Copyright 0 1991 by the Genetics Society of America

Repeated Use of GALl for Gene Disruption in Candida albicans

Jessica A. German,*" Winnie Chant9* and John W. Gorman* Departments of "Gene Expression Sciences and TAntiin.ctives, Smith Kline Beecham Pharmaceuticals,

King of Prussia, Pennsylvania 19406 Manuscript received August 17, 1990

Accepted for publication May 30, 1991

ABSTRACT A technique which has the potential to allow repeated use of the same selectable marker to create

gene disruptions in Candida albicans has been developed. In this approach, originally described for Saccharomyces cerevisiae, the selectable marker is flanked by direct repeats. Mitotic recombination between these repeats leads to elimination of the selectable marker. A module in which the GALl gene is flanked by direct repeats of the bacterial CAT gene was constructed and used to disrupt one copy of the URA3 gene in a gall mutant. Gal- revertants were selected by plating on 2-deoxy-~- galactose (2DOG). The frequency of 2DOG-resistant colonies recovered was 20 times higher than that obtained with a similar construct not flanked by direct repeats. Of these, 20% had lost the GALl gene by recombination between the direct repeats. The GALl gene was used again to disrupt the remaining wild-type copy of the URA3 gene of one of these gall isolates, resulting in a stable ura3 mutant. This technique should be generally applicable to derive homozygous gene disruptions in this diploid organism.

T HE dimorphic yeast Candida albicans is a diploid organism which lacks a sexual cycle. Therefore,

induction and analysis of specific mutations has been difficult. In the last few years, many of the molecular genetic techniques used to study Saccharomyces cerevis- iae have been successfully adapted for use with C. albicans.

With the development of a transformation system and the demonstration of homologous recombination between the inserted fragment and the genome (KURTZ, CORTELYOU and KIRSCH 1986), it became possible to use gene replacement techniques to create specific mutations in the genome of C. albicans. The one-step gene disruption procedure of ROTHSTEIN (1 983), in which a selectable marker is inserted into the cloned gene of interest, has been used most com- monly. However, as a single replacement event alters only one of the two alleles, it is then necessary to mutate the second copy to obtain a homozygous mu- tant. Several techniques have been employed to in- duce homozygosity. For example, UV-induced mitotic recombination has been used to obtain a homozygous ura3 mutant from a heterozygous gene disruptant (KELLY et al. 1987). This method generally requires extensive screening to recover auxotrophs. In addi- tion, chromosomal translocations appear to be rela- tively common in many strains of C. albicans (C.

' Current address: Department of Microbial Molecular Biology, Bristol- Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543. ' Current address: Department of Macromolecular Sciences, Smith Kline

Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, Pennsylvania 19406.

Genetics 129 19-24 (September, 1991)

BINGHAM and J. A. GORMAN, manuscript submitted for publication). If the two alleles of the gene are situated on nonhomologous chromosomes, the prob- ability of recovering a homozygote by mitotic recom- bination would be extremely low. A more convenient technique is sequential gene disruption, where a sec- ond selectable marker is used to disrupt the second copy of the gene (KURTZ and MARRINAN 1989). This, however, requires the use of a doubly auxotrophic mutant host.

As the number of defined auxotrophic mutants of C. albicans suitable for use as hosts for transformation is currently very limited, it would be convenient to be able to use the same selectable marker for a series of replacement events. One approach is to use cotrans- formation, in which the nonselectable disrupted gene is introduced with a selectable plasmid which subse- quently can be lost (SILICIANO and TATCHELL 1984). This technique has been used to create a LEU2/leu2 heterozygote of C. albicans (KELLY, MILLER and KURTZ 1988). However, the same method was not repeated to derive a leu2 homozygote.

We have explored the feasibility of using a method described by ALANI, GAO and KLECKNER (1987). In this procedure, the selectable marker is flanked by direct repeats of a heterologous sequence. This cas- sette is then used to disrupt a cloned gene. A linear fragment containing the disrupted gene is integrated into the yeast genome by homologous recombination. Mitotic recombination between the direct repeats will lead to deletion of the selectable marker, leaving one copy of the repeat sequence within the disrupted gene.

