INTRODUCTION

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Wangiella dermatitidis is a polymorphic, dematiaceous (melanized) fungus that traditionally is most associated with cutaneous and subcutaneous phaeohyphomycosis (16). Recently, this pathogen has become a paradigm for this emerging mycosis, because invasive and central nervous system phaeohyphomycosis is also being diagnosed with greater frequency in both immunocompetent and immunocompromised patients (17, 22). In vivo, this fungus produces a variety of dark-walled, vegetative forms, such as budding yeast, various hyphal types, and septate and nonseptate, isotropically enlarged bodies that resemble the sclerotic cells of chromoblastomycotic fungi in subcutaneous lesions (11, 16). In vitro, W. dermatitidis is easily manipulated in ways that produce each growth form in relatively homogeneous populations (29, 30). This inherent polymorphism allows it to serve as an exquisite model for the more than 100 other dematiaceous pathogens of humans (31). Cells induced to carry out interconversions from one growth form to another often exhibit dramatic changes in cell wall chitin and 1,8-dihydroxynaphthalene (DHN)-melanin contents (9, 12, 28, 29). Although the DHN-melanin has been shown to contribute to the virulence of W. dermatitidis (12), the role of chitin has not been established. However, it is known that in yeast cells chitin is mainly localized in septal regions, whereas in hyphal and isotropic forms it is also found throughout the cell wall (14). Furthermore, inhibitors of chitin synthases (polyoxins) have greater effects on cells in morphological transition than on yeasts growing by budding (9). These results imply that chitin, like melanin, is also important in the pathogenicity and virulence of W. dermatitidis.

The chitin synthases (Chs) responsible for chitin polymerization are primarily associated with the plasma membranes of fungi (6). In Saccharomyces cerevisiae, three Chs-encoding genes (CHS) have been cloned and characterized, and the isozyme product of each has been found to be involved in different aspects of yeast development (4, 8, 24). Many other Chs-encoding genes have been identified in fungi, including a number of pathogens of humans. For example, Candida albicans also has three CHS genes (20), but other pathogens such as W. dermatitidis and Aspergillus fumigatus have four and seven CHS genes, respectively (1, 3, 18, 19, 30). Based on derived amino acid sequences the chitin synthases are currently classified among a minimum of five classes (3, 18). The gene products of the CHS genes of S. cerevisiae and C. albicans represent class I, II, and IV chitin synthases, whereas W. dermatitidis and A. fumigatus have one and two additional CHS genes, respectively, which encode class III chitin synthases (3, 18, 19, 30, 32).

It is suspected that chitin synthases in pathogenic fungi should be important to pathogenicity and virulence, although reduced virulence has only been firmly documented for class III disruption mutants of A. fumigatus (19). In spite of this finding, little is known about class III chitin synthases and what is known comes from studies with mutants of obligately filamentous fungi (19, 33, 34). In this study, we report the cloning, characterization, and disruption of WdCHS3, a gene that encodes a class III chitin synthase in a vegetatively more versatile fungus. Our findings showed that high expression of WdCHS3 and high production of WdChs3p are related to a variety of environmental stresses, including the shift of cells to high temperatures. However, our results also showed that this high expression is not responsible for inducing changes in cellular morphology and that decreased chitin contents, abnormal phenotypes, or loss of virulence could not be detected in wdchs3 disruption strains, although these mutants had significantly reduced chitin synthase activities. These results suggested that the contribution of WdChs3p to the growth, survival, and reproduction of W. dermatitidis is redundant with another WdChsp at 37°C or resides elsewhere in its poorly understood life cycle. Nonetheless, the present study presents the first evidence that the transcription of a chitin synthase gene is likely regulated by a negative regulatory element in its 5' upstream sequence.

