INTRODUCTION

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Dimorphic fungi exhibit either a yeast-like form or a (pseudo)hyphal form mainly in response to environmental conditions, and the switch from a yeast-like form to a filamentous form often correlates with their pathogenicities. Candida albicans is a typical dimorphic fungus which can cause life-threatening infections in immunocompromised patients. In this organism, the dimorphic transition is thought to be critical for pathogenesis, as an elongated hyphal form facilitates tissue penetration (47-49). Environmental conditions for a dimorphic transition have also been studied in the related species Candida tropicalis (52). Saccharomyces cerevisiae also shows a dimorphic transition (12, 20, 23, 43, 57), which is induced mainly by nitrogen starvation (12). A number of genes have been shown to participate in the dimorphism of C. albicans and S. cerevisiae. In S. cerevisiae, it has been reported that a mitogen-activated protein kinase (MAPK) is involved in pseudohyphal development and that nitrogen starvation and activated Ras proteins stimulate pseudohyphal growth through both MAPK-dependent and MAPK-independent pathways (7, 23, 25, 29). In addition, several genes have been reported whose regulation influences pseudohyphal growth (2, 3, 9, 11, 56). Furthermore, a variety of genes, such as PHR1, HYR1, CPH1 (ACPR), CST20, HST7, EFG1, RBF1, and TUP1, have been shown to affect dimorphism in C. albicans (1, 4, 15, 18, 21, 22, 24, 26, 30, 45, 50). In spite of these investigations, the mechanisms of dimorphism are still not clear.

Candida maltosa is a dimorphic fungus with a diploid genome (14, 19, 31), and it has been shown by phylogenetic analysis to be closely related to C. albicans (34). For recombinant DNA technology, host-vector systems have been constructed in our laboratory (13, 16, 35-37, 51). Recently, we found that a part of the centromeric DNA (CEN) region, when present on a plasmid, induces pseudohyphal growth in this yeast. It is suggested that some trans-acting factors which interact with a sequence essential for centromeric activity, GGTAGCG, are involved in the regulation of the transition from the yeast form to the pseudohyphal form (31). To help understand the mechanism of this transition, we screened a gene library of C. maltosa for DNA sequences which could suppress the pseudohyphal growth that is induced by additional copies of the CEN sequence.

	MATERIALS AND METHODS

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Strains and media. C. maltosa IAM12247 (the wild-type strain) and its derivatives, CHA1 (his5 ade1) (16) and CHAU1 (his5 ade1 ura3) (37), were used. The media for C. maltosa were YPD (1% yeast extract, 2% Bacto Peptone, 2% glucose), SD (pH 4) [0.17% yeast nitogen base without amino acid or ammonium sulfate (Difco), 0.5% (NH₄)₂SO₄, 2% glucose, appropriate nutrients], SG (pH 4) (SD in which the glucose was replaced with 2% galactose), and hexadecane medium (pH 4) (the same as SD and SG, except that n-hexadecane, which is supplied as vapor, was the sole carbon source). The SD (pH 7), SG (pH 7), and hexadecane (pH 7) media were buffered with 150 mM HEPES and adjusted to pH 7.0. When necessary, agar was added to a concentration of 2%.

