A System for Gene Cloning and Manipulation in the Yeast Candida Glabrata

GENE 07830

A system for gene cloning and manipulation in the yeast Candida glabrata

(AIDS; Immunocompromised; mutant; pathogenic fungi; plasmid; Torulopsis; transformation)

Pengbo Zhou, Mark S. Szczypka, Rachel Young and Dennis J. Thiele

Department qf Biological Chemistry, University of‘h4iehignn Medico! School, Ann Arbor, MI 48109-0606, USA

Received by J.A. Gorman: 4 April 1993; Revised/Accepted: 22 September/5 October 1993; Received at publishers: 10 January 1994

SUMMARY

The opportunistic pathogenic yeast, Ca~dida (Tor~~u~s~s)gZabrata, is an asexual imperfect fungus that exists largely as a haploid. Besides being a clinically important pathogen, this yeast also provides a model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription. To facilitate molecular genetic studies in C. glabrata, we isolated a strain auxotrophic for uracil biosynthesis. The ura- mutation could be functionally compIemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C. glabrata URA3 gene in this strain. We also found that the centromere-based S. cereuisiae plasmid pRS316 could stably transform and replicate in multiple copies in C. glabrata. In contrast, high-copy-number S. cerevisiae plasmids containing the 2~ circle autonomous replication sequence were not able to replicate productively in C. glabrata. We cloned the C. glubrata URA3 gene, encoding orotidine-S-phosphate decarboxylase, by complementation of a ura3- strain of S. cereuisiae. The deduced amino-acid sequence is highly similar to that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provides a genetic locus for targeted gene integration in C. glabrata. Integrative plasmids were constructed based on the cloned C. g~abrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination.

INTRODUCTION Although less virulent than Candida albicans, C. glabrata has been demonstrated to be the pathogen of opportunis- Candida (Torzdopsis) glabrata is among several clini- tic infections such as vaginitis, endophthalmitis and per- cally isolated yeast species of the Candida genus. sistent Torulopsis glabrata fungaemia (Redondo-Lopez et al., 1990; Odds, 1987; Damani and Webb, 1988). Corresp~~ndence to: Dr. D.J. Thiele, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI Clinically, this species has been found in many human or 4~109-0606, USA. TeI. (l-313) 763-5717; Fax: (1-313) 763-4581; e-mail: animal organs including the gastrointestinal and genito- Dennis_Thiele~UM.CC.UMICH.EDU urinary tracts, stomach, skin and urinary tract (White, gtabrata Abbreviations: A, absorbance (1 cm); aa, amino acid(s); Ap, ampicillin; 1976; Frye et al., 1988; Sinnott et al., 1987). C. ARS, autonomously replicating sequence(s); bp, base pair(s); C., is the second most frequently isolated species from vagina Candida; E., Escherichia; EMS, ethyl methanesulfonate; 5-FOA, in both asymptomatic females and patients suffering from 5-fluoroorotic acid; GCG, Genetics Computer Group (Madison, WI, yeast vaginitis (Redondo-Lopez et al., 1990). Recently USA); K., Kluyueromyces; kb, kilobase or 1000 bp; MCS, multiple cloning site(s); MT-lla, tandemly amplified genomic locus encoding there have been increasing concerns over C. glubrata one of the MT-11 isoforms of the C. glabruta metallothionein gene because it can cause systemic infections in immuno- family; nt, nucleotide(s); ORF, open reading frame; S., Saccharomyces; suppressed patients, where it may be life-threatening. C. SC, synthetic complete media; SC-ura, SC lacking uracil: URA3, gene glabrata infections were reported to be a factor in the encoding orotidine-5’-phosphate decarboxylase; wt, wild type; XGal, 5-bromo-4-chloro-3-indol~l-~-~-ga~actoside; YPD, rich media contain- death of diabetes (Hickey et al., 1983) and cancer patients ing 1% yeast extract/ 2% peptone/ 2% dextrose. (Aisner et al., 1976), and Moniaci et al. (1988) reported

