Isolation of Candida glabrata Homologs of the Saccharomyces cerevisiae KRE9 and KNH1 Genes and Their Involvement in Cell Wall β-1,6-Glucan Synthesis

Abstract

The Candida glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a Saccharomyces cerevisiae mutant with the disrupted KNH1 locus and the KRE9 gene placed under the control of a tetracycline-responsive promoter. Although a cgknh1Δ mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of CgKRE9 resulted in several phenotypes similar to those of the S. cerevisiae kre9Δ null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of β-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in C. glabrata of the cognate CgKRE9 promoter with the tetracycline-responsive promoter in a cgknh1Δ background rendered cell growth tetracycline sensitive on media containing glucose or galactose. cgkre9Δ cells were shown to be sensitive to calcofluor white specifically on glucose medium. In cgkre9 mutants grown on glucose medium, cellular chitin levels were massively increased.

Candida (Torulopsis) glabrata, an imperfect fungus, is a haploid yeast of the genus Candida and has been demonstrated to be a pathogen of opportunistic yeast infections (1). There are increasing concerns over C. glabrata, because it causes not only mucocutaneous but also systemic infections in transplant and immunosuppressed patients (21, 58, 59). Moreover, the extensive use of topical and systemic antifungal drugs has resulted in the appearance of azole-resistant infections with Candida species, including C. glabrata (41, 59). Thus, there is a need to develop new antifungal drugs with novel modes of action and broad spectra.

Fungal cell wall biosynthesis is one possible target for new antifungal drugs, since it is essential for fungal viability and does not occur in mammals (18, 19). Fungal cell wall biosynthesis has been studied quite extensively in Saccharomyces cerevisiae (11, 14, 30) and Candida albicans (5, 11, 19, 36, 37) but not in C. glabrata. However, in addition to the advantage of its haploidy in genetic manipulation, recent progress on the molecular biology of C. glabrata, including development of host-vector systems (28, 29, 60), a controllable gene expression system (40), and the isolation of several structural sequences (17, 28, 35, 44), provides us with an opportunity to study cell wall biosynthesis in this organism.

β-1,6-Glucan is a component of fungal cell walls, where it occurs as a polymer covalently attached to glycoproteins (26, 38) and to other cell wall structural polymers such as β-1,3-glucan and chitin (14, 30). In S. cerevisiae, many genes involved in β-1,6-glucan synthesis were isolated through mutations (kre [killer resistant] mutations) that confer resistance to the K1 killer toxin, which kills sensitive yeast cells following binding to this β-1,6-glucan polymer (4, 6, 8, 15, 34, 48, 49). While other genetic studies have identified additional genes affecting cellular levels of β-1,6-glucan (24, 25, 46, 55), it still remains unclear how these genes, including the KRE genes, are concerned in β-1,6-glucan biosynthesis. Among them, KRE9 and its homolog KNH1, genes encoding cell surface O glycoproteins, are required for β-1,6-glucan synthesis in S. cerevisiae (6, 8, 15). The S. cerevisiae kre9Δ null mutant shows several phenotypes: resistance to K1 killer toxin; slow growth, especially on glucose media; an 80% reduction of alkali-insoluble β-1,6-glucan; and defects in cell separation. Overexpression of KNH1 can partially suppress these phenotypes of a kre9Δ null mutant (15). Although a knh1Δ null mutant showed no obvious phenotype, disruption of both KRE9 and KNH1 was synthetically lethal (15). Further, the SKN7 gene encoding a yeast homolog of bacterial two-component regulators has also been isolated as a multicopy suppressor of the slow-growth phenotype of the kre9Δ null mutant (7, 9). Recently, a homolog of the KRE9 gene has been isolated from C. albicans (33).

Here we report isolation of the KRE9 and KNH1 homologs in C. glabrata and several lines of evidence, including the first analysis of cell wall components in C. glabrata, suggesting evolutionary conservation of these molecules as essential components of β-1,6-glucan synthesis.

MATERIALS AND METHODS

Strains, growth media, and procedures.