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xyl-5 mtl-1 mcrA⁺ mcrB

Plasmid construction and transformation. The construction of pUAH2A and pUAHHH1 was described previously (31). Ligation of DNA ends that were not cohesive with each other was done after blunt ending them with T4 DNA polymerase. The DraI-EcoT22I fragment of URA3 of C. maltosa, the AatII-ClaI fragment containing ARS (13, 51), and the 208-bp HincII-HindIII fragment of the CEN sequence were ligated into the NaeI site, the NspI site, and the EcoRI site, respectively, of pBluescript II KS(+) to yield the plasmid pBLU1. The AatII-ClaI fragment of ARS and the DraIII-SspI fragment of ADE1 (16) of C. maltosa were ligated into the EcoO109I-SspI site and the SalI site, respectively, of pUC119 to yield the plasmid pUAA10. The XbaI-XbaI fragment containing EPD1 (see Fig. 4) was ligated into the XbaI site of pUC119, and then the DraIII-SspI fragment of ADE1 (16) was inserted into the PvuII site of EPD1 to yield the plasmid pEPD1::ADE1. The same XbaI-XbaI fragment containing EPD1 was ligated into the XbaI site of pUC119, and then the SalI fragment containing HIS5 (13) was inserted into the PvuII site of EPD1 to yield the plasmid pEPD1::HIS5. The EcoRI-BstPI fragment containing the full-length EPD1 (see Fig. 4) and the XbaI-XbaI fragment of the GAL promoter (39) were ligated into the SmaI site and the XbaI site of pUAA10, respectively, to yield the plasmid pUAAEB5. The EcoRI-BglII fragment of the amino-terminal part of Epd1p (see Fig. 4) and the XbaI-XbaI fragment of the GAL promoter (39) were ligated into the SmaI site of pUAA10 to yield the plasmid pUAAEBG3. The EcoRI-SalI, XbaI-EcoRV, and SalI-XbaI fragments of pPHS1 (see Fig. 2) were ligated into the SmaI site of pUAA10 to construct plasmids pPHS1ES, pPHS1XE, and pPHS1SX, respectively.

Construction of genomic and subgenomic libraries of C. maltosa. Total DNA was prepared from C. maltosa IAM12247 as described previously (36). After partial digestion with Sau3AI, the total DNA was size fractionated by sucrose density gradient centrifugation. The DNA fragments of 6 to 8 kb were ligated to BamHI-digested and phosphatase-treated pUAA10 and transformed into E. coli DH5. A subgenomic library on pUC119 was constructed as follows. Total DNA of C. maltosa IAM12247 was digested with EcoRI and electrophoresed through a 0.6% agarose gel. A piece of the gel containing EcoRI fragments of around 4.5 kb was cut out, and the DNA contained in it was eluted, purified and ligated with EcoRI-digested and phosphatase-treated pUC119. E. coli MV1190 was transformed with the ligated DNA, and approximately 5,000 ampicillin-resistant colonies were selected and used for screening by colony hybridization.

Deletion of EPD1. pEPD1::HIS5 was digested with ApaLI and used to transform C. maltosa CHAU1. Stable His⁺ transformants were selected to obtain the strain UEP11. pEPD1::ADE1 was digested with ApaLI and used to transform strain UEP11. Stable His⁺ and Ade⁺ transformants were selected to obtain the strain UEP21 (epd1/epd1). Disruption of the EPD1 gene in these strains was confirmed by Southern hybridization and PCR.

Glucan analysis. The alkali-insoluble and -soluble 1,3- and 1,6--D-glucans were isolated and quantified as described by Popolo et al. (40).

Southern blot analysis. Total DNA of C. maltosa was isolated from a 10-ml culture grown for 15 h in selective medium as described previously (36). Southern blot analysis was performed by using the Amersham ECL direct nucleic acid labelling and detection system according to the instructions of the supplier. A 548-bp EcoRV-PvuII fragment of EPD1 was used as a probe. Colony hybridization was carried out with a Hybond-N⁺ membrane (Amersham) as follows. An EcoRI-XbaI fragment from the insert of pPHS1SX was labelled with [-³²P]dCTP (Amersham) by use of a random primer labelling kit (Takara) and used as a probe. Hybridization and membrane washing were carried out by the method suggested by the membrane supplier.

Northern blot analysis. Total yeast RNA from various culture conditions was extracted by the method of Schmitt et al. (46), separated by agarose gel electrophoresis, blotted on a Hybond-N membrane (Amersham), and analyzed. Northern hybridization was carried out as described previously (38).

DNA sequence analysis of EPD1. Appropriate restriction fragments of the EPD1 gene were subcloned into the pUC series plasmids, and DNA sequencing was carried out with an automated DNA sequencer (LI-COR model 4000L).