20 J. A. Gorman, W. Chan and J. W. Gorman

ECORV

A. Hlndlll Xbrl Xhol EcoRV EcoRl Xbd Pstl I I I I

rvvu

lkb - 9.

BamHI X k l BwHI Ncol

" 1 " '

FIGURE 1.-Schematic diagram of the C. ulbi- cans genes employed. A, The 6.2-kb Hind111 frag- ment of pYK40 containing the URA3 gene. The short arrows indicate the regions for which DNA sequence data was obtained. B, The 4.5-kb frag- ment of pGalS-20 containing the GALZ gene. The wavy arrows indicate the location of the coding sequence and the direction of transcription. Open blocks, C. albicuns sequence; closed block, vector sequence.

The same selectable marker can then be used to disrupt the second copy of the gene.

The GAL1 gene of C. albicans provides a good selectable marker for this purpose, as both forward and reverse selection can be achieved. Using a gall mutant, transformants are selected by growth on ga- lactose. Excision events, leading to the Gal- pheno- type, can be directly selected by plating on 2-deoxy- D-galactose (2DOG), as gall mutants are resistant to this compound (J. A. GORMAN, J. W. GORMAN and Y. KOLTIN, manuscript submitted for publication). We have constructed a module in which the GALl gene is flanked by direct repeats of the bacterial chloram- phenicol acetyltransferase (CAT) gene. The usefulness of this module for creating gene disruptions in C. albicans has been examined.

MATERIALS AND METHODS

Strains: The C. albicans strain employed, 792P1-45, is a galactokinase negative (gall) mutant derived from the adel strain B792P1 (J. A. GORMAN, J. W. GORMAN and Y. KOL- TIN, manuscript submitted for publication). The rate of spontaneous reversion to Gal+ is less than 1 X lo-'. Esche- richia coli strains HBlOl and TB1 were used for plasmid transformations and amplification.

Growth media: Yeast minimal medium contained yeast nitrogen base without amino acids (YNB, Difco) supple- mented with adenine (20 pg/ml) and either 2% glucose (YNBGlu) or 2% galactose (YNBGal), plus 2% agar for solid medium. For selection of Gal+ transformants, the plates were supplemented with 0.01% glucose. 2DOG YNB plates (YNBDOG) contained 2% glycerol, 0.4% casamino acids, adenine and 0.4% 2DOG. When required, plates were sup- plemented with uracil (1 mg/ml) or uridine (50 pg/pl). E. coli were grown in LB broth or LB agar plates (MILLER 1972) supplemented with ampicillin (75 pg/ml) as required.

Plasmids: The cloned C. albicans genes employed were isolated from the genomic library described by ROSENBLUTH et al. (1985). Plasmid pYK40, kindly provided by Y. KOLTIN, contains a 9-kb insert which complements S. cerevisiae ura3 mutants. Subcloning of the insert demonstrated that the C. albicans URA3 gene was within a 6.2-kb HindIII fragment (Figure 1A) which had a restriction map similar to that of the URA3 clone isolated from a different strain of C. albicans by GILLUM, TSAY and KIRSCH (1984). DNA sequencing of portions of this fragment and comparison of the deduced amino acid sequences with that of the S. cerevisiae URA3 gene product (ROSE, GRISAFI and BOTSTEIN 1984) indicated the approximate location of the coding region and the direction of transcription (see Figure 1A). The complete

sequence of the URA3 gene, subsequently published (LOS- BERGER and ERNST 1989) confirmed this tentative localiza- tion. Plasmid pYSK209 contains a 13-kb C. albicans insert which complements a gall mutant of S. cerevisiae (MAGEE et al. 1988). A 4.5-kb BamHI-XhoI fragment was found to contain the GALl complementing activity. This fragment was subcloned into BamHI-SaZI-digested pUC 18 (giving pGalS-20) and the entire fragment sequenced (J. A. GOR- MAN, P. KMETZ and w. CHAN, manuscript in preparation). The location of the coding sequence is shown in Figure 1B.

