ORIGINAL PAPER

X. Ye á B. Feng á P.J. Szaniszlo

A color-selectable and site-speci®c integrative transformation systemfor gene expression studies in the dematiaceous fungusWangiella (Exophiala) dermatitidis

Received: 12 March / 18 June 1999

Abstract To explore potential virulence factors in thedematiaceous (melanized) fungus Wangiella dermatiti-dis, we established a gene expression system withproperties of homologous transformation and coloridenti®cation. Using a polyketide synthase gene(WdPKS1) fragment for targeting, we found that 52%of transformants became albinos easily distinguishablefrom nonspeci®c transformants. Southern analysis con-®rmed that the integrations were at the WdPKS1 locus,which however did not a�ect transformant growth. Witha heterologous promoter, P-glaA, enhanced expressionof lacZ was found at 37 °C. Our results indicated thatthis system allows the e�cient production of isogenicstrains for gene function analysis in W. dermatitidis.

Key words Wangiella á Exophiala á Transformation áGene expression á Polyketide synthase

Introduction

The zoopathogenic fungus Wangiella (Exophiala) de-rmatitidis is one of many form-species of the Fungi Im-perfecti, which are darkly pigmented (dematiaceous)owing to the deposition of melanin in their cell walls (Geiset al. 1984; Taylor et al. 1987; Kwon-Chung and Ben-nette 1992). This fungus has recently become betterknown as a paradigm for studies of the causative agents

of pheohyphomycosis and other emerging de-matoymycoses of humans, because of its increasing de-tection as a systemic pathogen in both immunocompetentand immunocompromised patients (Matsumoto et al.1993, 1994). Moreover, because W. dermatitidis has awell-de®ned polymorphic nature and awell-characterizedcell-wall chemistry, it serves as an excellent model for themore than 100 other dematiaceous fungal pathogens ofhumans (Szaniszlo et al. 1993;Montijn et al. 1997). It hasbeen shown that the cell-wall component 1,8-dihy-droxynaphthalene (DHN) melanin contributes signi®-cantly to the virulence of W. dermatitidis (Dixon et al.1987, 1992; Cooper and Szaniszlo 1997), perhaps becauseit enhances resistance to killing in the phagolysosome ofneutrophils (Schnitzler et al. 1999).

The polymorphism of W. dermatitidis, which is alsosuspected to contribute to its virulence, is expressed asthree basic vegetative morphologies: yeasts, hyphae andsclerotic-body like multicellular forms (Szaniszlo et al.1983; Karuppayil and Szaniszlo 1997). Taking advan-tage of the fact that this fungus grows as a budding,haploid yeast in most rich media, genetic transforma-tions of W. dermatitidis were ®rst achieved usingprotoplasted yeast cells and an integrative vector car-rying a hygromycin B phosphotransferase gene (hph) asa dominant selectable marker (Peng et al. 1995).Transformation e�ciencies were improved subsequentlyby the electroporation of intact yeast cells and the uti-lization of other vectors and selectable markers (Kwon-Chung et al. 1998; Zheng and Szaniszlo 1999).

In this communication, we report a novel integrativetransformation-based gene expression system that gen-erates isogenic strains of W. dermatitidis, which areblack/white color-selectable. Our approach was initiatedwith the cloning of a fragment of the polyketide synthasegene (WdPKS1), which is required for melanin biosyn-thesis in this dematiaceous fungus (Feng and Szaniszlo1998). Disruption of this nonessential gene by a targetedintegrative transformation produced an albino pheno-type from the wild-type strain. In the resulting albino

Curr Genet (1999) 36: 241±247 Ó Springer-Verlag 1999

X. Ye á B. FengDivision of Biological Sciences,The University of Texas at Austin, Austin, Texas 78712, USA

P.J. Szaniszlo (&)Department of Microbiology, and Institute of Cell andMolecular Biology, The University of Texas at Austin,Austin, Texas 78712, USAe-mail: pjszaniszlo@mail.utexas.eduTel.: +1-512-4713384Fax: +1-512-4717088

Communicated by K. Esser

transformant background, a heterologous glucoamylasepromoter, P-glaA, was evaluated by using the reportergene lacZ to monitor expression level. The results con-®rmed that our transformation-expression strategy wassuccessful and useful for gene overexpression analysisin W. dermatitidis.