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB005130.

	RESULTS

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Isolation of a multicopy suppressor of pseudohyphal growth induced by additional copies of the CEN sequence. Previously, we found that the CEN region on a plasmid could induce pseudohyphal growth when it was introduced into C. maltosa (Fig. 1A) (31). To analyze the mechanism of induction, we carried out screening for genes that can suppress pseudohyphal growth under these conditions. First, we confirmed that the host strain does not show pseudohyphal growth on SG agar plates at both pH 4 and pH 7 but that the host strain containing pUAHHH1 carrying the truncated CEN sequence does show it on SG agar plates at both pHs, although the pseudohyphal morphology is more evident at pH 4. Then the latter strain was transformed with a C. maltosa genomic library constructed in the high-copy-number vector pUAA10. After 3 days, about 35,000 transformants were obtained on SG (pH 4) agar plates and examined microscopically. Most colonies showed pseudohyphal growth, but four showed reduced pseudohyphal growth. Plasmids were isolated from these four colonies and reintroduced into C. maltosa CHA1 containing pUAHHH1, and the suppression of pseudohyphal growth by these plasmids was confirmed. One of the plasmids was designated pPHS1 (Fig. 1B). Restriction enzyme analyses and subcloning of the insert of pPHS1 were performed as shown in Fig. 2. The plasmid pPHS1SX, which carried the smallest fragment, suppressed pseudohyphal growth at the same level as pPHS1 (Fig. 1C). Only a part of one open reading frame (ORF) and its possible promoter region were found in the inserted DNA of pPHS1SX. The deduced amino acid sequence of the ORF is homologous to the N-terminal parts of the S. cerevisiae protein Ggp1/Gas1/Cwh52p and of the C. albicans proteins Phr1p and Phr2p. In the other three isolated plasmids, the HIS5 gene was cloned. The transformants with these YRp-type plasmids carrying ADE1 and HIS5 had lost pUAHHH1 carrying HIS5 and the truncated CEN sequence, and hence, they did not show pseudohyphal growth. We next investigated whether this newly isolated plasmid (pPHS1) could suppress the pseudohyphal transition induced by n-hexadecane, which we found to be a strong inducer of the transition (see Fig. 6 and 7). We did not observe suppression by the plasmid under these conditions (data not shown).

View larger version (97K): [in this window] [in a new window]   FIG. 1.   Colony morphology of C. maltosa CHA1 containing plasmids pUAHHH1 and pUAA10 (vector alone) (A), pUAHHH1 and pPHS1 (suppressor) (B), and pUAHHH1 and pPHS1SX (subclone of the insert of pPHS1) (C). The cells were grown on SG agar plates (pH 4) for 3 days.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Delimitation of the inserted C. maltosa DNA of pPHS1. Colony morphology was observed after 3 days on SG agar plates (pH 4). +, suppression of the pseudohyphal growth induced by additional copies of the CEN sequence; , no suppression of the pseudohyphal growth.

Isolation of the entire EPD1 gene. To clone the full-length ORF, the EcoRI-XbaI fragment from the insert of pPHS1SX was used as a hybridization probe. Southern blot analysis on EcoRI digests of C. maltosa IAM12247 genomic DNA was carried out under conditions of high stringency, and one positive band at 4.5 kb was detected (data not shown). Then, the C. maltosa subgenomic DNA library that contained a DNA fragment of about 4.5 kb was constructed in pUC119 and screened by colony hybridization with the same probe. One positive clone was obtained, and the isolated plasmid was designated pPCO10. Subcloning and sequencing of the inserted DNA in this plasmid revealed a single, uninterrupted ORF of 1,650 nucleotides, which could encode a 549-residue protein. We designated this gene EPD1. The plasmid pPHS1SX contained the promoter region and the region encoding amino acids Met-1 to Asn-126 of Epd1p. The deduced amino acid sequence of the entire Epd1p is shown in Fig. 3.