Plasmid constructions: Plasmid pHW92, containing the C. albicans URA3 gene was constructed by inserting a 6.2- kb HindIII fragment of pYK4O into the HindIII site of pUC18ARI (pUC18 with the EcoRI site deleted) in the orientation such that the PstI site downstream of the URA3 gene was proximal to the PstI site in the polylinker of pUCl8ARI (Figure 1A). This plasmid was subsequently digested with PstI and religated to remove the EcoRV site present in the fragment derived from the original cloning vector, giving plasmid pWC10.

Plasmid pWC25 was constructed in several steps. A syn- thetic oligodeoxynucleotide adapter (see Figure 2) was in- serted into XmaI cut pUC 18 to provide additional restriction sites for cloning (pUC18-PL). The 4.5-kb BamHI-XhoI frag- ment of pGalS-20, which contains the C. albicans GALl gene, was treated with Klenow fragment to create blunt ends and ligated into the StuI site of pUC 18-PL. A plasmid with the GALl gene in the orientation shown in Figure 2 was selected (pWC21). A '126-bp HindIII-XbaI fragment of the bacterial CAT gene derived from the plasmid HIVcatBGH (VALERIE et al. 1988) was similarly inserted into the StuI site of pUC18-PL, following fill-in to create blunt ends. The plasmid with CAT in the orientation shown in Figure 2 was selected (pUC-CAT2). Next, the CAT gene was isolated from pUC-CAT2 as a 733-bp MluI-BssHII fragment and inserted into MluI cut pWC21. Plasmid with the CAT gene in the orientation which regenerates the MluI site adjacent to GALl was isolated. The same CAT fragment was then inserted into the BssHII site of this plasmid. The construct in which the BssHII site was regenerated adjacent to GALl was selected. The final 6.0-kb CAT-GALl-CAT module in pWC25 is shown in Figure 2. The large GALl insert was subsequently replaced with a smaller GALl frag- ment to reduce the size of the module for gene disruption. The 2.75-kb Xbal fragment of pGalS-20 was treated with Klenow to create blunt ends and inserted into the StuI site of pUCl8-PL in the opposite orientation as that shown in Figure 2. The BssHII-MluI fragment of this construct was substituted for the 4.5-kb BssHII-MluI fragment of pWC25. Plasmid with the GALl gene in the same orientation as pWC25 (see Figure 2) was recovered. In this construct, pWC30, the BssHII and MluI sites were not regenerated.

T o disrupt the C. albicans URA3 gene with the CAT- GALl-CAT construct, the 4.2-kb module was isolated from

Gene Disruption in

cCCGGGGCGCGCAGGCCTACGCGTCccggg gggccCCGCGCGTCCGGATGCGCAGGGCCc

Smal BssHll Stul Mlul Smal ""-

/ \ ' f

\

GALl - Smal BssHll Mlul Smal

FIGURE 2.-Construction of pWC25. The sequence of the syn- thetic oligonucleotide adapter inserted into pUCl 8ARI is shown at the top. The CAT and GAL1 genes were inserted into the StuI site i n the orientation shown. The lower diagram shows the final 6.0 CAT-GALI-CAT module.

pWC30 by digestion with SnaI and inserted into the URA3 gene in pWC10, which had been digested with EcoRI and RcoRV and treated with Klenow to create blunt ends. The resulting plasmid was designated pWC32. A similar disrup tion of URA3 with only the GALl gene was made by inserting the 4.5-kbBamHI-XhoI fragment of pYSK209 into the same cut vector, generating pWC12. For transformation, the plasmids pWC12 and pWC32 were digested with PstI and XhoI, and the linear disrupted URA3 fragments were isolated by agarose gel electrophoresis and electroelution.

Yeast transformation: C. albicans was transformed by a modification of the standard spheroplast transformation procedure used for S. certwisiae (SHERMAN, FINK and HICKS 1986), using Zymolyase 100 T (0.25 mg/ml). As C. albicans can utilize sorbitol as a carbon source, the top agar used for plating the spheroplasts contained 0.5 M MgS04 as an os- motic stabilizer. The cells were plated on YNBGal-uridine and incubated at 30" for 5 days to recover Gal' transform- ants. Single colonies of all transformants were isolated be- fore further analysis.