	MATERIALS AND METHODS

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Strains, culture, and transformations. The laboratory wild-type strain 8656 (ATCC 34100), and the temperature-sensitive Mc3 (wdcdc2) mutant strain (ATCC 38716) of W. dermatitidis used in this study have been extensively characterized (10, 27), whereas the temperature-sensitive Hf1 strain has been described only preliminarily (N. D. P. McIntosh, R. J. Rennard, S. M. Karuppayil, and P. J. Szaniszlo, Abstr. 95th Gen. Meet. Am. Soc. Microbiol. 1995, abstr. F30, p. 92, 1995). Routine propagation of these strains was in the rich medium YPD and all transformations were carried out as previously described (32, 36). To determine whether the transition of yeast cells to hyphae or isotropic forms in W. dermatitidis was dependent upon increased WdCHS3 gene expression, two experimental systems were used as follows. In system 1, log-phase yeast cells of wild-type, Mc3, and Hf1 grown in YPD at 25°C were used to inoculate prewarmed YPD at a density of 10⁶ cells/ml and grown for 24 h at 37°C, which is considered the restrictive temperature for the temperature-sensitive mutants. Yeast, isotropic forms, and hyphae were obtained, respectively. In system 2, log-phase wild-type yeast cells grown at pH 6.5 in the nutrient-poor medium MCD (Bacto Czapek Dox broth [Difco] plus 0.1% yeast extract) were used to inoculate pH 2.5 MCD at an density of 10⁶ cells/ml and then cultured with shaking at 25°C. By 72 h, nearly 100% of the yeast inoculum had converted to isotropically enlarged cells and multicellular forms. The log-phase, wild-type yeast cells were also used to inoculate the totally synthetic pH 6.5 SM medium (10) containing different concentrations of EGTA or devoid of nitrogen. By 24 h, most yeast cells cultured in the presence of the 0.5 mM EGTA had converted to isotropically enlarged cells or less frequently to multicellular forms; those cultured in the presence of 5 or 20 mM EGTA were arrested in their cell cycles either as normal-size yeast cells without buds or with tiny buds (15, 29), and those cultured without nitrogen had initiated yeast-to-hyphal transitions. The XL1-Blue and SOLR (Strategene, La Jolla, Calif.) strains of Escherichia coli used for the construction of genomic and cDNA libraries, subcloning, and plasmid preparation were grown in Luria-Bertani medium supplemented with 100 µg of ampicillin per ml.

Preparation and analysis of nucleic acids. Methods for the isolation of genomic DNA and total RNA, labeling of DNA fragments (25 ng) used for probes in library screens and in Southern and Northern analysis, DNA sequencing, sequence analysis, and PCR amplifications were as described previously (32, 36). The two nested primers designed to amplify a highly specific 570-bp fragment from a previously cloned replication form of M13 PCR-WdCHS3 (21) had the following sequences: CHS3-1 (5'-TAACGAGGACAAGGTCTTAACGGC-3') and CHS3-2 (5'-CCTTCCAAAAACGCCGCCGGGTCC-3'). Primers designed to amplify 5' upstream sequences were as follows: Prev, 5'-TGTCCCGGGCGCAACTGCGA-3' SmaI P240, 5'-CTCAGGGCCCACCTTGAACATA-3' ApaI P555, 5'-AAGGGCCCAGTAGTTGCAGT-3' ApaI