SSDl 0378-1119(94)00041-P 136 a case of C. glabr(l~a infection in a patient with AIDS. To obtain a genetically defined locus for gene targeting Studies of the body’s defense against Ca~~d~d~~infection by homologous recombination, a genetic selection reveal that the human tumor neurosis factor ~1(TNF-cr) approach was implemented to isolate the C. glabruta can enhance the fungicidal activity against C. glahrata, a URA3 gene by complementation of a S. cerevisiae uru3- process mediated by human neutrophils (Ferrante, 1989). strain. This selection is based on the suggestion that C. However, the molecular mechanism of TNF-cl. in stimu- glabrata and S. cerevisiae are very close evolutionarily lating this potential antifungal activity of neutrophils re- (Barns et al., 1991) and that C. glubrata genes can func- mains to be determined. Because of the metabolic tion properly in S. crrevisiae (Zhou and Thiele, 1991; similarities between fungi and their eukaryotic hosts, Zhou et al., 1992). A C. glabrata genomic DNA library efforts to develop efficient antifungal agents are largely was constructed and was transformed into the recipient hampered. S. cerez~isiue strain DTY7 (IeuZ-3, -112 and uru3-52). A In order to study the molecular mechanisms under- plasmid, designated Ul (b), was recovered based on its lying pathogenic as well as other biological processes of ability to complement the urn3- phenotype of DTY7. C. giahrata, the development of genetic tools and molecu- Restriction analysis indicated that Ul( b) contains a lar biology methods is essential. The advantage of using 3.7-kb insert. Furthermore, subcloning and complemen- C. glabrata to study the pathogenicity of the Candidu tation studies identified a l.O-kb XhoI-SspI fragment species is that this organism is unicellular and exists as within the 3.7-kb insert that enables DTY7 cells to grow a haploid (Whelan, 1987), rendering it more tractable for on SC-ura media, suggesting that the functional C. gla- the isolation of mutants and for manipulation of the C. brata U RA3 gene is located within this fragment (Fig. 1a). glahvata genome. Recently, a gene cloning system using The nt sequence of the C. glabrata URA3 gene was subse- a DNA fragment carrying a C. g~ab~atu autonomous rep- quently determined and a continuous ORF was identified lication function has been described (Mehra et al., 1992). using the GCG computer program {Fig. lb). The C. gfa- The aim of the present study was to develop genetic brata URA3 gene encodes a polypeptide of 2.56 amino tools that can be used for gene manipulation in C. gla- acids that shares extensive homology with the URA3 bratu, and these include (1) the isolation of a C. glabrata gene product from S. cerevisiue, demonstrating 82% pro- mutant strain harboring a uracil biosynthetic mutation tein sequence identity (Rose et al., 1984). Fig. 2 shows in the ura3 locus, (2) the isolation and sequencing of the the aa sequence comparison between C. glabratu and S. C. glabratu URA3 gene, which provides a genetic locus cerevisiae URA3 proteins. Furthermore, the C. glabraru for targeted gene integration as demonstrated with the URA3 protein is 8.5% and 74% identical to the function- C. glabrata AMTI gene and (3) the use of episomal plas- ally analogous proteins from the yeasts Kluyveromyces mids that can conveniently shuttle between C. glabrata, lartis and C. albicam, respectively (Shuster et al., 1987; S. sereaisiae and Es~her~c~~ia coli. Ernst and Losberger, 1989). This protein sequence sim- ilarity provides a basis for effective complementation of the S. eerez+sicle ura3- auxotroph by C. glab~at~~ URA3 EXPERIMENTAL AND DISCUSSION gene product and vice versa (Zhou et al., 1992).