The S. cerevisiae and C. glabrata strains used in this study are listed in Table 1. YPD and YPGal are complex yeast media with 2% glucose and 2% galactose, respectively, and YNB is a synthetic medium with either 2% glucose or 2% galactose and supplemented for auxotrophic requirements. Yeast transformations were carried out by the modified lithium acetate method (20, 23) and the one-step transformation method (12). Tetracycline assays were carried out as previously described (39). Seeded-plate assays for killer toxin sensitivity were performed as previously described (8). Spotting assays were performed as previously described (31). 5-Fluoro-orotic acid, G418 (Geneticin), and calcofluor white (CFW) were purchased from PCR Inc. (Gainesville, Fla.), GIBCO BRL (Grand Island, N.Y.), and Polysciences Inc. (Warrington, Pa.), respectively. Plasmid DNA was propagated in Escherichia coli XL-1-blue cells (Stratagene, La Jolla, Calif.).

TABLE 1.

Yeast strains used in this study

Strain	Genotype or description	Source or reference
S. cerevisiae
SEY6210	MATα leu2-3,112 ura3-52 his3-Δ200 lys2-801 trp1-Δ901 suc2-Δ9	S. D. Emr
HAB813	MATα kre9Δ::HIS3 in SEY6210	6
FAHAP4	MATa ade2-101 his3-Δ200 leu2-Δ1 lys2-801 trp1-Δ63::TRP1-tetRHAP4AD ura3-52	39
SNB50-1	MATa Kanr-97t-KRE9 in FAHAP4	This work
SNB54-5	MATa knh1Δ::hisG in SNB50-1	This work
C. glabrata
2001HTU	cgura3Δ cgtrp1Δ cghis3Δ	28
ACG22	cgtrp1Δ::CgTRP1-TAGAL4 in 2001HTU	40
SNBG1-7-7	cgkre9Δ::CgTRP1 in 2001HTU	This work
SNBG2-26	cgknh1Δ::CgHIS3 in 2001HTU	This work
SNBG3-10	URA3-97t-CgKRE9 in ACG22	This work
SNBG4-49	cgknh1Δ::CgHIS3 in SNBG3-10	This work
SNBG5	cgkre9Δ::CgTRP1; suppressor from SNBG1-7-7	This work

Manipulation of DNA.

Techniques for manipulation of DNA were performed as previously described (52). Yeast genomic DNA was prepared as previously described (51). Southern blots were performed by using nylon membranes (Hybond N; Amersham Canada Limited, Oakville, Ontario, Canada) and following the instructions of the manufacturer. A PCR fragment harboring the entire coding sequence for S. cerevisiae KRE9 was used as a probe. DNA sequencing was performed by the dideoxy method (53) on an ABI 373A sequencer with Bluescript universal and reverse primers and synthetic oligonucleotides complementary to specific regions of CgKRE9 and CgKNH1.

Plasmids.

A 0.7-kbp HindIII fragment harboring the tetO-HOP1 chimeric promoter and a 1.4-kbp NotI fragment harboring the kanamycin resistance gene (Kan^r) were excised from p97t (39) and pKanMX2 (57), respectively, and systematically cloned into Bluescript SKII+ (Stratagene) to generate p97tKan. A 0.4-kbp SpeI-SacII fragment of pMPY-ZAP (54), harboring the hisG sequence, was blunted with T4 DNA polymerase (GIBCO BRL) and cloned into the EcoRV site of Bluescript SKII+ to construct phisG+ and phisG−. The latter plasmids have their hisG sequences in opposite orientations. A 0.4-kbp SmaI-EcoRV fragment of phisG+, a 1.1-kbp SmaI-HindIII fragment of pMPY-ZAP (harboring the S. cerevisiae URA3 gene), and a 0.4-kbp HindIII-SmaI fragment of phisG− were systematically cloned into Bluescript SKII+ to generate pSNZAP3, harboring a modified hisG-URA3-hisG module.