Construction of cDNA, partial genomic libraries, and plasmids. To obtain RNA for cDNA library construction, wild-type yeast cells were grown at 25°C for 36 h, shifted to 37°C, and then incubated for an additional 12 h. The cDNA library was constructed by using the ZAP-cDNA synthesis kit (Stratagene). The BglII partial genomic library was constructed as previously described (36). The WdCHS3 disruption vector pWD3-33 was constructed by cloning a BamHI-HindIII fragment (1 kb) of the WdCHS3 coding region from pZW122 into pAN7-1 (25). Prior to transformation, this plasmid was linearized with EcoRV. The WdCHS3-myc epitope tagging plasmid pZW9712 was constructed as follows. The ~260-bp DraI-BamHI insert from pJR1265 (provided by R. W. Schekman, University of California, Berkeley) (7) was inserted at the SnaBI-BamHI site (near the N terminus of WdChs3p) of pES900, which was derived by cloning an EcoRI-SacII insert from pZW122 in pBluscript KS(+). The resulting plasmid pZW978 was completely digested with SacII and partially digested with EcoRI to release a 1.1-kb fragment, which together with a 2.5-kb fragment released from pZW122 with SacII and XbaI, was then ligated into the EcoRI-XbaI site of pCB1004 (32). The resulting plasmid, pZW979, encoded a WdChs3p with six tandem repeats of the myc epitope after Tyr65, but without the WdCHS3 promoter. Meanwhile, the 2.5-kb XhoI-HindIII fragment from pWZ122 was cloned into pCB1005 in which the cloning sites between EcoRV and XbaI in pCB1004 were deleted to produce pZW971. After the EcoRI-SacI fragment in pZW971 was replaced by a 3.4-kb EcoRI (partially digested)-SacI insert from pZW979, pZW9712 was obtained, which contained the WdCHS3 complete coding region with a 6-myc tagging sequence and the WdCHS3 promoter.

Chitin content, chitin synthase, and -galactosidase assays. Chitin contents and chitin synthase activities were measured as previously described (32). Differences in chitin and chitin synthase activities among groups were evaluated for statistical significance by the parametric one-way analysis of variance Newman-Keuls test for paired data. The analysis was performed by using PRISM version 2.0 software (GraphPad Software, Inc., San Diego, Calif.). Probability values of <0.05 were considered significant. Specific activities of -galactosidase were assayed according to the method used with S. cerevisiae (2).

Immunoblotting and indirect immunofluorescence microscopy. Proteins in cell-wall-free extracts and in isolated cell membranes were prepared for polyacrylamide gel electrophoresis with 8% acrylamide by first breaking cells with glass beads as previously described (32) and then denaturing by mixing with an sodium dodecyl sulfate-reductive loading buffer, followed by heating at 100°C for 5 min (2). Western blotting was performed with primary anti-myc monoclonal antibody (1:7,500) (Invitrogen, San Diego, Calif.), secondary peroxide-labeled anti-mouse antibody (1:3,000) and the ECL Western Blotting Analysis System (Amersham, Piscataway, N.J.). Cells for indirect immunofluorescence localization of WdChs3p-myc were prepared as outlined by Pringle et al. (26). After blocking with 5% goat serum (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) and 0.05% Tween 20-phosphate-buffered saline (PBS) for 1 h, the cells were incubated with 1:500 anti-myc monoclonal antibody (Invitrogen) for 1 h and with 1:300 fluorescein isothiocyanate (FITC)-conjugated Affinipure goat antimouse immunoglobulin G (H+L; Jackson Immunoresearch Laboratories, Inc.) for 1 h at room temperature. Nuclear staining of cells was done with DAPI (4',6'-diamidino-2-phenylindole; 1 µg/ml in PBS) for 2 min (26). Samples were visualized by Nomarski phase-contrast and fluorescent microscopy with an FITC filter cassette and the ICM 405 ZIESS Photoinvertoscope (Carl Zeiss, Inc., Oberkochen, Germany).

Nucleotide sequence accession number. The GenBank accession number of WdCHS3 is AF053314.