(a) Isolation of a ura3- auxotrophic strain and cloning of (b) Episomal plasmids for gene transfer in C. gfabrata the C. glabrata URA3 gene The ability to stably introduce genetic information into To generate a C. glabrata strain suitable for genetic C. glabrata by plasmid-mediated transformation is transformation, a strain bearing a mutation in the gene critical to the development of this organism as a facile encoding orotidine S- phosphate decarboxylase was iso- experimental system. We observed that the low-copy lated. The C. glabrata strain (851038) was described pre- centromere-based S. cerevisiae plasmid pRS316 (Sikorski viously (Zhou and Thieie, 1991) and used as the parental and Hieter, 1989) can effciently transform C. gl~rb~at~~Q wt strain (a gift from Dr. P. Magee). One stable ura- (ura3-) strain, forming large colonies on SC-ura media mutant was identified after EMS mutagenesis, purified after one to two days of incubation. This plasmid con- for single colonies and designated ‘Q’ (Fig. 1). The ura- tains the ARS4, URA3 and CEM6 sequences of S. cerevis- phenotype of the Q mutant strain can be complemented iue and exists as a single or low-copy episomal plasmid by the S. cerevisiue URA3 gene on an episomal plasmid in baker’s yeast (Sikorski and Hieter, 1989). Plasmid pRS316 as described in section b, or in a single integrated pRS316 can be readily recovered from C. glabrata by copy (Zhou et al., 1992), consistent with a mutation in transforming total genomic DNA of the C. glabrata recip- the C. glabratu orotidine-S-phosphate decarboxylase- ient cells into E. coli and isolating plasmid DNA from encoding gene, which is functionally equivalent to URA3 individual Ap-resistant E. coli transformants. This sug- from S. cereuisiue (Rose et al., 1984). gests that pRS316 exists in C. glabruta Q cells as an extra- 137

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36 YRKAGWDAX~KRIGN C. g&&rata URA3 265 12 IIIIIl:lll:I.I. YRKAGWEAYLRRCGQQN S. cerevisiae URA3 267 Fig. 2. Amino-acid sequence comparison between the URA3 proteins 156 52 from C. glabruta (265 aa) and S. cereuisiue (267 aa) (Rose et al., 1984) using the GAP comparison of the GCG program. A vertical line indi- 216 72 cates identical aa: a colon (:) or a period (.) indicate aa with an evolution- 276 ary comparison values greater than 0.5 or 0.1-0.5, respectively. P2

336 112

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516 genomic DNA were grown to prepare plasmid DNA (Ausubel et al., 112 1987), and the size of the inserts was determined by digestion with 576 EcoR1-t Xhul to release the cloned fragments, and electrophoretically 192 fractionated on 1% agarose gels. The ligation mix used for yeast trans- 636 212 formation had a 40% frequency of DNA fragment insertion and the average size of the inserts was approx. 3 kb. Transformation of the 696 232 ligation mixture into the recipient S. cereuisiue DTY7 strain (Ieu2-3,-

