment, including YKU80-ILV2 regions, was amplified by PCR using primer pair preKu80HindIII and preILV2HindIII and genomic DNA of tADE2-38 as a template. After the PCR, the product was digested with HindIII and inserted into pBluescriptII SK(+) cleaved by HindIII to yield the plasmid pKUILV2-1.

pILV2-7. PCR was carried out with pKUILV2-1 and the primer pair of preKuR2 and preILV2F2. The primers anneal to sites flanking the open reading frame (ORF) of YKU80 toward the outside of the ORF in pKUILV2-1. The PCR product was circularized by self-ligation, and the ORF of YKU80 was removed from the plasmid to produce pILV2-7.

pBS-SF.4. PCR was carried out with the primer pair of pSFS2AF6(Bgl-Kpn) and pSFS2AR5(Nhe-Sac) and plasmid pSFS2A (34) as a template DNA, including the SAT1 flipper. The PCR product was digested with KpnI and SacI and inserted into pBluescriptII SK(+) that had been cleaved by KpnI and SacI to generate pBS-SF.4.

pKU-self.5. The plasmid pKU-self.5 was constructed to add BglII and NheI sites on YKU80. PCR was carried out with the primer pair of pYKU80F2 (Bgl) and pYKU80R2 (Nhe) and plasmid pKUILV2-1 as a template. The PCR products were treated with T4 polynucleotide kinase and circularized by self-ligation to construct pKU-self.5.

pKU-self(ORF2). PCR was carried out with the primer pair pYKU80ORF2.F (Bgl) and pYKU80ORF2.R (Nhe-Bgl) and plasmid pKUILV2-1 as a template. PCR products were digested and circularized by self-ligation.

pKUSF-18 and pKUSForf2-10. pBS-SF4 was digested with BglII and NheI and the inserted DNA, including the SAT1 flipper, was isolated from the plasmid. The isolated fragment was then ligated with plasmids pKU-self.5 and pKU-self(ORF2) cleaved by BglII and NheI to yield pKUSF-18 and pKUSForf2-10, respectively.

Genomic DNA isolation from C. glabrata and DNA manipulation. Cells were suspended in 40 to 70 µl of TE (10 mM Tris, pH 7.5, 1 mM EDTA), and an equal volume of 0.5-mm glass beads was added to the 2-ml collection tube. The cells were disrupted with Multi-Beads Shocker (Yasui Kikai, Inc.) by using five cycles of 2,700 rpm for 15 s with a 3-s brake time and then boiled for 10 min. From the resulting suspension, 1 µl was added for a 25-µl PCR mixture.

DNA amplification was performed using KOD-plus-DNA polymerase (Toyobo), GoTaq green master mix (Promega), or ready-to-go PCR beads (Amersham Biosciences) according to the manufacturers’ protocols. BigDye Terminator cycle sequencing kits (version 3.1; Applied Biosystems, Inc.) and an Applied Biosystems model 3100 automated capillary sequencer were used for nucleotide sequencing. Other standard recombinant-DNA procedures were performed as described previously (15, 36).

RNA manipulations for quantitative real-time RT-PCR. For RNA extraction, the hot-phenol method was performed as described previously (18). For quantitative real-time reverse transcription-PCR (RT-PCR), mRNA was purified from total RNA extracted with the Oligotex-dT(Super) mRNA purification kit (TaKaRa, Japan) according to the manufacturer's protocol. To quantify mRNA, we used the Syber RT-PCR kit (TaKaRa) for the synthesis of cDNA and real-time PCR. The real-time PCR for ACT1 was performed with primers ACT1 59F and ACT1 249R; that for YKU80 was performed with primers YKU80 622F and YKU80 798R (see Table S1 in the supplemental material) using an ABI PRISM 7000 (Applied Biosystems).