	RESULTS

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Cloning of WdCHS3, which encodes a class III chitin synthase. The highly specific 570-bp WdCHS3 PCR fragment amplified by the CHS3-1 and CHS3-2 nested primer set and labeled with [-³²P]dATP identified three positive cDNA clones among 15,000 plaques screened in a W. dermatitidis cDNA library. One of these, pWZ103, containing a 3.0-kb WdCHS3 cDNA insert, was rescued in E. coli. A genomic clone of WdCHS3 was also isolated from a partial genomic library of W. dermatitidis constructed and screened by colony hybridization by using a 2.0-kb BamHI fragment of the WdCHS3 cDNA as a probe, and one positive clone pWZ122 was then isolated. Restriction enzyme mapping of a 4.8-kb insert confirmed its identity to the cDNA gene (Fig. 1A). The nucleotide sequences of WdCHS3 and its cDNA (data not shown) revealed a single open reading frame of 2658 bp interrupted by two introns (positions 52 to 104 and positions 2642 to 2690), which encoded a putative protein of 885 amino acids with a calculated mass of 99.4 kDa and a pI of 8.47. The 5' end of the cDNA was present 238 bp from the start codon and was preceded by a highly pyrimidine-rich sequence in the genomic DNA. The 3' end of the cDNA was 266 bp downstream of the stop codon, and the polyadenylation signal sequence AAAAATAAAA was present 20 bp ahead of the poly(A). However, neither a TATA box nor a CCAAT box was identified in the promoter region. Comparison of the deduced WdChs3 protein with other deduced chitin synthases indicated highest identities to chsG (75.2%) of A. fumigatus (19), chsB (72.5%) of A. nidulans (33), and chs1 (65.6%) of N. crassa (34), respectively (Fig. 1B). Thus, the WdChs3p represents a class III chitin synthase as defined by Bowen et al. (3). Three amino acid regions (I [415 to 447], II [454 to 493], and III [517 to 536]), which are very highly conserved in all chitin synthases and hypothesized to be critical sites for catalytic activity (23), were also identified in the derived WdChs3p sequence (Fig. 1B). Hydropathy analysis indicated that WdCHS3 encodes a transmembrane enzyme with a hydrophilic region located near its amino terminus, a neutral region at its center, and a hydrophobic region near its carboxy terminus, a profile similar to those of other class III chitin synthases (data not shown).

View larger version (5163K): [in this window] [in a new window]   FIG. 1.   Restriction map (A) and multiple protein sequence comparison of four class III chitin synthases developed by using CLUSTAL analysis (B). Restriction enzyme abbreviations: A, ApaI; B, BamHI; EcoRI; H, HindIII; P, PstI; S, SacII. The 2-kb BamHI fragment (hatched box) represents the probe used for Southern and Northern analyses, and the 1-kb HindIII-BamHI fragment (gray box) was used for the WdCHS3 gene disruptions. The conserved regions (I, II, and III) of chitin synthases are underlined. The class III enzymes compared were WdChs3p (W. dermatitidis), AfChsGp (A. fumigatus), AnChsBp (A. nidulans), and NcChs1p (N. crassa).

WdCHS3 is differentially expressed in response to stress, but expression is not a prerequisite for polymorphic transition. Northern analysis, with a 2-kb BamHI fragment of WdCHS3 as a probe, detected a single transcript of about 2.6 kb in yeasts, isotropic forms, and hyphae cultured at 37°C, but not in these strains grown as yeasts at 25°C (Fig. 2A). The probe also detected the same-size transcripts in the wild type grown at 25°C under the acidic (pH 2.5) conditions that initiated the development of isotropic forms (Fig. 2B), as well as in wild type grown at 25°C and subjected to Ca²⁺ limitation by increasing EGTA concentrations, which also initiated isotropic-form development, or to nitrogen starvation that initiated hyphal development (Fig. 2C). These particular results indicated that the growth of W. dermatitidis in one or the other of its alternative growth forms, or the transition of one form to another, was not dependent upon increased WdCHS3 gene expression. Instead, we suggest that the increased expression detected was a general response induced by the environmental factors tested. This suggestion is supported by the observations that although the wild type showed increased expression of WdCHS3 at 25°C when deprived of Ca²⁺ and NH₄ or when exposed to acidity, all of which induced phenotypic transitions from yeasts to hyphae or to isotropic forms, the wild type also showed increased expression when shifted to 37°C, even though it continued to grow as a yeast, whereas Mc3 and Hf1 converted to isotropic forms and hyphae, respectively.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Northern blot analysis of WdCHS3 expression. (A) Total RNA (20 µg) from wild type (grown at 37°C, lane 1, and 25°C, lane 4), Mc3 (grown at 37°C, lane 2, and 25°C, lane 5), and Hf1 (grown at 37°C, lane 3, and 25°C, lane 6) electrophoresed in a formaldehyde-containing 1.2% agrose gel before being transferred to nylon membrane and probed with the 32P-labeled 2-kb BamHI fragment of WdCHS3 and 0.6-kb PCR fragment of the actin gene (WdACT1) of W. dermatitidis, simultaneously. (B) Total RNA was from wild type (grown in MCD [pH 6.5], lane 1, or in MCD [pH 2.5], lane 2). (C) Total RNA was from wild type (grown in SM [pH 6.5], 0 mM EGTA, lane 1; 0.5 mM EGTA, lane 2; 5 mM EGTA, lane 3; 20 mM EGTA, lane 4; or SM without nitrogen, lane 5).