756 112 and ~~3-52) yielded a total of 40000 colonies which grew on 252 synthetic complete (SC) agar lacking leucine. This represents approx. 3 616 C. glabrcrta genome equivalents, based upon the average insert size, the 265 percentage of recombinants and the C. glubratu estimated genome size. 876 These transformants were replica-printed onto SC-ura agar plates and 936 8 transformants were identified for their ability to grow on this medium. 937 To rescue the plasmids from the transformants, total genomic DNA Fig. 1. C. glahrataur(13- mutant strain and the URA3 gene. C. glubruta was prepared by the rapid glass bead procedure (Ausubel et al., 1987) 85/038 was treated with EMS to 60% survival. The auxotrophic mutant and used to transform E. cob XL-1 blue competent cells to ampicillin cells were enriched by Nystatin treatment as described (Haas et al., resistance. Restriction analysis of plasmid DNA from one uracil protot- 1990) and were selected on 5-FOA plates for uracil auxotrophy roph indicated that this plasmid, designated Ul (b). contained a 3.7-kb (Ausubel et al., 1987). The UMJ- cells were purified to single colonies. fragment and that this plasmid can complement the uru3- phenotype Their ura3- phenotype was complemented by introducing a C. glabrata when re-transformed into DTY7 (MATa his6 leu-7-3, -112 ~~3-52). To episomal plasmid pRS316, which carries the S. ~~r~~lisi~~~LrRA3 gene locate the putative C. ~~~~ru~~ U&t3 gene within this fragment, frag- functional in C. glabt-urn (Zhou et al., 1992). Transformation of C. ments of the original 3.7-kb insert were subcloned into pRS425 and gLzhrmrrrwas carried out using the lithium acetate transformation pro- transformed into DTY7 for complementation analysis. A I-kb cedure, as described (Sherman et al., 1983). Competent cells for trans- Xhol i SspI fragment was identified that enabled DTY7 cells to grow formation were freshly prepared logarithmic phase cells grown in YPD on SC-ma media (a). To determine the nucleotide sequence of the C. medium (Sherman et al., 1983) with an A,,, between 2.0 and 3.0. glahrutu URA3 gene, fragments of the 1021-bp XkoI+Sspl insert were Routinely, we obtained approx. 1000 t~~nsformants using l.Opg of subcloned into the plasmid pBluescript SK(+) as indicated. Plasmid pRS316 plasmid DNA. To clone the C. glubrara URA3 gene. C. g/&&a DNA was prepared and dideoxy sequencing of the double-stranded genomic DNA was partially digested with Sau3A and 20.0 ug were used plasmid DNA was carried out using protocols described in the for ligation into the BamHl site of the S. cewuisiae high-copy plasmid Sequenase Version 2.0 guidebook from US Biochemical (Cleveland, pRS425 carrying the LECi2 gene as a selection marker (Sikorski and OH, IJSA). The primers used for sequencing both strands of DNA Hieter, 1989). 1% of the ligation mix was used to transform competent inserts are the Ml3 -20 primer (5’-GTAAAACGACGGCCAGT) and 0. coli strain XL-1 blue (Stratagene, La Jolla, CA, USA). and the percen- T3 primer (S-ATTAACCCTCACTAAAG), which hybridize to either tage of recombinant plasmids was determined by comparing the number ends before the inserted DNA fragments (see Stratagene catalog). of white colonies with the total number of transformants on LB plates Positions of the nt encompassing the C. ~l~br~rff UR.43 gene are num- containing XGal and 50 pg Ap/ml. We routinely obtained from 30% bered relative to the first nt (+ I ) in the URA3 ORF (b). The deduced to 50% insertion frequency. A total of 30 individual colonies containing aa are shown under the first nt of each codon. The GenBank accession the plasmid-borne Snu3A DNA restriction fragments of C. giahrnra No. is L13661. 138 chromosomal, self-replicating plasmid. Furthermore, 123 456 transformants with pRS3f6 can be cured of the plasmid by selection on 5-FOA medium. All isolates resistant to 5-FOA revert to uracil auxotrophy, as expected, consistent with a lack of integration of the plasmid into the C. kb kb glabruta genome (data not shown). The copy number of the pRS316 plasmid was deter- - ‘6.0 mined in two C. glabrata strains: Q and the amtl-l strain, 4.4 in which the C. glabrata AMTl copper-activated transcription factor gene was insertionally inactivated (Zhou - 2.5 et al., 1992). By comparing the counts @pm) of the Iinear- ized plasmid bands to the authentic AMTI gene DNA 2.1 restriction fragments in Southern blotting analysis, we - calculated the ratios from each comparison, which indi- 1.4 cate that pRS316 is maintained at from 10 to 31 copies in Q or umtl-1 cells (Fig. 3). These results confirm that the pRS316 plasmid retains episomal replication function 0.7 in C. glabrata, although it is currently unclear why the single copy S. cerevisiae plasmid, pRS316, exists as a high- copy plasmid in C. glubrata. Another episomal plasmid, pW25, that contains multiple copies of the MT-flu gene on the plasmid pRS426 (Sikorski and Hieter, 1989), was copy number constructed and also found to be able to replicate in the C. gfabrata Q strain. The existence of a weak autono- 20 31 10 22 mously replicating sequence ARS activity associated with Fig. 3. Southern blotting analysis to determine the copy number of the the MT-lla gene was recently reported (Mehra et al., pRS316 plasmid in C. gltrbratu. The C. glahruta Q (lane 1 and 4) and 1992). However, the small colony size of transformants ~~1-1 (Zhou et al., 1992) (lanes 2 and 5) strains were transformed with harboring only one copy of the MT-lla gene on pRS426 the pRS316 plasmid that contained a 613-bp BgIII-StyI fragment of the AMTI gene (5’ portion) which was fused in-frame to the E. coli /ucZ suggests that the MT-IZu gene has poor ARS function gene. Genomic DNA from the recipient cells, or from the Q cells without relative to that of pRS316. The S. cerevisiae high copy episomal plasmid (lanes 3 and 6) was prepared and digested to comple- plasmid pRS426, which contains 2~ plasmid DNA tion with the restriction enzymes Xhul +XhoI+BglII (lanes I-3) or sequences, was also transformed into the C. gl~~~r~t~~Q Psti t BgiII (lanes 4-6). Southern blotting ( 1.0% agarosc gel) was car- strain. The recipient cells grew as microcolonies on ried out as described (Ausubei et al., 1987) using a 613-bp 3ZP-labeled BglII-fryI fragment containing the 5’ portion of the AMTJ gene as the SC-ura plates, indicating that pRS426 replicates poorly hybridization probe (Zhou and Thiele, 1991). The copy number of the in C. glahrara. This suggests that the S. crrevisine 2~ se- plasmid was determined by comparing respectively, the cpm of “P quence can not foster efficient replication in C. gltrbrutu. associated with the 4.4-kh or 6.0-kb plasmid DNA fragments with that The existence of extra-chromosomal 2n-like circular of the chromosomal AMTl bands (0.7 or 1.4 kb in the Q strain, 2.1 or 2.5 kb in the umtl-I strain) using a Beta-gen scanner. The sizes of each DNA has not been reported in C. glabrata. fragment (in kb) corresponding to the extrdchromosomal plasmid and the authentic AMTl gene restriction fragments are indicated. The (c) Integrative transformation and homologous number below each lane represents the copy number of the pRS316 recombination at the C. glabmta uva3 locus plasmid, relative to the single endogenous AMTI locus. Since the plasmid Ul (b) can not self-replicate in C. glubr~t~~,we used it as an integrative plasmid to target integration of genes at the C. g~ubrut~ urn3 locus (Fig. 4a). fragments were tested for their efficiency of integrative Following integrative transformation using the AM TI transformation and homologous recombination. The gene carried on the linearized Ul( b) plasmid, our plasmid carrying the 3.7-kb Sau3A fragment [Ill(b)] Southern blotting analysis detected the appearance of the transformed more efficiently than that containing only a diagnostic 10.7-kb (PstI digestion) or 2.2-kb and 0.7-kb l.O-kb XhoI-Sspl fragment which only encompasses the (StuI + SpeI digestion) restriction fragments of the URA+ URA3 ORF. This observation suggests a positive correla- transformant (Fig. 4b, c), indicating that Ul (b) plasmid tion between the length of homologous DNA and efficient has successfully delivered the AMTl gene to the C. gla- chromosomal recombination in C. glabrata, however, fur- bratn ura3 locus. Two pRS425-based integrative plasmids ther experimentation must be conducted to ascertain the with the URA3 gene carried on different DNA restriction validity of this observation. 139