5' RACE. 5' RACE (rapid amplification of cDNA ends) was performed using the GeneRacer kit (Invitrogen). Briefly, total RNA from parent strain ACG4 was treated with calf intestinal phosphatase and then treated with tobacco acid pyrophosphatase to remove the 5' cap structure from intact, full-length mRNA. The GeneRacer RNA oligonucleotide, with a known priming site, was ligated to the 5' end of the mRNA by using T4 RNA ligase. The ligated mRNA was reverse transcribed into cDNA by random primers (N6). To obtain 5' ends, the first-strand cDNA strands were amplified using the GeneRacer 5' nested primer and primer kdyku80checkR1. Purified RACE PCR products were cloned into the pCR4-TOPO vector (Invitrogen) for sequencing.

C. glabrata transformation. The C. glabrata transformation protocol is a slightly modified version of the transformation protocol described in previous studies (20). Cells were streaked on a YPD agar plate and grown for 2 to 3 days at 37°C. A few colonies were then incubated in 10 ml of YPD liquid medium and cultured overnight with shaking at 37°C. The cells were resuspended in 10 ml of fresh YPD (starting optical density at 600 nm [OD600] of 0.4) and shaken to an OD600 of 1.0, approximately 2 to 3 h at 37°C. The cells were harvested by centrifugation at 6,000 x g for 5 min. The pellets were rinsed with 10 ml of TE buffer and harvested by centrifugation. The cells were resuspended in 10 ml of 0.15 M lithium acetate dissolved in TE buffer (LiOAc/TE) and shaken lightly for 1 h at 30°C. The cells were again harvested and resuspended in 400 µl of 0.15 M LiOAc. Sixty microliters of the suspension was dispensed to a new 1.5-ml tube, supplemented with 5 to 10 µg (3 µl) of the disruption cassettes and 20 µg (2 µl) of carrier DNA (salmon sperm DNA [Wako], boiled before use for 10 min and chilled for 10 min to denature double-strand DNA) and mixed gently. This solution was incubated for 30 min at 37°C and then added to 120 µl of 52.5% polyethylene glycol 4000-0.15 M LiOAc and mixed entirely by pipetting. The cells were incubated for 45 min at 37°C. After mixing carefully, the cells were heat shocked by incubating for 45 min at 42°C and then spread onto appropriate selection plates and incubated at 37°C for a few days. For NSTC-resistant strains like HETS202, the cells were harvested and resuspended in 1 ml of fresh YPD liquid medium after the 42°C heat shock and this suspension was shaken at 37°C for 12 or 24 h. The cells were then harvested, washed in YPD liquid medium, and resuspended in 400 µl of YPD liquid medium. The suspension was spread onto four YPD plates containing 100 µg/ml of NSTC, and the plates were incubated at 37°C for 1 day.

Strain construction. Strain tADE2-38, in which ADE2 expression is able to be controlled by the Tet-P, was constructed from ACG4 (26) by the integration of the Tet-P cassette into the ADE2 promoter locus (Table 1). The Tet-P cassette was amplified by PCR with the following primers and template: pCGHade2F2 and pCgTETade2R1 as primers and plasmid DNA p97CGH (26) as a template. tADE2-38 was screened on an SD plate without histidine and then streaked on an SD plate containing adenine and doxycycline to verify that the red colony color resulted from the knockdown of ADE2 expression. To confirm the correct integration of the Tet-P cassette, PCR was carried out using the primers pCgADE2checkF1 and pCgADE2checkR1.

A disruption cassette was amplified by PCR using pILV2-7 as a template and using preKU80HindIII and pILV2mR2 as primers (see Table S1 in the supplemental material). This cassette included the point mutation in ILV2 that changed amino acid 182 (proline) to leucine. The cassettes were used for the transformation of parent strain ACG4 and integrated into the YKU80-ILV2 locus by homologous recombination after the cassette entered into the cell. The transformant designated HET80 was screened on plates containing 20 µg/ml SM, and genomic DNA was extracted in order to check for the YKU80 deletion. The verification of YKU80 deletion was performed by PCR with three sets of primers; check PCR 1 used preKU80checkR2 and pKU80checkF2, check PCR 2 used pKU80checkR1 and pmILV2checkR1, and check PCR 3 used preKU80HindIII and Ku80checkmidR3.