WdCHS3 gene disruption lowers chitin synthase activities at 37°C. Site-specific integration of linearized pWD3-33 at the WdCHS3 locus was predicted to result in a tandemly arranged 5'- and 3'-truncated gene. Southern blot analysis of putative transformants of the wild type, Hf1, and Mc3 strains by using a WdCHS3 BamHI 2-kb fragment as a probe identified expected band shifts from 6 to 12.6 kb with KpnI-digested DNA and from 10 to 16.6 kb with XbaI-digested DNA and confirmed that these transformants were WdCHS3 disruptants (data not shown). Measurements of the chitin synthase activities of the three parent strains showed that all had significantly (P < 0.05) higher total zymogenic WdChs activities when grown in YPD at 37°C than when the same strain was grown at 25°C (Fig. 3A). Although this same trend was also true for total nonzymogenic WdChs activities, none of the differences between the same strain grown at the two temperatures were statistically significant (Fig. 3B). Nonetheless, all three wdchs3 strains grown at 37°C had significantly (P < 0.05) lower activities in both trypsin treatment (to activate zymogens) and non-trypsin treatment assays than did their nondisruption controls grown identically (Fig. 3), suggesting that WdChs3p contributed most of the additional WdChs activity associated with the shift of cells to 37°C. Support for this conclusion is provided by the fact that no matter what their morphology cells with a disruption in WdCHS3 and grown at 37°C had consistently reduced WdChs activities, with levels about equal to those associated with the same strain or its wdchs3 counterpart grown at 25°C.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   WdChs activities of the wild-type (wt), Mc3, and Hf1 strains (hatched bars) and of their wdchs3 mutants (open bars) incubated at 25 or 37°C and assayed after trypsin treatment (A) or without trypsin treatment (B). Results are derived from at least three independent experiments. Standard deviations are shown. Significantly different (P < 0.05) activities between controls (nondisruption strains) grown at 25°C and those grown at 37°C are indicated by one asterisk, whereas significant differences (P < 0.05) between controls and disruption strains at each temperature are indicated by two asterisks.

The 5' upstream sequence of the WdCHS3 contains a negative regulatory region. Fragments of various lengths from the 5' upstream region of WdCHS3 (Fig. 4A) fused with lacZ in pYEX303-gal (35) were specifically integrated into the wdpks1 locus of the wild type. Among 50 HmB-resistant transformants, about 40% produced white colonies, indicating that those constructs were integrated at the wdpks1 locus, which was subsequently confirmed by Southern blotting (data not shown). Two independently derived white transformants with each construct were then assayed for -galactosidase activity (Fig. 4B). The results showed that increased activity correlated with the higher WdCHS3 expression at 37°C determined by Northern analysis, and also that levels of -galactosidase activity in the 37°C-grown transformants with upstream sequences truncated to 780 and 240 bp were four- to sixfold higher than in those grown at 25°C. However, almost no -galactosidase activity was detected in transformants with either the intact integrated 1.6-kb upstream sequence or with none of the upstream sequence. The increased -galactosidase activities associated with the deletion of the 5' upstream sequences from 780 bp strongly indicated the presence of a negative regulatory element between 780 and 1600 bp. Furthermore, the increasing activities associated with the deletions from 780 to 240 bp may be indicative of additional negative regulatory elements.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Analysis of the 5' upstream sequence of WdCHS3 by expressing WdCHS3::lacZ reporter fusions in the wild-type strain. (A) Plasmids with WdCHS3 upstream sequences (at positions 1600, 780, 555, 240, and 0 bp) extending from the first codon fused to E. coli lacZ, the A. niger glaA gene terminator, and a fragment of WdPKS were integrated at the wdpks locus by transformation. (B) Two independent white strains from each transformation were grown in YPD broth at 25 or 37°C for 20 h and then assayed for -galactosidase activity.