b C spars P x s P spars x 12 34 kb kb %+ 10.6- - 3.6 - 2.6 - 2.2

spars P x s P S P spars

- 0.7

Fig. 4. Gene targeting at the C. glahrtrtuuru3 locus. (a) C. g/ubruTu integrative plasmid U 1(b). A 3.7-kb C. g/trhruftr genomic fragment containing the URA3 gene was cloned into the BunlHI site of the plasmid pRS425 to make the plasmid Ul (b) (10.6-kb). The restriction sites that can be used for subcloning genes of interest into the Ut(b) plasmid are indicated. The EcoRI, &oRV and Kpnl sites underlined are not unique but can also be used as cloning sites, resulting in the inactivation of the LECJZ gene. Prior to transformation, the plasmid may be linearized by digestion with Stul, which has a unique restriction site within the (lRA3 coding sequence, to facilitate homologous recombination. (b) Schematic representation of the URA3 locus prior and after plasmid integration. (c) Southern blotting verified the integration. Integrative plasmid U I (b), which carries the AMTI gene, was first digested to completion with SruI. The linearized plasmid was transformed into Q, Genomic DNA from Q or a URA+ transformant were digested to completion with PstI (lanes 1 and 2) or StuI+SprI (lanes 3 and 4), and fractionated on a 1% agarose gel. Southern blotting was performed (Ausubel et al., 1987) using a 1.5-kb “‘P-labeled XhoI + PstI DNA restriction fragment encompassing the (IRA3 ORF as a hybridization probe. The sizes of each DNA restriction fragment are indicated in kb. P, PsrI; S. S&I; Sp, SpeI; X, XhoI.