SAT1 flipper in C. glabrata. The SAT1 flipper module was 4.3 kb long and contained the following DNA elements: the NSTC resistance gene SAT1, FLP1 encoding a site-specific recombinase, and two FLP recombination target (FRT) (34-bp) sites that are recognized by Flp1 recombinase (34). To examine the utility of the module in C. glabrata, ACG4 was transformed with the SAT1 flipper, leading first to illegitimate integration. The transformant ACG4-SF was confirmed by PCR and grown under permissive conditions (YPD plate without NSTC). NSTC-sensitive clones were screened by subsequent testing for growth under restrictive conditions on YPD plates with NSTC. Those clones have lost the SAT1 flipper, as confirmed by PCR analysis. To examine the efficiency of the SAT1 flipper module in C. glabrata, cells of HETS202, in which the SAT1 flipper was integrated, were cultured in the permissive liquid medium overnight at 37°C, spread on YPD plates, and then replicated on the restrictive plates (YPD, including 100 µg/ml of NSTC). Only 0.5% of the colonies could grow on the restrictive medium. Twelve colonies that could not grow on the selective plates were checked by PCR, and all of the strains produced a PCR product that proved the loss of the SAT1 flipper.

Knockdown system using SAT1 flipper. To knock down the YKU80 gene, the YKU80 knockdown cassette (Y-kd cassette) was integrated just upstream of either the first or the second ATG codon, yielding HETS146 and HETS202, respectively. The two Y-kd cassettes comprising the SAT1 flipper flanked by long homologous sequences were amplified by PCR using the following templates and primers. For the amplification of the first Y-kd cassette, pKUSF-18 was used as a template, and kdYKU80-1 and kdYKU80-4 were used as primers. For the amplification of the second Y-kd cassette, pKUSForf2-10 was used as a template, and preKU80HindIII and pILV2mR2 were used as primers. After the selection of transformants on YPD plates containing 100 µg/ml NSTC, PCR analysis was performed to confirm the site-specific integration of these Y-kd cassettes. Check PCR 1 used primers pSFS-2AF2(PacI) and kdYKU80checkF1; check PCR 2 used primers preku80checkR2 and ku80checkmidR3. YKU80 knockdown strains for gene disruption, HETS202, KUE100, and KUE200, were constructed by using C. glabrata strains 2001H and 2001HT (21).

POP-SF1467 and POP-SF221 were isolated by the following procedure. Transformants derived from HETS146 and HETS202 were incubated in YPD liquid medium without NSTC at 37°C for 24 h with shaking. Under this condition, the FLP1-mediated excision of the SAT1 flipper occurs automatically. The cells were spread onto YPD agar plates without NSTC and incubated at 37°C for 24 h. The colonies were replicated to YPD plates with and without 100 µg/ml NSTC. Clones, which did not grow on the plate containing NSTC, were confirmed to have excised the SAT1 flipper by PCR: check PCR 3 used primers pFRT1 F1 and kdYKU80checkF1; check PCR 4 used primers kdYKU80checkF1 and kdYKU80checkR1. POP-SF1467 and POP-SF221 were derived from HETS146 and HETS202, respectively.

Determination of gene targeting efficiency. The inactivation of ADE2, which is one of the genes required for adenine biosynthesis, induces the color of colonies to turn red due to the accumulation of the intermediate metabolite phosphoribosyl aminoimidazole (19). On an agar plate containing doxycycline, the transformant (tADE2), in which the Tet-P was integrated into the promoter region of ADE2 via homologous recombination, forms colonies that turn red. On the other hand, transformants in which the Tet-P was integrated into any other region form colonies that remain white. The gene targeting efficiency (%) was calculated by the following formula: number of tADE2 strains obtained from the transformation/total transformants x 100%. For a list of the primer pairs used to attach homologous tags to the Tet-P cassette, see Table S1 in the supplemental material. The calculation of the targeting efficiency using the other loci was carried out with PCR using appropriate primer sets.