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Comparison of predicted amino acid sequences of Epd1p, Ggp1/Gas1/Cwh52p, Phr1p, and Phr2p. Identical and similar residues are indicated by asterisks and dots, respectively. The hydrophobic amino and carboxy termini are boxed. The proposed GPI attachment sites of Epd1p and Ggp1/Gas1/Cwh52p are indicated by bold letters, and the serine-rich regions are underlined. Amino-acid N-126, from which the EPD1 gene on plasmid pPHS1SX is truncated (see Results), is boxed inversely.

Disruption of EPD1. An epd1 null mutant was constructed to analyze the role of EPD1 in morphogenesis. Two EPD1 alleles in C. maltosa CHAU1 were inactivated by successive gene replacements with the insertion alleles epd1::ADE1 and epd1::HIS5 (Fig. 4A). Single and double EPD1 mutants were generated as described in Materials and Methods and designated UEP11 (EPD1/epd1) and UEP21 (epd1/epd1), respectively. Replacement of the EPD1 gene by homologous recombination was confirmed by hybridization to a genomic Southern blot (Fig. 4B) and by PCR (data not shown). The null mutants were viable, indicating that EPD1 is not essential for cell viability. The duplication times of strains CHAU1 and UEP21 during the exponential growth phase in SD (pH 4) were 80 and 120 min, respectively.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Disruption of EPD1. (A) Construction of the disrupted genes epd1::ADE1 and epd1::HIS5. The detailed methods for the disruption are described in Materials and Methods. (B) Southern blot analysis of the EPD1 mutants. Genomic DNAs from the indicated strains were digested with XbaI and hybridized with the labelled EPD1 DNA region indicated in panel A as a probe. The electrophoretic positions of DNA size markers are indicated on the left.

View larger version (71K): [in this window] [in a new window]   FIG. 5.   Effect of disruption of EPD1 on yeast form growth of C. maltosa CHA1 (EPD1/EPD1) (A), UEP11 (EPD1/epd1) (B), and UEP21 (epd1/epd1) (C). Cells were grown in SD (pH 4) until the early stationary phase.

View larger version (94K): [in this window] [in a new window]   FIG. 6.   Effect of disruption of EPD1 on colony morphology at pH 4. (A and B) CHAU1 bearing pBLU1 and UEP21 containing pBLU1, respectively, were grown on SG agar plates (pH 4) for 1 day. (C and D) CHAU1 and UEP21, respectively, grown on plates containing n-hexadecane as a sole carbon source (pH 4) for 3 days.

View larger version (108K): [in this window] [in a new window]   FIG. 7.   Effect of disruption of EPD1 on colony morphology at pH 7. CHAU1 carrying pBLU1 (A) and UEP21 bearing pBLU1 (B) were grown on SG (pH 7) plates for 1 day. CHAU1 (C) and UEP21 (D) were grown on hexadecane medium (pH 7) for 2 and 3 days, respectively.

View this table: [in this window] [in a new window]   TABLE 1.   -Glucan contents of epd1 mutant cellsa

Transcriptional regulation of EPD1. We also examined the transcriptional regulation of EPD1 by Northern blot analysis. Total RNA was isolated from CHA1 containing the plasmid pUAH2A, which does not induce pseudohyphal growth, or the centromere plasmid pUAHHH1, which induces pseudohyphal growth at various growth phases (early log phase and mid-log phase) in SG (pH 4). The transcriptional level was not influenced by the growth phase or the pseudohyphal morphogenesis, and the transcript was not detected in stationary-phase cells (data not shown). We also examined the effect of pH on the transcriptional regulation of EPD1. Total RNA was isolated at early log phase (optical densities at 600 nm [OD₆₀₀], 0.5 and 2) in SD (pH 4) and SD (pH 7). The transcripts were detected in all samples, and the level of EPD1 mRNA was reduced in the cells grown at pH 7 (Fig. 8). This result is similar to that found with PHR2 (30). However, the expression of EPD1 is not so strictly regulated by the environmental pH signal as is that of PHR2.