(d) Conclusions funded by a National Institutes of Health grant (1) An auxotrophic strain of C. glahrata, carrying a GM41840 and grant MOl-RR00042 to the General non-functional ura3 locus was isolated by EMS mutagen- Clinical Research Center, University of Michigan from esis from the wt strain 85/038. the NIH. P.Z. was supported in part by a Rackham (2) The C. glahrata URA3 gene was cloned and se- Predoctoral fellowship from the Horace H. Rackham quenced. It encodes a 265-aa polypeptide which is highly School of Graduate Studies, University of Michigan, and similar to the URA3 gene product from the baker’s yeast a Loeb Predoctoral fellowship from the University of S. cerevisiae and other yeasts. The URA3 gene provides Michigan Cancer Center. a defined genetic locus for targeted gene integration in C. glahratu. (3) The episomal plasmid pRS316, initially constructed REFERENCES as a low copy replicating plasmids in S. cerevisiae, was Aisner, J.. Schimpff, S.C., Sutherland, J.C., Young, V.M. and Wiernik, demonstrated to replicate efficiently in the C. glabratu P.H.: Toruhpsis ghhruru infections in patients with cancer. ura3- strain. This plasmid can shuttle between C. gla- Increasing incidence and relationship to colonization. Am. J. Med. hratu, S. wrrvisiae and E. coli, and should prove useful 61 (1976) 23-28. for gene manipulations or phylogenetic studies in these Ausubel, F.M., Brent. R., Kingston, R.E., Moore. D.D., Seidman, J.A., two yeast systems. Smith, J.A. and Struhl, K. (Eds.): Current Protocols in Molecular Biology. Wiley, New York, 1987. (4) Modified or wt genes of interest which are cloned Barns. SM., Lane, D.J.. Sogin, M.L., Bibeau, C. and Weisburg, W.G.: into the URA3-based integrative plasmids, such as Ul( b), Evolutionary relationships among pathogenic Ctrdidrr species and can be efficiently targeted to integrate at the C. glabratll relatives. J. Bacterial. 173 (1991) 2250-2255. ura3 locus for genetic and functional analysis. Gene Damani, N.N. and Webb, C.H.: Torulopsi.s glrrhrtrtrr fungaemia. Ulster Med. J. 57 (1988) 220-223. disruption, deletion and replacement can be achieved Ernst, J.F. and Losberger. C.: Sequence and transcript analysis of the by constructing integrative plasmids or specific DNA C. alhicms URA3 gene encoding orotidine-5’-monophosphate de- restriction fragments carrying genes of interest and the carboxylase. Curr. Genet. 16 (1989) 153-157. appropriate selection marker such as URA3. Ferrante A.: Tumor necrosis factor alpha potentiates neutrophil antimi- crobial activity: increased fungicidal activity against Torulopsis glrr- hrutrr and Cur&r/a ulhictrns and associated increases in oxygen radical production and lysosomal enzyme release. Infect. Immun. ACKNOWLEDGEMENTS 57 (1989) 2115m~2122. Frye, K.R.. Donovan, J.M. and Drach. G.W.: Torulopsis gltrhrcrtu urinary infections: a review. J. Ural. 139 (198X) 1245-1249. We thank Simon Knight for critical reading of the Haas, L.O.C., Cregg. J.M. and Gleeson, M.A.G.: Development of an manuscript and Paul Joyce and Nancy Martin for helpful integrative DNA transformation system for the yeast Cmrlida tropi- discussions and encouragement. This research was c~trlis. J. Bacterial. 172 ( 1990) 4571-4577. 140

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