Evaluation of DNA damage in {Delta}yku80 mutants in vitro. To measure the sensitivity of strains to DNA-damaging agents, we employed the microdilution method based on the CLSI (formerly NCCLS) standard M27-A (27). Drop titer tests and spread titer tests for sensitivity were also carried out. Cells from an overnight culture were harvested, rinsed with YPD, and then adjusted to an OD600 of 0.3. This suspension was diluted 10–3, and 3 µl of each dilution was dropped onto a plate containing a reagent. The plates were incubated at 37°C for 2 days. The spread titer test to measure the sensitivity to UV rays was performed as follows. Cells of an overnight culture were harvested, rinsed with YPD, and adjusted to an OD600 of 4. This suspension was diluted 10–4. Fifty microliters of the diluted suspension was spread onto two plates. These plates were exposed to 0, 20, 40, or 60 J/m2 254-nm UV rays (CL-1000 UV cross-linker [UV Products]) and then incubated at 37°C for 1 day. The CFU were determined on each exposed plate, and the survival rate (%) was calculated by the following formula: CFU on each exposed plate/CFU on the unexposed plate x 100%.

Infection experiment. Seven male CD-1 (ICR) mice, 5 weeks old and weighing 28.5 to 30.9 g, were used for virulence studies for each group. Cyclophosphamide (Shionogi & Co., Ltd., Japan) and hydrocortisone sodium phosphate (Banyu Pharmaceutical Co., Ltd., Japan) were concomitantly administered to the lateral tail vein at doses of 200 mg/kg of body weight and 125 mg/kg, respectively. Three days after the first administration, 100 mg/kg and 125 mg/kg of cyclophosphamide and hydrocortisone, respectively, and 1 x 107 cells of C. glabrata were administered into the lateral tail veins of the mice. The same amounts of the immunosuppressive agents were administered once every 4 days. The cells were suspended with saline to a final volume of 250 µl prior to their administration to the mice. Out of the seven mice allocated for each date, the heaviest and the lightest mice were removed and the five remaining mice were etherized and killed by cervical dislocation. Both kidneys were extirpated, homogenized, diluted with saline, and spread onto agar plates to determine the number of CFU of C. glabrata in the kidneys. This analysis was carried out at days 1, 7, and 14 after the injection of C. glabrata. The animal experiment complied with all relevant guidelines and policies of the Animal Welfare Committee of the Faculty of Medicine of Chiba University, Japan.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Length of homologous sequence required for efficient gene targeting to the correct locus in ACG4. The length requirement for the homologous sequence to induce correct gene targeting was measured in ACG4 (Fig. 1). The DNA cassette consists of the Tet-P, the HIS3 gene as a selection marker, and homologous sequences ranging in size from 40 to 1,000 bp on either side. When the flanking sequences were 40 bp, no correctly targeted integrants were identified. The highest efficiency of gene targeting was 63.5% when the length of the flanking sequences was 500 bp. The efficiency was decreased to 16.3% when the homologous sequence of the 5' side was 80 bp, even if the 3' side had a 500-bp homologous sequence (Fig. 1). Unexpectedly, when the length of the homologous sequences was 1,000 bp, the targeting efficiency was lower than the efficiency when the length was 500 bp. From those results, it was suggested that efficient gene targeting required a few hundred base pairs of homologous flanking sequence in ACG4.


Figure 1
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  		FIG. 1. Gene targeting efficiency depends on the length of the homologous flanking sequence. The numbers shown on the right side describe the number of correct integrants per total number of colonies tested. The lengths of homologous tags are shown on the left side. The designation 80-500 means that an 80-bp flanking sequence and a 500-bp flanking sequence were attached to the 5' and 3' sides, respectively, of the DNA cassette. The efficiency of gene targeting accompanied by homologous recombination was measured by determining the frequency of integration of a DNA cassette bearing a Tet-P into the endogenous promoter region of ADE2. When the Tet-P is integrated into the correct locus, the colonies turn red on plates supplemented with doxycycline. As in S. cerevisiae, red colony color is caused by inactivation of the ADE2 gene in C. glabrata (19). Closed and open boxes represent the gene targeting efficiency of HET80 and ACG4, respectively.