Materials and methods

Strains and growth media. The laboratory wild-type strain ofW. dermatitidis, strain 8658/ATCC 34100 (E. dermatitidis/CBS527.76), used in this study, and its growth conditions have beenextensively described previously (Cooper and Szaniszlo 1993). Thesemisynthetic complete medium CDY (Roberts and Szaniszlo1978) and the rich medium YPD (Ausubel et al. 1989) were usedfor the propagation of W. dermatitidis cells. Di�erent carbonsources (e.g., maltose or xylose) were substituted for dextrose inmedia for some experiments as speci®ed. In addition, hygromycin B(30 lg/ml, w/v) was added to certain media to select W. dermati-tidis transformants and X-gal (100 lg/ml, w/v) to detect b-galac-tosidase activity. The Escherichia coli XL1-Blue strain (Stratagene,La Jolla, Calif.) was used for the ampli®cation of plasmids and wasroutinely grown in Luria-Bertani (LB) broth medium (Ausubel

et al. 1989) supplemented with 25 lg/ml of chloramphenicol or100 lg/ml of ampicillin when required.

Plasmids. pCB1004 (Carroll et al. 1994), a pCB KS-derived plas-mid containing an E. coli hygromycin B phosphotransferase gene(hph) as a dominant selectable marker, was provided by Dr. J.Sweigard (Dupont, Wilmington, Del.). pGPT-pyrG1 (Ward et al.1993), carrying a cassette of the Aspergillus awamori glucoamylasepromoter (P-glaA) and the Aspergillus niger glaA terminator(T-glaA), was obtained from Dr. M. Ward (Genencor, Palo Alto,Calif.). pCaSpeR(AUG)b-gal (Thummel et al. 1988) having theE. coli lacZ structural gene was provided by Dr. R. Brenner(University of Texas, Austin).

Cloning and DNA sequence analysis. Standard procedures for re-combinant-DNA manipulations were essentially performed as de-scribed by Sambrook et al. (1989) and Ausubel et al. (1989). Toclone the WdPKS1 gene, a 772-bp PCR product (see Fig. 1 A) was®rst ampli®ed from W. dermatitidis genomic DNA based on theconserved b-ketoacyl synthase motif (starting from amino-acid549) of polyketide synthase 1 of Colletotrichum lagenarium (Ta-kano et al. 1995). Plasmid pBF9, containing the PCR fragment ofWdPKS1 in pCB1004, was then used for targeted transformationof W. dermatitidis strain 8656 (Feng and Szaniszlo 1998). Formarker rescue, the SpeI-digested 12-kb fraction of the WdPKS1disruptant's genomic DNA, containing both a 5-kb integrated

Fig. 1 A restriction map of the7-kb WdPKS1 gene fragment.The box represents the openreading frame, in which thegray solid region corresponds tothe 772-bp PCR sequence andthe hatched region is the 2-kbBglII fragment that was used asa targeting sequence inpYEX303 and as a probe forSouthern analysis. The thicksolid line ¯anking the box is the3¢ untranslated region of thegene fragment contained inpBF35. B comparison of thededuced amino-acid sequencesof four PKS homologous frag-ments. Reference numbering ofthe sequences is according tothat of the partial WdPKS1gene product (Pkswd; GenBankaccession: AF130309). Theother polyketide synthasesequences used for comparisonare from Aspergillus fumigatus(Pksaf; GenBank accession:AF025541), Aspergillus nidulans(Pksan; SWISS-PROTaccession: Q03149) andColletotrichum lagenarium(Pkscl; PIR accession: S60224).Amino acids are designated bythe single-letter code. ``.''symbols arti®cially introducedspaces for alignment

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pBF9 and a 7-kb WdPKS1 sequence, was isolated and transformedinto E. coli XL1-blue cells. Finally, plasmid pBF35 from the bac-terial transformants was subjected to DNA sequencing by thedideoxy chain-termination method using an ABI Prism-377 DNAsequencer (Perkin Elmer, Norwalk, Conn.). Sequence data wereanalyzed using the GCG software package (Genetics ComputerGroup, Inc., Madison, Wis.).