A 1.0-kbp EcoRI-HindIII fragment harboring the entire CgKRE9 sequence was generated by PCR from C. glabrata genomic DNA with a pair of primers (5′-AAAGAATTCGGATCCAACACGCCTGTTGTG-3′, 5′-TTTCTCAAGCTTTTGGAAGATGGGAGGAC-3′), cloned into pUC118, and subjected to replacement of the region between KpnI and SalI sites with the C. glabrata TRP1 (CgTRP1) sequence (28) to generate pCGK9ΔT (Fig. 1A). The 5′ portion of the CgKNH1 sequence was generated by PCR with a pair of primers (5′-ATATGGTACCAATCAAATGCTCTCG-3′, 5′-CGTTGGGCCCGACACTCTGCGACACTTC-3′) as a 0.3-kbp KpnI-SmaI fragment. The 3′ portion of the CgKNH1 sequence was generated by PCR with a pair of primers (5′-ATATGGATCCTTACGGGGAACAGAACGG-3′, 5′-AAGAGAGCTCAGTAAGTAGAGTGAATATAC-3′) as a 0.4-kbp BamHI-SacI fragment. These two fragments and a 1.0-kbp XhoI fragment harboring the C. glabrata HIS3 (CgHIS3) gene (28) were cloned into Bluescript SKII+ to generate pCGK1ΔH (Fig. 1B). A portion of the CgKRE9 sequence including the start codon was generated by PCR with pSB2-1 as a template and a pair of primers (5′-CCATCGATGAATTCATGCTGCTGCTGGCTATACTGCTATC-3′, 5′-TTTCTCAAGCTTTTGGAAGATGGGAGGAC-3′) as a 0.3-kbp EcoRI-KpnI fragment. This fragment and a 1.4-kbp SacI-BamHI fragment of pSB2-1 were cloned into p97t (39) to generate pCGK9tetAB (Fig. 2A).

FIG. 1.

Disruption of CgKRE9 and CgKNH1 and morphological effects of the deletions. (A) Disruption of CgKRE9. A PCR-amplified fragment (double-headed arrow) from pCGK9ΔT (Materials and Methods) was used for the one-step gene replacement. (B) Disruption of CgKNH1. A KpnI-SacI fragment of pCGK1ΔH (Materials and Methods) was used for the one-step gene replacement. Homologous recombination between the two regions (hatched boxes) resulted in disruption of the chromosomal copy. The wild-type strain, 2001HTU (C), cgkre9Δ deletion strain SNBG1-7-7 (D), and cgknh1Δ deletion strain SNBG2-26 (E) as viewed by Normarski optics are shown. Cells precultured on galactose medium were cultured on glucose medium.

FIG. 2.

Construction of a tetracycline-sensitive mutant of CgKRE9 (Tet^s CgKRE9). (A) Scheme for replacement of the cognate CgKRE9 promoter region with the tetracycline-responsive promoter. A PCR-amplified fragment (double-headed arrow) from pCGK9tetAB (Materials and Methods) was used for the one-step gene replacement. The solid arrow indicates the ORF of CgKRE9. Open and shaded boxes indicate the S. cerevisiae URA3 gene and the tetracycline-responsive promoter, 97t, respectively. Homologous recombination between the two regions (hatched boxes) resulted in generation of the Tet^s CgKRE9 mutant. (B) Growth inhibition by tetracycline on the Tet^s CgKRE9 mutants. A total of 10⁴ cells were inoculated and were cultured on YPD (solid bars) or on YPGal (open bars) for 20 h at 30°C. Growth of cells with tetracycline (50 μg/ml) is expressed as percent of optical density at 600 nm of cells without tetracycline. As the wild type (WT), strain ACG22 (Table 1) was used. Error bars, standard deviations.

pRS424 (13) was used to clone fragments for deletional analysis of the inserts of pSB2-1 and pSBG9-1. A 4.4-kbp PstI fragment of pSB2-1 (Fig. 3A) and a 3.2-kbp SacI-EcoRI fragment of PstI fragment-deleted pSBG9-1 (Fig. 3B) were used for construction of plasmids derived from pRS316, pRS416 (56), pCgACT-14, and pCgACH-3 (29).

FIG. 3.

Restriction maps and deletional analysis of inserts of C. glabrata genomic DNA on pSB2-1 and pSBG9-1. Open bars indicate the inserts on pSB2-1 (A) and pSBG9-1 (B). Fragments used for deletional analysis are represented by solid bars. The presence and absence of complementation activity in Tet^s KRE9 knh1Δ cells are indicated as + and −, respectively. Arrows indicate ORFs of CgKRE9 (A) and CgKNH1 (B). Hatched bars indicate regions with homology to the syntenic S. cerevisiae genes.

Construction of tetracycline-sensitive mutants of S. cerevisiae KRE9 (Tet^s KRE9).