 
Effect of carrier DNA concentration on transformation efficiency. Denatured salmon sperm DNA is used as carrier DNA for experiments with S. cerevisiae and K. lactis (14, 22). The requirement for the amount of carrier DNA for efficient gene targeting in C. glabrata was investigated in this work. The efficiency was measured by the integration rate of the Tet-P cassette into the ADE2 promoter with a 200-bp homologous sequence. The highest efficiency was identified when 20 µg of carrier DNA was used. About two-times-higher efficiency was achieved when using this amount of carrier DNA than when no carrier DNA was used.

Construction of the YKU80 deletion mutant for efficient gene targeting. A search of the C. glabrata genomic sequences in Genolevures (http://cbi.labri.fr/Genolevures/) revealed that one gene has homology to S. cerevisiae YKU80. It is designated YKU80 (CAGL0K03443g) in C. glabrata. The amino acid sequences deduced from these genes shared 39% identity and 58% similarity. A DNA cassette was designed to delete YKU80 and simultaneously confer resistance to sulfometuron methyl. Recipient strain ACG4 was transformed with the cassette to construct yku80 mutant HET80. To confirm construction of the yku80 deletion, PCR analysis was performed using three sets of primers (Fig. 2).


Figure 2
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  		FIG. 2. Construction of YKU80 deletion mutant using SM resistance. (A) Procedure for the construction of the YKU80 deletion strain, HET80, with a PCR-mediated cassette. On chromosome K in C. glabrata, YKU80 (CAGL0K03443g) is adjacent to ILV2 (CAGL0K03465g), followed by 1 kb of intergenic space. A point mutation in Ilv2p, in which proline 182 is replaced by leucine, confers resistance to SM in S. cerevisiae (41). The cassette DNA fragment contained the 5' homologous flanking region of the YKU80-ILV2 locus and a part of the ILV2 gene carrying a point mutation that changed proline182 to leucine. Integration of the cassette removes YKU80 and induces the point mutation in Ilv2p, which confers SM resistance. The parent strain ACG4 was transformed with this deletion cassette, and transformants were selected on plates containing SM. The asterisks indicate the point mutation. (B) PCR analysis of the yku80 deletion strain with the three sets of primers. Lanes a and h show results for ACG4 and HET80, respectively. The lengths of the expected PCR products are shown on the right-hand side of panel A.

 
Gene targeting efficiency with short homologous sequences in HET80. The yku80 deletion mutant HET80 was evaluated for gene targeting efficiency (Fig. 1). The DNA cassette, including the Tet-P with the homologous flanking sequences ranging from 40 to 200 bp, was integrated into the promoter region of ADE2. The efficiency of gene targeting was 5.1% with 40 bp of homologous flanking sequence in HET80, while this length of homologous sequences provided no correct integration in ACG4. Likewise, with 80-bp flanking homologous sequences, the efficiency of gene targeting was 8.6% in HET80, approximately sevenfold more efficient than in ACG4. The efficiency rose to 80% with 500 bp of homologous flanking sequence in HET80. The relative increase in the rate of gene targeting efficiency caused by YKU80 deletion was greater with the shorter length of homologous flanking sequence in HET80 (Fig. 1).

To evaluate the targeting efficiency for various targets in C. glabrata, DNA cassettes for targeting to 27 different loci were produced and introduced into HET80. Between 5 and 30 colonies of the transformants were checked for the correct integration site by PCR. The average efficiency was 13% with 60 bp of homologous flanking sequence. These results suggest that gene targeting in HET80 was effective at various loci.

Susceptibilities of yku80 mutant to DNA-damaging agents. To characterize the yku80 deletion mutant of C. glabrata, the effects of temperature and DNA-damaging agents were examined in HET80. Since C. glabrata grows well at 37°C, 37°C and 42°C were chosen as permissive and restrictive temperatures, respectively. Whereas the growth seemed slightly delayed at 42°C in HET80, no distinct growth defect relative to the wild-type strain was identified. No valid differences between HET80 and ACG4 were recognized in the mortality of cells exposed to UV irradiation, MMS, H2O2, or hydroxyurea (Fig. 3). These results suggest that the YKU80 deletion mutant of C. glabrata is not disabled in general cellular functions or DNA repair.