DNA isolation and techniques: Restriction enzymes, T4 ligase and Klenow fragment of DNA polymerase I were purchased from New England Biolabs or Pharmacia and used according to the manufacturers' specifications. Plasmid isolation, DNA manipulations and Southern blotting were as previously described (MANIATIS, FRITSCH and SAMBROOK 1982). Yeast DNA was isolated according to the procedure of SHERMAN, FINK and HICKS (1986). followed by phenol extraction. DNA probes for hybridization were labeled with [T-'~P]~CTP, using random primers (Prime Time C, Inter- national Biotechnologies, Inc.).

RESULTS AND DISCUSSION

Construction and testing of a module containing a selectable marker flanked by direct repeats: A vector containing the GAL1 gene flanked by direct repeats of the CAT gene (pWC25) was constructed as shown in Figure 2. This module was constructed such that other genes which might be used as selectable markers could be easily substituted for the CALI gene. As long as the gene of interest does not contain either

Candida albicans

1 2 3 4 5 6 7 8

9.4 -

6.6 -

4.4 -

a a

00"o-a

21

2.3 - 2.0 -

FIGURE 3.-Southern blot analysis of genomic DNA of GAL1 disrupted strains. Total genomic DNA was digested with Xhol and Psf l , and run on a 0.8% agarose gel. The blots were probed w i t h the 4.5-kb Xhol-Psfl LIRA3 fragment of pWCI0. Six of the ten colonies tested are shown. Lanes: 1 , the homozygous URA3 strain 792P1-45; 2. the heterozygous LIRA3 deletion strain WCC2-I: 3- 8, 2DOG' clones derived from WCC2-I. Hind111 restricted X-DNA was used as size marker.

a BssHII or MluI site, it can be inserted into the StuI site of pUC18-PL, then isolated as a BssHII-MZuI fragment and substituted for the GAL1 gene of

To test the potential of this construct, the SmaI fragment of pWC30, a derivitive of pWC25 contain- ing a smaller G A L l fragment, was used to disrupt the URA3 gene of C. albicans. T h e 5' end of the URA3 gene and some upstream sequence (EcoRV-EcoRI, see Figure 1A) was replaced by the CAT-GALI-CAT mod- ule, generating pWC32. T h e disrupted URA3 gene was isolated as a 7.5-kb XhoI-PstI fragment which contained homology to URA3 flanking sequence at both ends. As a control, a plasmid (pWC12) in which the URA3 gene was disrupted in the same way with the G A L l gene only (not flanked by direct repeats) was employed. This disrupted URA3 gene was isolated as a 7.9-kb XhoI-PstI fragment.

Each of these fragments was used to transform a homozygousgull mutant of C. albicuns t o Gal+. Trans- formants containing the URA3 gene disrupted with G A L l only were designated with the prefix WCC2 and those disrupted with CAT-CALI-CAT, WCC8. To confirm that the Gal+ colonies contained the expected insertional deletion in one of the two chromosomal URA3 genes, Southern blot analysis was performed. As shown for the representative transformants WCC2-I (Figure 3, lane 2) and WCC8-2 (Figure 4, lane 2), bands of the expected sizes were found. Each contained the 4.2-kb band of the wild-type nondis- rupted copy of the gene (Figures 3 and 4, lane l), and a larger band (7.9 for WCC2 and 7.5 for WCC8) indicative of the disrupted allele.

To determine whether the presence of the flanking repeat sequences would lead to loss of the GALl selectable marker, the frequency of gal- derivitives

p w c 2 5 .

22

A

1 2 3 4 5 6 7 8

2.3 - 2.0 -

J. A. Gorman, W. Chan and J. W. Gorman

B 1 2 3 4 5 6 7 8

TABLE 1

Frequency of 2DOG resistant isolates

FIGURE 4.-Southern blots of genomic DNA of CAT-CALI-CAT disrupted strains. Total genomic DNA was digested with Xhol and Pstl and run on duplicate gels. Six representative colonies are shown. The blots were probed with A, the URA3 fragment (see legend to Figure 3) or B, the 0.75-kb Smal fragment of pUC-CAT2 containing the CAT gene. Lanes: 1, 792P1-45; 2, WCC8-2; 3-8, 'LDOG resistant clones isolated from WCC8-2.

was determined. Single isolated colonies of WCC2-1 and WCC8-2 were grown in selective medium (YNBGal) and plated quantitatively on YNBDOG plates (Table 1). All 2DOG-resistant (2DOG') colonies were isolated and tested for growth on galactose; all were Gal-.