Southern hybridization. Total DNA of W. dermatitidis was isolatedby spheroplasting yeast cells in a 1.5-ml sorbitol solution with 20U of zymolyase-20T (ICN, Costa Mesa, Calif.). After incubationwith RNase, the cell lysate was extracted by phenol/chloroformand then DNA was recovered by ethanol-precipitation (Momanyand Szaniszlo 1994). After restriction enzyme digestion and sub-sequent separation in a 1% agarose gel, Southern blotting ofDNA samples was carried out under a high stringency conditionthat consisted of 50% formamide, 6 ´ SSPE, 200 lg/ml of dena-tured DNA, 5 ´ Denhardt's solution and 0.75% SDS. Probeswere prepared using the Prime-A-Gene labeling system (Promega,Madison, Wis.). Hybridizations and washes were performed at42 °C.

Transformation of W. dermatitidis. For transformation of W. der-matitidis, mid-log phase yeast cells were prepared from a YPDculture grown at 25 °C, which were washed three times with cold10% glycerol at 4 °C, and then resuspended in the same solution toapproximately 4 ´ 108 CFU/ml. Prior to transformation, plasmidswere usually linerized, re-puri®ed to remove salts, and then addedto 0.2-ml cell suspensions at a ratio of 1 lg DNA per 1 ´ 108 cells.Electroporation of the cell suspensions was carried out with a GenePulser electroporation system in 0.2-mm cuvettes (Bio-Rad, Rich-mond, Calif.) at a setting of 1.45 kV, 25 lF and 200 W (with a timeconstant about 4.5 ms). After a 2±4-h regeneration period in 0.5 mlYPD, the cell suspensions were spread on CDY-xylose agarcontaining 30 lg/ml of hygromycin B and incubated at 25 °C for5 days.

b-galactosidase activity detection. For detection of lacZ reportergene expression, two methods were used. In one 100 lg/ml of X-galwas added as a b-galactosidase chromogenic substrate to a CDYagar medium for monitoring in vivo b-galactosidase activity.Growth of W. dermatitidis cells on the X-gal medium required 2days incubation at 37 °C to see a color change. In the other, anin vitro b-galactosidase activity assay (Ausubel et al. 1989), log-phase transformant cells were harvested and permeablized withchloroform and SDS. These treated-cells were then incubated at30 °C for 30 min after adding the chromogenic substrate o-nitro-phenyl-b-D-galactoside (ONPG) at ®nal concentration of0.67 mg/ml. After stopping the reaction with 1 M Na2CO3, thesupernatant was used for spectrophotometric assay.

Results

Isolation and sequence characterization of WdPKS1

We initiated this study with cloning of the polyketidesynthase gene of W. dermatitidis, WdPKS1, by a mark-er-rescue approach (see Materials and methods). One ofsubclones, pBF35 (Fig. 1 A), containing a 7-kb insertwas isolated and sequenced. The deduced amino-acidsequence of the insert con®rmed it was homologous tomost of the C-terminal regions of other fungal polyke-tide synthases, and also shared similarity in the con-served domains for b-ketoacyl synthase, acetyl/malonyltransferase and acyl carrier protein (data not shown). A2-kb BglII fragment in this region (Fig. 1 A; GenBankaccession: AF130309) was shown to have deduced ami-no-acid sequence identities of 34±37% and similarities of53±56% when compared to the polyketide synthasesfrom C. lagenarium, Aspergillus fumigatus and Asper-gillus nidulans (Fig. 1 B), suggesting that this polyketidesynthase gene fragment is highly conserved among ®la-mentous ascomycetous fungi.