Figure 3
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  		FIG. 3. Phenotypic characterization of the {Delta}yku80 mutant using DNA-damaging agents. The experiment was conducted by measuring the OD600 at each time point. Closed circles and open circles indicate results for ACG4 and HET80 ({Delta}yku80), respectively, in all panels. (A) The results of the growth rate analyses at 37°C and 42°C are indicated by solid and dotted lines, respectively. The vertical axis is represented by a logarithm scale. Sensitivity to each reagent and UV radiation was evaluated by determining the cell survival ratio and described as a percentage compared to cells grown without DNA-damaging reagents or UV radiation. The panels were assigned for sensitivities against (B) MMS, (C) hydrogen peroxide, (D) UV radiation, and (E) hydroxyurea. The sensitivity to UV radiation was calculated from three independent experiments and is shown as means ± standard deviations.

 
Construction of YKU80 knockdown strain with insertion of SAT1 flipper. To employ the beneficial effects of YKU80 inactivation on gene targeting without any complicating effects, including possible synthetic sickness or lethality, it is necessary to construct a strain in which the expression of YKU80 is restored following transient knockdown during transformation. To realize such a convenient strain, the module SAT1 flipper was integrated just before the first ATG of the annotated open reading frame of YKU80, creating the strain HETS146. Unfortunately, the gene targeting efficiency was greatly decreased in this strain (Fig. 4 and 5). This was an unexpected result, and we constructed another strain, HETS202, in which the SAT1 flipper was integrated just before the second ATG, which is 81 nucleotides downstream from the first ATG on the same frame. During incubation under nonselective conditions, HETS146 and HETS202 lost the SAT1 flipper and produced POP-SF1467 and POP-SF221, respectively.


Figure 4
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  		FIG. 4. Insertion and pop-out of SAT1 flipper. The first ATG of YKU80 was shown in Genolevures (http://cbi.labri.fr/Genolevures/). A second ATG has been identified 81 bp downstream from the first ATG codon in the same frame. To construct yku80 knockdown strains (HETS strains), the SAT1 flipper was integrated into sites 5' of either the first or the second ATG of the YKU80 ORF to yield HETS146 and HETS202, respectively. The SAT1 flipper was removed from the HETS146 and HETS202 strains, producing POP-SF1467 and POP-SF221, respectively, which were generated under the following permissive conditions: YPD medium without NSTC with overnight shaking at 37°C. The 34-bp of palindrome DNA, the FRT, which is presented as two arrowheads face to face, remains behind in POP-SF1467 and POP-SF221.

 

Figure 5
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  		FIG. 5. Gene targeting efficiency in the yku80 knockdown strains and revertant strains. The determination of the gene targeting efficiency in each construct was measured as shown in Fig. 1. Targeting efficiency with 200 bp of homologous flanking sequence corresponding to the ADE2 promoter region was measured.

 
Evaluation of gene targeting efficiency in the YKU80 knockdown strains. The efficiency of gene targeting was measured by the integration into the promoter region of ADE2 of a DNA cassette containing the Tet-P with a flanking 200-bp homologous sequence. With the use of HETS146 and POP-SF1467, the efficiency rates were 1.97 and 27.4%, respectively, unexpectedly lower when the recipient included the SAT1 flipper than after it lost it. On the other hand, using HETS202 and POP-SF221, the rates were 40.3 and 25.0%, respectively. These results indicate that the expression of YKU80 was repressed when the SAT1 flipper was located before the second ATG and restored when it was lost (Fig. 5).

Transcription initiation site of YKU80. To confirm the site of the promoter region of YKU80, the initiation site of transcription of YKU80 was investigated by a 5' RACE experiment. The sequences of nine clones, including the 5' RACE products, were determined. This showed that all transcription was initiated downstream of the first ATG (data not shown). These results indicated that the first ATG is not an initiation codon of YKU80.

Evaluation of the yku80 knockdown effect on gene targeting to various loci. The gene targeting efficiency was 3.5% (4/114) in HETS202 with 40 bp of homologous sequence corresponding to the ADE2 promoter region. To extend these results to other loci, the gene targeting efficiency was measured at 15 loci using KUE200 in which the SAT1 flipper integrated upstream of the second ATG. The DNA cassettes included, in all cases, 56 bp of homologous flanking sequence on either side of the HIS3 selective marker. After each transformation, 3 to 30 colonies were checked by PCR. The efficiency of gene targeting was approximately 5 to 10% in three genes and higher than 30% in other genes. The results suggest that gene targeting with short homologous sequences in yku80 knockdown strains is effective at multiple loci.