Replacement of the cognate KRE9 promoter with the tetracycline-responsive promoter, 97t (39), was achieved by the one-step gene replacement method (3, 54) with slight modifications. A DNA fragment was amplified by PCR using p97tKan as a template and a pair of primers (5′-GAATAGAACAGGAGTCTCAAAGCATTCTTGAAGCCAGATTGCAACAGCTATGACCATG-3′, 5′-AAAGCACATATGATGGAATTTCTTTGTAAACGCATTATGAATTCT TTTCTGAGATAAAG-3′) and subsequently was used for transformation of the S. cerevisiae strain FAHAP4, which harbors the tetR-HAP4AD fusion activator gene (39). After selection on G418-containing plates, the correct integration was confirmed by PCR and the strain was designated SNB50-1. Disruption of the KNH1 gene in SNB50-1 was achieved by using a DNA fragment amplified by PCR using pSNZAP3 as a template and a pair of primers (5′-CTGATAGTATTATTCTTAACATTATTTTGTTCGGTAGTGTTCCGTAAAACGACGGCC AGT-3′, 5′-CATTATCTGTGCCTCAAAGCATTAACTTTTCTTGCAGTCAGAGAAACAGCTATGACCATG-3′). The correct integration was confirmed by PCR. The strain was subjected to 5-fluoro-orotic acid selection and finally designated SNB54-5 after the elimination of the URA3 gene was confirmed by PCR.

Cloning of C. glabrata KRE9 and KNH1 genes.

SNB54-5 cells were transformed with a pRS424-based C. glabrata subgenomic bank, harboring EcoRI 4- to 7-kbp fragments of C. glabrata genomic DNA, and spread onto both YNB-glucose and YNB-galactose plates containing tetracycline (50 μg/ml). After incubation at 30°C for 3 days, colonies appeared on the plates, cells were collected, and plasmid DNA was recovered from them.

Disruption of CgKRE9 and CgKNH1 and construction of tetracycline-sensitive mutants of CgKRE9 (Tet^s CgKRE9).

Disruption of CgKRE9 in strain 2001HTU was achieved by using a DNA fragment amplified by PCR using pCGK9ΔT as a template and a pair of primers (5′-CCATCGATGAATTCATGCTGCTGCTGGCTATACTGCTATC-3′, 5′-CAACTGGACAAATATCTAAC-3′) (Fig. 1). The correct integration was confirmed by PCR, and the strain was designated SNBG1-7-7. A KpnI-SacI fragment of pCGK1ΔH was used to disrupt CgKNH1 (Fig. 1) in strain 2001HTU. The correct integration was confirmed by PCR, and the strain was designated SNBG2-26.

A KpnI-ClaI fragment harboring target sequences for CgKRE9 and S. cerevisiae URA3 was excised from pCGK9tetAB and used for replacement of the CgKRE9 promoter region with the tetracycline-responsive promoter, 97t (39, 40), in the C. glabrata strain ACG22 (40) (Fig. 2A). After the correct integration was confirmed by PCR, the strain was designated SNBG3-10. To construct SNBG4-49, a KpnI-SacI fragment of pCGK1ΔH was used to disrupt CgKNH1 in SNB3-10. The correct integration was confirmed by PCR.

Cell wall component analysis.

The levels of cell wall alkali-insoluble β-glucan were determined as previously described (15). The alkali-soluble and alkali-insoluble Zymolyase-resistant cell wall fractions were subjected to a dot blot analysis by using anti-β-1,6-glucan antibody as previously described (33) with standardization by cell wall dry weight. The content of cellular chitin was determined as previously described (10) with Streptomyces griseus chitinase (Sigma, St. Louis, Mo.) and standardization by cell dry weight.

Sequence analysis and homology search.

Sequence analysis was performed by using GeneWorks (Intelligenetics, Mountain View, Calif.) and GeneJockey (Biosoft, Cambridge, United Kingdom) software. A homology search for C. glabrata sequences against S. cerevisiae sequences was performed by using the WU-BLAST2 program in the Saccharomyces Genome Database (Stanford University).

Nucleotide sequence accession numbers.

The nucleotide sequence data reported in this paper have been submitted to the GenBank database. The accession numbers of the C. glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes are AF064251 and AF064252, respectively.

RESULTS

Construction of tetracycline-sensitive mutants of the S. cerevisiae KRE9 gene.