Development of an integration-expression vectorfor site-speci®cally targeting to the WdPKS1 locus

Although a previously constructed plasmid pBF9 canintegrate at the WdPKS1 locus (Feng and Szaniszlo1998), its homologous transformation e�ciency was low(Table 1). This was perhaps due to the insu�cient lengthof the homologous sequence (772 bp) in the plasmid.Also, pBF9 lacks a unique restriction enzyme-site in thetargeting sequence, which limited the usefulness of thisplasmid as an integrative vector. Therefore, a site-spe-ci®c integration-based expression vector was developedby subcloning the 2-kb BglII fragment of WdPKS1(Fig. 1 A) from pBF35 into pCB1004, to which a 3.5-kbXhoI-HindIII fragment of a P-glaA promoter-terminatorcassette from pGPT-pyrG1 was added to produce thenew vector, pYEX303 (Fig. 2). To evaluate whether thisvector and the heterologous P-glaA promoter would befunctional in W. dermatitidis, a 3.5-kb fragment con-taining the E. coli lacZ as a reporter gene from pCAS-

Table 1 Transformation e�-ciency with pBF9- andpYEX303-derived plasmids

Plasmid DNAa Totaltransformantsb

Albinotransformants

Blacktransformants

Frequency ofspeci®ctransformation

pBF9 53 5 48 9.4%pYEX303 13 6 7 46.2%pYEX303-lacZ 12 9 3 75.0%Othersc 30 14 16 46.7%

aPlasmid DNA was linearized at the targeting sequence by SacII for pBF9- and NarI for thepYEX303-derived plasmidsb Total transformants recovered after electroporation of W. dermatitidis strain 8656 with approxi-mately 1 g of the linearized plasmid DNAc Six other pYEX303-derived plasmids carrying WdCDC42-1, a W. dermatitidis RHO-type GTPasegene, or its mutant alleles

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peR(AUG)b-gal was inserted into the BglII-XbaI sitedownstream from P-glaA to generate pYEX303-lacZ(Fig. 2). Before transformation of W. dermatitidis cells,pYEX303 and its derived plasmids were linearized byrestriction digestion at the unique NarI site in theWdPKS1 sequence to enhance the e�ciency of site-speci®c integration.

Transformation e�ciency with vector pYEX303and its derived plasmids

Transformation of wild-type yeast cells by electropo-ration with the pYEX303, pYEX303-lacZ and otherpYEX303-derived plasmids produced an average of ninetransformants per microgram of the linearized plasmidDNA (Table 1). Transformant cells in a hyromycin B-containing agar medium formed either albino coloniesor parental strain-like brown colonies (Fig. 3 A), in

which the independent albino transformants averaged52% of the population (Table 1). Because the albinophenotype was expected to result from plasmid inte-gration at the WdPKS1 locus and disruption of melaninprecursor synthesis in the transformants, 13 of them,including four of the pYEX303-lacZ transformants(Fig. 3 B; other data not shown), were analyzed bySouthern blotting. The results showed that all the albinotransformants contained a single insertion of pYEX303or its derived plasmids at the WdPKS1 locus, suggestingthat the pYEX303 vector was highly speci®c for ho-mologous recombination in its target site and that theresulting albino phenotype was a reliable indicator ofsite-speci®c transformation.

E�ects of disruption of WdPKS1 on cell growthand cellular morphology

The albino strains derived from transformation with theintegrative vectors pBF9 or pYEX303 were character-ized preliminarily under various growth conditions. Theresults showed that their growth rates were not distin-guishable from the wild-type strain both at 25 °C and37 °C (Fig. 4). Also, these transformants exhibited cel-lular morphologies similar, if not identical, to those ofthe parental strain. These results suggested that thedisruption of the secondary metabolism pathway leading

Fig. 2 Site-speci®c integration with pYEX303 and its derivedplasmids. The plasmids were constructed from parental vector pBC-SK())-based pCB1004, in which an E. coli hygromycin B phospho-transferase gene (hph) was used as a dominant selectable marker, andsequentially a wdpks1 targeting sequence and the glaA promoter-terminater cassette were incorporated into the new vector. Selectedstructural genes, such as lacZ orWdCDC42, were then inserted undercontrol of the glaA promoter for expression studies. The plasmidswere linearized at wdpks1 by NarI and delivered into log-phase yeastcells by electroporation