Effect of the remaining FRT on repression of YKU80 transcription. After the SAT1 flipper is removed from the genome, the FRT sequence, which is a 34-bp short palindrome, remains in the sequence upstream of the second ATG of YKU80 in POP-SF221. The effect on gene expression caused by the FRT remaining in the 5' end of the YKU80 ORF was quantified by real-time PCR. Since much more mRNA of YKU80 is transcribed during stationary growth phase than during exponential growth phase (K. Ueno et al., unpublished data), mRNA was harvested from the cells at stationary phase. The amount of mRNA relative to ACG4 is shown in Fig. 6. The YKU80 mRNA level was 0.38-fold in HETS202, but POP-SF221 had 1.09-fold, whereas no mRNA was identified in HET80. Thus, no effect of FRT on transcription was identified in POP-SF202 in comparison to its wild-type progenitor.


Figure 6
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  		FIG. 6. Relative quantification of YKU80 transcription by real-time RT-PCR. mRNA was harvested from the cells of overnight culture at 37°C. Data are shown as means ± standard deviations from three independent experiments. Transcription was not detected in HET80, the yku80-deleted strain. *, P was 0.042 versus HETS202 for that particular condition. #, P was 0.581 versus ACG4 for that particular condition.

 
Mutation rate measured by spontaneous 5-FOA resistance in HETS202. It is an important criterion of a transformation host strain that the strain be genetically stable, without elevated mutation frequency occurring during maintenance. In order to confirm the genetic stability of the YKU80 knockdown strain, the mutation rate of HETS202 was investigated. The mutation rates in HETS202 and ACG4 (as a control) were measured as the frequency of 5-fluoroorotic acid (5-FOA) resistance. The strains were subcultured on YPD plates containing 100 µg/ml NSTC 10 times, and the number of CFU was determined on YPD plates containing 0.2% 5-FOA. The mutation rates of ACG4 and HETS202 were (2.97 ± 1.38) x 10–7 and (4.15 ± 1.08) x 10–7 (P = 0.31), respectively, indicating no significant difference in mutation rate caused by the repression of YKU80.

In vivo survival of stains carrying FRT in YKU80 promoter. To evaluate the virulence of strains in which FRT remains before the ORF of YKU80, an in vivo experiment was carried out. Cells of ACG4 and POP-SF221 and KPOP11538 were administered into the lateral tail veins of mice. KPOP11538 has a cyb2 mutation and was used as a control because growth defects have been previously identified in vitro (Ueno, unpublished). The fungal burdens of ACG4 and POP-SF221 were transiently decreased at day 7 and increased at day 14, while the fungal burden of KPOP11538 gradually decreased and eventually fell below the limit of detection. The pattern of the survival curve was not different for ACG4 and POP-SF221 (Fig. 7). These results indicate that FRT located in the YKU80 promoter region has no effect on the survival of C. glabrata in vivo.


Figure 7
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  		FIG. 7. Effect of FRT sequence for survival ratio in vivo. A total of 1 x 107 cells of each strain were administered into the lateral tail veins of mice. Fungal burden on the kidney was measured at days 1, 7, and 14 after the infection. Outliers were statistically dismissed using the Thompson test for colony counting, and P values of less than 0.05 were considered significant. ACG4, POP-SF221, and KPOP11538 are indicated by closed circles, open circles, and open squares (cyb2 was a positive control), respectively. Error bars indicate standard deviations.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To facilitate systematic C. glabrata mutant construction in a high-throughput manner, a new recipient strain that permits efficient gene targeting is required. As a first step, we demonstrated that the targeting efficiency with short homologous sequences is increased in a yku80 deletion mutant. This made the strain useful for efficient gene targeting, but leaving the gene crippled could give misleading results in disruptants. In S. cerevisiae, many genetic interactions of YKU80 with other genes, including synthetic lethal combinations, have been reported (29, 33). There may be some effects, including unknown synthetic sickness or lethality, induced by YKU80 inactivation. Therefore, YKU80 should be restored to full activity for the analysis of the specific function of each gene after the mutant construction. Cotransformation of the DNA cassette for Tet-P integration and a DNA cassette for YKU80 restoration did not succeed (data not shown), so we decided to construct a strain in which the expression of YKU80 could be repressed during transformation for optimal gene targeting and the expression easily restored after the gene targeting.