To isolate the S. cerevisiae KRE9 homolog from C. glabrata, we performed complementation screening. As convenient hosts for the screening, tetracycline-sensitive mutants of the S. cerevisiae KRE9 gene (Tet^s KRE9) were constructed. The KRE9 promoter region was replaced with a tetracycline-responsive promoter in a strain, FAHAP4, harboring the tetR-HAP4AD fusion activator gene for tetracycline-controllable gene expression (39). As shown in Fig. 4, addition of tetracycline (50 μg/ml) inhibited growth of cells of Tet^s KRE9 mutant strain SNB50-1 on glucose medium but not on galactose medium. These observations resemble and are consistent with the finding that an S. cerevisiae kre9Δ mutant grows extremely slowly on glucose medium while growing somewhat better on galactose medium (15) and suggest that the concentration of tetracycline used in the present study is sufficient to repress the expression of KRE9 driven by the tetracycline-responsive promoter. The tetracycline sensitivity of the Tet^s KRE9 mutant was complemented by introduction of an extragenic copy of KRE9 on pRS316 (6) (data not shown). Disruption of KNH1 in a Tet^s KRE9 mutant rendered cell growth tetracycline sensitive on glucose or galactose media (Fig. 4). This result is consistent with the known synthetic lethality between kre9Δ and knh1Δ mutations in S. cerevisiae (15). This Tet^s KRE9 knh1Δ mutant strain, SNB54-5, was used for complementation cloning of a C. glabrata homolog(s).

FIG. 4.

Growth of S. cerevisiae Tet^s KRE9 knh1Δ cells harboring either pSB2-1 or pSBG9-1. About 10⁴ cells were inoculated and cultured on YNB-glucose for 20 h (solid bars) or on YNB-galactose for 40 h (open bars) at 30°C. Cells were grown with or without tetracycline (50 μg/ml), and growth on tetracycline is expressed as the percentage of optical density at 600 nm of cells grown without tetracycline. The strain FAHAP4 (Table 1) was used as the wild type (WT). Error bars, standard deviations.

Cloning of C. glabrata KRE9 and KNH1 genes.

By genomic Southern hybridization using the S. cerevisiae KRE9 sequence as a probe, 5- and 6-kbp EcoRI fragments of C. glabrata genomic DNA were shown to contain sequences hybridizing to S. cerevisiae KRE9 (data not shown). This result allowed us to make a subgenomic C. glabrata bank harboring EcoRI fragments ranging from 4 to 7 kbp to assist in their cloning by functional complementation.

After screening Tet^s KRE9 knh1Δ cells transformed with the subgenomic bank on plates containing glucose as a carbon source and tetracycline (50 μg/ml), pSB2-1 harboring the 6-kbp EcoRI fragment was isolated as a plasmid which allowed the mutant cells to grow as well as wild-type cells. However, plasmids harboring the 5-kbp EcoRI fragment, which also gave a hybridization signal in Southern analysis, were not isolated. Since the expression of KNH1 is induced by galactose in S. cerevisiae (15), we screened a population of transformed cells for growth on plates containing galactose as a carbon source. In this way, pSBG9-1, a plasmid harboring the 5-kbp EcoRI fragment was isolated, as well as pSB2-1. As shown in Fig. 4, while the tetracycline sensitivity of Tet^s KRE9 knh1Δ cells was complemented by pSBG9-1 partially on glucose medium but completely on galactose medium, pSB2-1 completely complemented the tetracycline sensitivity of Tet^s KRE9 knh1Δ cells on both media.

Deletional analysis of the inserts of the two plasmids demonstrated that a 1.4-kbp BamHI-PstI fragment of pSB2-1 and a 3.0-kbp PstI-EcoRI fragment of pSBG9-1 were sufficient for the complementation activity (Fig. 3). DNA sequencing determined that the two plasmids harbored distinct open reading frames (ORFs). The ORF on pSB2-1 was predicted to encode a protein (276 amino acids) similar to S. cerevisiae Kre9p with 53% overall identity, and the protein (265 amino acids) deduced from the ORF on pSBG9-1 revealed 48% overall identity with S. cerevisiae Knh1p (Fig. 5). We designated the genes on pSB2-1 and pSBG9-1 CgKRE9 and CgKNH1, respectively. Both predicted gene products showed features characteristic of their S. cerevisiae counterparts: putative N-terminal signals for secretion, a high proportion of serine/threonine residues (22% in both proteins) that could be potential sites for O glycosylation, and C termini rich in basic amino acid residues (Fig. 5).