Fig. 3 A Selection of speci®c transformants. Albino transformantsresulting from the integration of pYEX303 (i.e., ALB303 strain) wereinitially selected on a hygromycin B-containing CDY agar medium.The plates were incubated at 25 °C for 7 days before photography.Note examples of two albino colonies as indicated by the white arrowsand a parental-like black/brown colony as indicated by the blackarrow. B Southern analysis of transformant DNA with a WdPKS1probe. When digested by BamHI, the DNA fragment containing thepYEX303-lacZ insertion in WdPKS1 was expected to be 20 kb (lanes3 to 6), whereas the wild-type band was 6 kb (lane 1). When digestedby BamHI, the DNA fragment containing the pYEX303-lacZinsertion in WdPKS1 was expected to be 20 kb (lanes 3 to 6),whereas the wild-type band was 6 kb (lane 1). A pigmentedtransformant (lane 2) was included in the analysis as a control toshow both the wild-type band and an ectopic integration band(>13 kb)

244

to melanin biosynthesis in W. dermatitidis did not sig-ni®cantly a�ect cell physiological properties under ourlaboratory conditions. Generally, all the albino strainswere very stable in subcultures; only very low frequen-cies of genetic reversion were observed in nonselectivemedium and even on aged plates, where black-coloredcolony sectors could be immediately identi®ed.

Functional characterization of P-glaAin albino transformants

For characterization of the heterologous glucoamylasepromoter P-glaA in W. dermatitidis, the transformantscarrying pYEX303-lacZ were grown on CDY agar mediacontaining X-gal for monitoring b-galactosidase activity.The results showed that the albino transformants turneda blue color within 2 days during incubation in maltose-containing medium at 37 °C, whereas the same trans-formants cultured in xylose-containing medium or inmaltose-containing medium at 25 °C remained whiteuntil after prolonged incubation for more than 7 days(data not shown). Also, in vitro b-galactosidase-activityassays showed that enzyme activities of cell lysates from37 °C cultures were dramatically higher than those from25 °C cultures (Fig. 5), although by this assay little dif-ferences was found between the cells from the maltosemedium and those from xylose medium (Fig. 5). Theresults suggested that P-glaAwas functional in the albinotransformant of W. dermatitidis. However, the inducibleproperties were not same as those described for Asper-gillus (Fowler et al. 1990; Van den Hondel et al. 1991).

Discussion

W. dermatitidis represents a useful model for the study offungal growth, cellular di�erentiation and cell-wall vir-ulence factors among a broad range of melanized

pathogens of humans (Szaniszlo et al. 1993). After es-tablishment of genetic transformation and gene disrup-tion protocols (Peng et al. 1995; Zheng and Szaniszlo1999), we recognized a need to develop a gene expressionsystem for this fungus. The system described here wasbased on a unique integrative transformation, which usedboth a drug-resistance marker and a color-selectablephenotype for the identi®cation of speci®c transformants.The isogenic strains derived by this method preventspotential problems associated with random integrationsinto the genome, which may raise questions about thevalidity of the cellular e�ect of an introduced gene or of aspeci®c gene mutation, versus a side e�ect produced bynonspeci®c insertional mutagenesis. Therefore, this geneexpression system provides an extremely useful tool forfuture gene functional studies in W. dermatitidis.

Transformations of ®lamentous fungi mostly dependon integrative vectors due to limited successes in thedevelopment of automously replicating plasmids. Re-cently, the utilization of plasmids bearing the repliconAMA1 from A. nidulans (Gems et al. 1991) was fruitfulin the transformation of several species ofAspergillus andPenicillium (Clutterbuck et al. 1994; Aleksenko et al.1995; Fierro et al. 1996), but the AMA1 replicator wasfound not to be functional in W. dermatitidis in prelim-inary experiments (data not shown). To improve inte-grative transformation e�ciencies in ®lamentous fungi,homologous sequences like repetitive ribosomal RNAgenes (rDNA) (Tsuge et al. 1990) and numerous homol-ogous markers, such as pyrG (Weidner et al. 1998), arg2(Baek and Kenerley 1998), and ura3 and ura5 (Bergesand Barreau 1991), have been isolated and used in inte-grative vectors, which can restore recipient cell proto-trophic phenotypes by homologous recombination at thetarget locus (Baek and Kenerley 1998; Weidner et al.1998). However, their speci®c integrations are usuallynot immediately distinguishable from gene conversionsor those with ectopic integrations, and depend solely onsubsequent Southern analysis (Weidner et al. 1998).