View larger version (49K): [in this window] [in a new window]   FIG. 8.   Northern blot analysis of the EPD1 transcript. C. maltosa CHA1 was incubated in SD (pH 4) and SD (pH 7) media. RNA was isolated, and Northern blot hybridization was performed as described in Materials and Methods. Lane 1, OD600 of 0.5 in SD (pH 4); lane 2, OD600 of 0.5 in SD (pH 7); lane 3, OD600 of 2 in SD (pH 4); lane 4, OD600 of 2 in SD (pH 7). The upper panel displays the results of hybridization with EPD1, and the lower panel displays the results of ethidium bromide (EtBr) staining of rRNA. The positions of 25S and 18S rRNA are shown on the right.

The amino-terminal part of Epd1p has a dominant-negative effect on the pseudohyphal growth induced by the CEN sequence. The 5' end of EPD1 was initially isolated as a suppressor of the pseudohyphal growth induced by additional copies of the CEN sequence. To confirm that this phenotype was due to the truncated EPD1 gene, we did the following experiments. Plasmids pUAAEB5 and pUAAEBG3, which express full-length and truncated EPD1, respectively, under the control of the GAL promoter (39), were constructed and transformed into the wild-type strain CHA1 containing pUAHHH1. After 3 days of incubation on SG (pH 4), transformants were examined under the microscope. CHA1 containing both pUAHHH1 and pUAAEB5 showed pseudohyphal growth, whereas CHA1 containing both pUAHHH1 and pUAAEBG3 did not (Fig. 9). This result and the initial isolation of the 5' end of EPD1 suggest that the overexpression of only the amino-terminal region of Epd1p in a multicopy plasmid has a dominant-negative effect on pseudohyphal growth induced by the extra CEN sequence. On the other hand, overexpression of the full-length EPD1 using the GAL promoter in CHA1 did not induce pseudohyphal growth on SG agar plates (pH 4) (data not shown).

View larger version (64K): [in this window] [in a new window]   FIG. 9.   Effect of GAL promoter-induced expression of EPD1 on colony morphology. CHA1 containing pUAHHH1 and pUAAEB5 with full-length EPD1 (A) and pUAHHH1 and pUAAEBG3 with the amino-terminal part of EPD1 (B) were grown on SG agar plates (pH 4) for 3 days.

	DISCUSSION

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Cells of yeasts and fungi are surrounded by walls that are responsible for the mechanical strength of the cell (17) and are essential for maintenance of the cell shape (58). Fungal cell walls are composed primarily of three classes of polysaccharides: mannoproteins, -glucans, and chitins (5). The -glucans constitute about 60% of the cell wall carbohydrate and are believed to be the most important elements in determining cell morphology in S. cerevisiae (58) and probably in C. albicans and C. maltosa. The deduced protein product of EPD1 isolated from C. maltosa in the present work contains several conserved structural features of the GPI-anchored proteins, such as a hydrophobic signal sequence at the N terminus, a C-terminal hydrophobic domain, and a GPI attachment site (8, 10, 28, 32). These and other structural similarities suggest that Epd1p is modified by the addition of GPI and that it is a homolog of the S. cerevisiae GGP1/GAS1/CWH52 gene product and the C. albicans PHR1 and PHR2 gene products. Although the primary function of this family of genes is currently unknown (30, 40, 45), it may be essential for the assembly of -glucan, since the lack of EPD1 in C. maltosa and of GGP1 in S. cerevisiae (40) reduces the level of -glucans (Table 1).