Synthesis of WdChs3p is enhanced by incubation at 37°C. A myc epitope encoding six tandem repeats was introduced at a position close to 5' end of WdCHS3. Transformation of the HindIII-linearized vector, pZW9712, into W. dermatitidis was expected to result in a tandemly arranged wdchs3-myc copy and a WdCHS3 copy separated by vector sequence. Southern analysis of 4 of 200 HmB-resistant transformants of wild type showed that 3 had the required site-specific integrations (Fig. 5A). Western analysis of proteins, either in cell-wall-free extracts or in isolated membranes of the wdchs3-myc transformants grown at 37°C, using monoclonal anti-myc antibody, detected a dominant protein band at about 115 kDa and a broad very high-molecular-mass band in two strains (Fig. 5B). The dominant band, postulated to be WdChs3p-myc, was somewhat larger than the calculated WdChs3p molecular size (99.4 kDa), probably because of the 6-myc insertion and posttranslational modification. In contrast, no signals were detected in any nonmembrane fractions, suggesting that WdChs3p-myc was integrated in the plasma membrane or existed in membrane-bound structures. That the WdChs3p-myc localized in the latter, as well as the former, was indicated by immunofluorescent microscopic detection of this protein at high levels in the cytoplasms of the wild-type, Mc3, and Hf1 strains incubated at 37°C but not at 25°C (data not shown). However, in a third strain, only an ~70-kDa protein band was detected in both the cell extracts and membranes, suggesting that a recombination error during the site-specific integration had caused an open reading frame sequence shift, leading to the translation of a truncated protein. The chitin synthase activity of the wdchs3-myc1 transformant grown at 37°C was measured and shown to have consistently higher enzyme activity than that of the wild-type strain, which indicated that the extra WdChs activity came from the expression of a functional wdchs3-myc in this strain (data not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Construction of WdChs3p-myc strains. (A) Southern analysis of site-specific integrations of pZW9712. DNA from wild type and the three transformants, wdchs3p-myc1 (lane 1), wdchs3p-myc2 (lane 2), and wdchs3p-myc3 (lane 3), digested with BglII or KpnI, and hybridized with a 2-kb WdCHS3 BamHI fragment. (B) Western analysis of membrane proteins (M) and cell-wall-free extracts (E) from wild type and the same three wdchs3p-myc transformants in the same order grown at 37°C for 20 h and hybridized with anti-myc monoclonal antibody.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Western analysis of WdChs3p-myc production. Cell-wall-free extracts (20 µg of protein/lane) prepared from wdchs3-myc1 cultures grown at 18, 25, 30, 37, and 42°C for 24 h (A) or from wdchs3-myc1 cultures after switching from 25 to 37°C at 0, 3, 6, 10, and 24 h (B) were hybridized with anti-myc antibody.