Fig. 4 Comparison of growth rates of an albino transformant and theblack parental strain. Cells were grown in YPD broth at 25 °C or37 °C and cell numbers were measured microscopically by hemacy-tometer counting. The growth rates were also determined spectro-photometrically atD500 and by colony counting after plating (data notshown)

Fig. 5 Detection of b-galactosidase activity in albino transformantsgrown on CDY broth containing either xylose or maltose as the solecarbon source at 25 °C or 37 °C for 12 h. Assays of cell lysates werecarried out spectrophotometrically after reactionwith substrateONPGfor 30 min at 30 °C. The enzyme activities represent the average valuesof two experiments and were calculated with the equation:U = 1000 ´ [(D420) ) (1.75 ´ D550)]/30 ´ 0.1 ´ D500 (Ausubel et al.1989)

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In this study, we used a 2-kb WdPKS1 gene fragmentas a targeting sequence in vector pYEX303 to enhancehomologous transformation. The signi®cant advantageof using this particular sequence is that Wangiella cellsare constitutively melanized, and thus subsequent iden-ti®cation of speci®c transformants was made very con-venient by visually distinguishing black-to-white colorloss. Also, the low frequency of genetic reversions couldbe directly monitored by their white-to-black colorchanges, although most transformants derived by thismethodology were very stable in subcultures. Moreover,our phenotypic characterization of these albino strainsshowed that WdPKS1 disruptant cells grew normallylike those of the parental strain under all laboratoryconditions. However, this was not surprising, becausepolyketide synthase is involved in a secondary metabo-lism pathway and does not directly a�ect fungal cellvegetative growth, although WdPKS1 disruptants havebeen shown to have signi®cantly lower virulence thanthe wild-type strain in a mouse test (data not shown),which is consistent with our previous ®ndings withchemically derived melanin-de®cient mutant strains(Geis et al. 1984; Dixon et al. 1987, 1992).

For gene overexpression in W. dermatitidis, theheterologous glucoamylase promoter, P-glaA, was em-ployed for the control of expression activity. Expressionunder the control of P-glaA is reported to be regulated bya speci®c trans-acting regulatory factor(s) (Verdoes et al.1994). In A. niger, P-glaA-mediated gene expression isinducible in the presence of maltose or starch as solecarbon sources, whereas it is repressed by xylose (Fowleret al. 1990). P-glaA is thus now widely used for theoverexpression of homologous and heterologous genes invarious ®lamentous fungi (Van den Hondel et al. 1991).Our results indicated that the P-glaA promoter was alsofunctional in W. dermatitidis. However, the e�ects ofcarbon source seemed inconsistent with observationsmade by di�erent detection methods. Also, an elevatedtemperature of incubation could enhance expression ac-tivities in W. dermatitidis, suggesting that some tran-scriptional factors involved in the regulation of thispromoter activity were probably di�erent or missing inthis fungus. More recently, this established transforma-tion and expression system has been used to examinegene overexpression of WdCDC42-1, a W. dermatitidisRHO-type GTPase gene (Ye and Szaniszlo 1996), and itsmutant alleles in this fungus (data to be reported else-where). These latter studies con®rmed and signi®cantlyextended the results presented here, and further provedthat this system can be very useful for the analysis ofdominant gene e�ects and mutational responses in thispathogenic fungus and probably in other members of theform-family Dematiaceae of the Fungi Imperfecti.

Acknowledgements We thank Drs. M. Ward, J. Sweigard and R.Brenner for providing plasmids used in this study. This researchwas supported by a grant to P.J. Szaniszlo from the National In-stitute of Allergy and Infectious Diseases (AI 33049).

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