It was reported that C. albicans phr1/phr1 mutants display defects in the formation of germ tubes and apical growth of hyphal forms at the restrictive pH. Prolonged incubation of wild-type C. albicans at higher pH resulted in the mixture of yeast form and pseudohyphal-form cells, but incubation of the phr1/phr1 null mutant did not result in pseudohyphal-form cells (45). The effect of EPD1 disruption is similar to the results obtained by Saporito-Irwin et al. (45), and the function of EPD1 in the transition from yeast form to pseudohyphal form may be similar to that of PHR1 in the morphological transition of C. albicans. But, in the case of the induction of pseudohyphae by n-hexadecane, the disruption of EPD1 affected pseudohyphal morphology at both pH 4 and 7. Although the growth rate of the homozygous null mutant, UEP21, was lower than that of the wild-type strain in SD at pH 4 in the yeast form, it was almost comparable to that of the pseudohypha-forming wild-type strain in hexadecane medium at pH 4, in contrast to the failure of growth of the disruptant of PHR2, a homolog of EPD1. The incapability of strain UEP21 to show pseudohyphal growth, therefore, cannot be attributed to its growth defect. UEP21 cells were round and larger than wild-type cells in SD (pH 4), as was also observed for ggp1, phr1, and phr2 mutants (30, 40, 45), which could be due to a lack of cell wall integrity caused by defects in -glucan assembly. All these results suggest that the function of Epd1p may not be identical, but is quite similar, to those of the GGP1/GAS1/CWH52, PHR1, and PHR2 products.

We conclude that the increased expression of EPD1 may not be required for pseudohyphal growth, not only because overexpression of the full-length EPD1 did not affect the transition but also because Northern blot analysis showed that the expression level of EPD1 was not affected by the pseudohyphal morphogenesis. EPD1 expression seems constitutive during the early and mid-log phases, in contrast to the expression of HYR1, a C. albicans gene encoding a GPI-anchored surface protein which is activated in response to hyphal development (1). The EPD1 gene product may have a more general function in the transition from yeast form to pseudohyphal form. We also examined the effect of pH on EPD1 expression and found that the expression of EPD1 is regulated by pH, similar to but not as strictly as PHR2 (30). In C. maltosa, pseudohyphal growth is not as pronounced at pH 7 as it is at pH 4. This might be explained by the lower level of expression of EPD1 at pH 7. These results suggest that EPD1 works mainly at pH 4, and partially at pH 7. In addition, on hexadecane plates, UEP11 (EPD1/epd1) showed reduced pseudohyphal growth and UEP21 (epd1/epd1) did not show pseudohyphal growth even at pH 7. Pseudohyphal growth on hexadecane may require greater amounts of Epd1p than it does on SG agar plates.

The most interesting finding in this work was that the amino-terminal part of Epd1p has a dominant-negative effect on the pseudohyphal growth induced by the CEN sequence. It may inhibit the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence in the cells. As the total glucan content of the wild-type strain CHA1 that contains pUAAEBG3 bearing the truncated EPD1 downstream of the GAL promoter was identical to that of strain CHA1 containing vector pUAA10 (data not shown), the expression of the amino-terminal part of Epd1p might cause an abnormal architecture, but not an abnormal composition, of the cell wall. However, the expression of the amino terminus of Epd1p by its own promoter had no suppressive effect on the pseudohyphal growth induced by n-hexadecane as a sole carbon source. These differential phenotypes might be due to the differences between the cell wall architectures of the pseudohypha-forming cells induced by n-hexadecane and those induced by the CEN sequence.

There are several examples suggesting that the dose of a gene affects the filamentous growth of yeast. For example, hyphal formation is sensitive to the dose of the MAPK cascade components in C. albicans (18). Our previous data indicated that three copies of YCp-type plasmid induce more marked pseudohyphal growth than the single copy in C. maltosa (31), and the present results indicate that a heterozygous disruptant of EPD1 shows only a slight transition from a yeast form to a pseudohyphal form induced by n-hexadecane. These results taken together suggest that the copy numbers of some cellular components are important for the induction of pseudohyphal growth.

EPD1 is not essential for growth, but it is essential for the pseudohyphal transition. Defining the precise role of Epd1p will be helpful for understanding the mechanism of the dimorphism of fungi.