The SAT1 flipper is an FLP1-mediated pop-out system that is efficiently removed from chromosomal sites in C. albicans (34). In this work, we have used the SAT1 flipper for the repression of YKU80 expression in C. glabrata. The SAT1 flipper has not previously been reported to be useful for gene manipulation in C. glabrata. The maltose promoter is not likely to regulate transcription in C. glabrata as well as in C. albicans, because the DNA cassette SAT1 flipper was frequently lost in medium, including glucose. Even when the cells were cultured in YPD, including NSTC, 26.3% of them lost the SAT1 flipper. The high frequency of the pop-out is convenient for this work, since a step in which the strain is plated on maltose medium to induce the pop-out can be skipped. For other uses, however, the promoter needs to be replaced with a controllable promoter in order to obtain better regulation of FLP1. These results suggest that the MAL2 promoter in the SAT1 flipper cassette can't be repressed in medium, including glucose, since it is adequately leaky to generate derivatives in which the SAT1 flipper is removed.

In yku80 mutants of S. cerevisiae, a severe growth defect at 37°C had been identified due to telomere shortening, among other causes (1, 5, 9, 11). These cells also exhibit hypersensitivity to DNA-damaging agents, such as MMS, but not to UV radiation or hydroxyurea (5, 16, 37). In the {Delta}yku80 mutant of C. glabrata, no obvious growth delay was identified at 37°C or 42°C. No sensitization to MMS, H2O2, UV radiation, or hydroxyurea was identified in this study (Fig. 3). These results suggest that DNA repair and other essential cell functions are not critically affected by the deletion of yku80 in C. glabrata. It seems likely that DSB repair is supported by another pathway(s) independent of YKU80, such as microhomology end joining (23). In Schizosaccharomyces pombe, although sensitivity to MMS did not occur in Ku single mutants, NHEJ was almost suppressed, which was verified by a plasmid repair assay (24, 39).

The single deletion of yku80 in C. glabrata had no obvious effect on the growth rate, DNA repair ability, and mutation rate (Fig. 3). The SAT1 flipper could be maintained in YKU80 knockdown strains under selection with 100 µg/ml of NSTC and then automatically lost from the cells under nonselective conditions. This construct allows efficient gene targeting and restoration of the expression of YKU80 by a single transformation. Although the FRT has been left at the promoter region of YKU80 after the SAT1 flipper was excised from chromosome, the FRT has no detectable effect on the survival of C. glabrata in vivo (Fig. 7). The YKU80 knockdown strain, HETS202, is a new recipient strain that enables rapid construction of desired recombinants via high-efficiency gene targeting; furthermore, strains deploying NHEJ regulation, e.g., HETS202, will facilitate gene manipulation and contribute to comprehensive gene function analyses, elucidating pathogenicity and facilitating the identification of antifungal drug targets in C. glabrata.


   ACKNOWLEDGMENTS
 
We thank P. T. Magee (University of Minnesota) for critical reading of the manuscript, O. Reuss (Universitat Wurzburg, Germany) for the gift of plasmid pSFS2A, and K. Kitada for sharing C. glabrata strains 2001H, 2001T, and 2001HT.

This work was supported by a grant-in-aid for scientific research for the following priority areas, infection and host response, genome biology and microbial genome, from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan. Phone: 81-43-226-2792. Fax: 81-43-226-2486. E-mail: chibana{at}faculty.chiba-u.jp Back

{triangledown} Published ahead of print on 18 May 2007. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back


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Eukaryotic Cell, July 2007, p. 1239-1247, Vol. 6, No. 7
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