The average frequency of loss of the Gal+ pheno- type in WCC2-1 cultures was about 2 per IO' cells (see Table 1). These gal- colonies could arise by mutation in the inserted GALI gene, or by deletion of the disrupted allele by gene conversion or mitotic recombination. T o distinguish between these possibil- ities, 10 independent gal- colonies derived from WCC2-1 were analyzed by Southern blotting, using URA3 as probe. Figure 3 shows data for six of these. Eight of the ten showed only a single 4.2-kb band, indicating that they were homozygous for the wild- type URA3 allele, and thus arose by mitotic recombi- nation. The other two one of which is shown in lane 8) retained the 7.9-kb band, indicating that these still contained the disrupted URA3 gene. These probably represent mutations in the inserted GALI gene.

The frequency of gal- derivatives found with WCC8-2 was considerably higher, with an average in the range of 3 per lo5 cells (see Table 1). Therefore, the presence of the direct repeats flanking the GALI gene increased the appearance of gal- colonies 15- fold. While gal- colonies could have arisen by mitotic recombination or mutation, as with WCC2-1 deriva- tives, it seemed probable that recombination between the CAT repeats occurred, leading to physical loss of the GALl gene. T o confirm this, 10 independent gal- colonies were analyzed. Data for six representative isolates is shown in Figure 4. Using either the URA3 (Figure 4A) or the CAT (Figure 4B) gene as probe,

No. Strain Total 2DOG' Frequency

WCCP-I 2.2 X 10' 83 3.7 x 2.0 X lo7 39 1.9 x 4.4 X lo7 64 1.4 X lo-" 5 . o ~ lo7 51 1.0 x lo-" 5.2 X lo7 56 1.1 x lo-"

Average = 1.7 (.C 1.2) X 1 0-6

WCC8-2 1.9 X 10" 38 2.0 x lo+ 3.8 X 10" 166 4.4 x lo+ 1.8 x 10'' 27 1.5 X 3.9 x 10" 199 5.2 X 1.7 X 10" 50 2.9 x 1 .9x 10" 44 2.3 x 1.8 X 10" 36 2.0 x lo+ 2.1 x 10" 37 1.8 X

Average = 2.8 (k1.3) X

Single colonies were picked from YNBGal plates, grown to mid- log phase in YNBGal, diluted and plated on YNBDOG to determine the frequency of 2DOG' colonies or on YNBGlu for total colony count.

identical results were obtained. One of the isolates (see lane 5) showed the hybrization bands expected if the GALl gene had been "looped-out" by recombina- tion between the two CAT repeats; the 7.5-kb band was absent, and a new 4.0-kb band was present. Eight appeared to be identical to WCC8-2, while one (see lane 7) contained a larger band. The size suggested that this contained a duplication of the inserted mod- ule. In light of the unexpectedly high number of gal- colonies that appeared to retain the 7.5-kb band in- dicative of the intact CAT-CALI-CAT module, an ad- ditional 25 single colonies obtained on the 2DOG plates (from two different single colony isolates of WCC8-2) were examined. Of these, six showed hy- bridization bands of 4.2 and 4.0 kb, and thus had looped out the GALl gene (data not shown). The remainder appeared to be identical to WCC8.

T o check the stability of the gal- phenotype, the same 2DOG' isolates were patched onto YNBGal plates. While all were initially gal-, after 4 or 5 days, several Gal+ colonies arose in each of the isolates which had not looped-out the CAT-GALI-CAT mod- ule. Thus, the alteration resulting in the Gal- pheno- type was revertable. As would be expected, all the loop-out isolates were stable.