	DISCUSSION

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Protein database comparisons of the predicted sequence of WdChs3p revealed that genes and gene fragments encoding class III chitin synthases have been isolated mainly from filamentous fungi. Gene disruptions in N. crassa and Aspergillus species (19, 33, 34) have suggested that class III enzymes may be essential for normal hyphal growth in these filamentous ascomycetes. However, disruption of a gene encoding a class III chitin synthase in the dimorphic basidiomycete Ustilago maydis had no effect on yeast cell or hyphal growth and morphology, although chitin synthase activities were reduced (13). These results with a dimorphic plant pathogen are similar to our results with the polymorphic human pathogen W. dermatitidis, because no growth or morphological defects were detected in wdchs3 mutants grown as yeasts, hyphae, or isotropic forms. To date, our in vitro research has only demonstrated a reduction in the chitin synthase activities associated with the membranes of wdchs3 mutants of these phenotypes cultured at 37°C. However, we suspect that the contribution of WdChs3p is redundant with that of another WdChsp at 37°C, a possibility supported by our recent finding that double-mutant strains with both WdCHS3 and WdCHS2 deleted, which appear to grow normally like the wild-type and the wdchs3 and wdchs2 single-deletion strains, are less virulent in our acute mouse model, even though the wdchs2 and wdchs3 single deletion strains showed no loss of virulence in the same model (data to be published elsewhere). The observation that significant reductions in WdChs activities of the wdchs3 mutants did not affect chitin contents is also similar to the situation in A. fumigatus (19), where mutants with defects in one or both of its class III-encoding CHS genes had the same amount of chitin as the wild type. This also indicates that another chitin synthase probably compensates for the loss of class III isozymes in both species by increasing either the production or the zymogenic activation of at least one other WdChsp.

Our studies of chitin synthase activities in W. dermatitidis demonstrated that (i) total activity is stimulated by trypsin treatment; (ii) cells grown at 37°C have more activity than cells grown at 25°C; (iii) WdChs activities of the temperature-sensitive mutants Hf1 and Mc3 growing as hyphae or as isotropic forms, respectively, at the nonpermissive temperature (37°C) are higher than those of wild-type yeasts grown identically; and (iv) WdChs3p contributes most of the additional activity associated with cells shifted to the higher temperature. These observations are consistent with the facts that most fungal chitin synthases are zymogenic (5) and that WdChs3p is synthesized as a zymogen that is posttranslationally activated by an unknown factor before carrying out chitin biosynthesis. However, because no decrease in chitin was detected in any wdchs3 mutant compared to its parental strain, it appears that the added zymogenic activity detected in vitro under the conditions of our experiments, was not activated in vivo. In contrast, studies of the overexpression of WdCHS3 cDNA in S. cerevisiae showed that its gene product had a very high activity in the absence of trypsin activation, which was stable over a broad range of pH and temperatures and was inhibited by polyoxin D and nikkomycin Z (unpublished data). Also the expression of WdCHS3 cDNA did not complement the defect of the chs1/chs2 double mutant of S. cerevisiae (unpublished data). Taken together, these observations suggest that posttranslational regulation of WdChs3p in its native host and in a host not having a homolog of a class III Chs is different and worthy of further investigation.

The differential expression of WdCHS3 in response to stress indicated that this gene is not only posttranslationally regulated but also transcriptionally regulated, which was confirmed by analysis of 5' upstream sequences fused with the lacZ reporter gene. Site-specific integrations of the reporter gene constructs at the wdpks1 locus showed that a negative regulatory element exists between 1600 and 780 bp in the 5' upstream sequence of WdCHS3 gene. Because the Northern blotting showed that WdCHS3 was highly expressed at 37°C but the expression of lacZ with the 1.6-kb upstream sequence was repressed at this temperature, it is implied that there are even more complicated regulatory factors to be defined at other regions. Furthermore, our finding that WdChs3p-myc production is also affected by temperature and time further confirmed that synthesis of WdChs3p is directly correlated with WdCHS3 gene expression.

The 6-myc epitope tagging of WdChs3p at the N-terminal region of WdChs3p did not appear to affect its function. However, not every site-specific integrative transformant expressed a full-length WdChs3p-myc, suggesting that errors during the homologous recombination possibly caused an open reading frame shift. Therefore, it will be important to confirm the size and function of any epitope-tagged WdChsp before performing additional protein purification and localization experiments. Also, the nature of the smeared high-molecular-weight band in the WdChs3p Western blots, which probably represents aggregated protein, needs to be investigated further to be certain that this material does not confound the results of activity assays planned for the future. Nonetheless, the use of anti-myc antibody may eventually allow the detection, separation, and purification of WdChs3p-myc-containing fractions by density gradient centrifugation or Rotofor isoelectric focusing. The latter has been used previously and suggested that WdChsp activities might be associated with four proteins (30).