Frequency of intrachromosomal recombination and mutation: Of the 35 gal- colonies derived from WCC-8 which were examined, 7 had deleted the GALl gene and one of the CAT repeats. Thus, the "loop-out" event occurred at a frequency of approxi- mately 5.5 X IO-' (20% of the total 2DOG' colonies). This is considerably lower than the frequency re- ported for similar intrachromosomal recombination in a S. cereuisiae haploid (ALANI et al. 1987). While

Gene Disruption in Candida albicans 23

this might indicate dissimilar recombinational mech- anisms in the two yeast, it also might simply be a reflection of differences related to the size of the repeat sequences, the distance between them or the selection system employed. Alternatively, the chro- mosomal location of the particular gene disrupted might affect the rate of recombination. The C. albi- cans URA3 gene is located on chromosome 5 [MAGEE et al. (1988), referred to as chromosome 3 in this reference]. It has been noted that, although consid- erable variation in chromosome size is seen between different isolates of C. albicans (reviewed in SCHERER and MAGEE 1990), this chromosome shows almost no length heterogeneity (IWAGUCHI, HOMMA and TANAKA 1990; C. THRASH-BINGHAM and J. A. GOR- MAN, manuscript submitted for publication). In addi- tion, a repeat sequence, designated as Ca3 or 27a, which frequently undergoes internal changes to pro- duce polymorphisms (SCHERER and STEVENS 1988) is present in all chromosomes except chromosome 5 (SADHU et al. 199 1). Thus, the URA3 gene may be situated in a chromosomal region that is suppressed for recombination. Disruption of genes located on chromosomes other than chromosome 5 should give insight into this question.

The remaining Gal- colonies isolated from WCC8- 2 showed no apparent alteration of the inserted GAL1 gene. If these 2DOG‘ colonies arose by spontaneous mutation in the GALl gene, the frequency would be approximately 2.2 X This is at least 60-fold higher than the apparent mutation frequency of 3.4 X 10” found for the GALl gene in WCC2-I, where only 20% of the total Gal- isolates examined showed no change by genomic blot, Therefore, the presence of the flanking heterologous repeat sequences dra- matically increased the frequency of mutation to Gal-. In the filamentous ascomycete Neurospora crassa, se- quences duplicated by integration of transforming DNA are subject to mutations associated with exten- sive cytosine methylation (for a review, see SELKER 1990). This process, limited to the premeiotic stage, is called RIP for repeat induced mutation. Ascobolus immersus undergoes a similar process of premeiotic methylation of duplicated sequences (GOYON and FAUCERON 1989). However, here the inactivation ap- pears to be due to methylation only and is revertable during mitotic growth (FAUGERON et al. 1990). Clearly, a mechanism causing inactivation of unlinked duplicated genes cannot be active in asexual diploid yeast such as C. albicans. However, it is possible that the presence of tandemly repeated sequences or het- erologous sequences triggers some mechanism of lo- calized gene inactivation, conceivably one involving reversible methylation. Further analysis of the inacti- vated GALl allele in these Gal- isolates may reveal a

novel process of selective gene inactivation in this diploid species.

Use of the GALl gene to create a second gene disruption: In order to see if the GALl gene could be reused to derive a double disruption of URA3, one of the stable gal- loop-out strains (GALoop4) was trans- formed with either the GALl disrupted URA3 gene from pWC12 or the CAT-GAL-CAT disrupted gene from pWC32. The resulting Gal+ colonies were re- plated on YNBGal-uracil plates to obtain single colo- nies, then tested for uracil auxotrophy. In each case, approximately half the transformants were Ura+ and half Ura-. This indicated that the transforming frag- ment integrated into either the wild type or the dis- rupted copy of URA3 with equal frequency. Southern blot analysis of several Ura- Gal+ colonies confirmed that integration had occurred at the remaining wild- type URA3 allele (data not shown).

It should be straightforward to use the CAT-GALI- CAT construct to create multiply marked strains. First, it will be necessary to derive a Gal- derivative of the doubly disrupted Ura- strain still containing one copy of the CAT-GALI-CAT module by selection on 2DOG plates. A homozygous ura3::CAT diploid should be characterized by a stable gal phenotype. This strain could then serve as a host for disruption of another gene again using GALl as the selectable marker. The use of the module described or similar constructs employing other selectable markers for which both forward and reverse selection are possible should fa- cilitate gene replacement in C. albicans.

We would like to thank YICAL KOLTIN for plasmid pYK40, DAN SYLVESTER for DNA sequencing of URA3 subclones and GORDON ROBINSON and CATHRINE THRASH-BINGHAM for helpful comments on the manuscript.

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Communicating editor: N. KLECKNER

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