FIG. 5.

Sequence comparisons of CgKre9p and CgKnh1p with their S. cerevisiae counterparts. (A) Alignment of the putative Kre9p and Knh1p amino acid sequences deduced from the C. glabrata (CgKRE9 and CgKNH1) and S. cerevisiae (KRE9 and KNH1) nucleotide sequences. The residues with conserved identity in all proteins are underlined in the consensus sequence. The putative N-terminal signals for secretion are underlined in each protein. Gaps (shown as dashes) were introduced to improve alignment. (B) Sequence identities between Kre9p and Knh1p proteins.

Extensive sequencing on 3′ flanking regions of both CgKRE9 and CgKNH1 identified additional regions similar to the genes flanking the KRE9 and KNH1 genes in the S. cerevisiae genome. On pSB2-1, two sequences homologous to the RFA3 and CPS1 genes, respectively, which are located in the 3′ region of the KRE9 locus on chromosome X of S. cerevisiae, were found (Fig. 3A). A sequence homologous to the YLA1 gene, located in the 3′ region of the KNH1 locus on chromosome IV, was found on pSBG9-1 (Fig. 3B).

Complementation activity of either CgKRE9 or CgKNH1 on a yeast centromeric plasmid, pRS416 (56), was also examined in the Tet^s KRE9 knh1Δ mutant. The tetracycline sensitivity of the mutant cells on glucose or galactose medium was complemented by introducing a plasmid, CgKRE9-pRS416, whereas CgKNH1-pRS416 complemented the sensitivity only on galactose medium (Fig. 6), suggesting that expression of CgKNH1 is induced by galactose in S. cerevisiae.

FIG. 6.

Growth of S. cerevisiae Tet^s KRE9 knh1Δ cells harboring a single copy of either CgKRE9 or CgKNH1. About 10⁴ cells were inoculated and cultured on YNB-glucose for 20 h (solid bars) or on YNB-galactose for 40 h (open bars) at 30°C. Growth of cells with tetracycline (50 μg/ml) is expressed as percent of optical density at 600 nm of cells without tetracycline. FAHAP4 (Table 1) was used as the wild-type. Error bars, standard deviations.

Complementation of the killer phenotype of the S. cerevisiae kre9 mutant by CgKRE9 and CgKNH1.

Mutations in KRE9 confer resistance to the K1 killer toxin in S. cerevisiae (6, 8). In order to test whether multiple copies of CgKRE9 and CgKNH1 could complement this phenotype, pSB2-1 and pSBG9-1 were transformed into the S. cerevisiae kre9Δ null mutant strain HAB813 (Table 1) and the killer sensitivities of the transformants were examined by measuring zones of killing in a seeded-plate assay (8). The kre9Δ mutant cells are known to show no killer zone in the assay, since the mutant has an 80% reduction of β-1,6-glucan, which is necessary for the toxin binding. As shown in Table 2, cells harboring pSB2-1 formed killer zones when grown on glucose or galactose plates while cells harboring pSBG9-1 did so only when grown on galactose plates. The killer zone sizes, however, were smaller than those of wild-type strain SEY6210 cells, suggesting that the complementation was partial. We also examined complementation activity of either CgKRE9 or CgKNH1 on a single-copy plasmid as assayed via the killer resistance. Cells harboring CgKRE9-pRS416 formed killer zones in the seeding assay on glucose or galactose plates to the same extent as those harboring multiple copies of CgKRE9 (Table 2), whereas cells harboring CgKNH1-pRS416 failed to form killer zones (data not shown). To show that the partial complementation of the killer phenotype of kre9Δ mutant was due to restoration of β-1,6-glucan levels, alkali-insoluble β-1,6-glucan levels in the mutant cells harboring either pSB2-1 or pSBG9-1 were determined. As shown in Table 2, although cells harboring pSBG9-1 showed no restoration, in cells harboring pSB2-1, the alkali-insoluble β-1,6-glucan level was partially elevated over that of the mutant when the cells were grown on glucose medium.

TABLE 2.

Killer phenotypes of alkali-insoluble β-glucan levels of the S. cerevisiae kre9 cells harboring either CgKRE9 or CgKNH1^d

Strain	Allele at KRE9 locus	Plasmid	Alkali-insoluble glucan(s)a		Killer zone sizeb (cm) on:
			β-1,6-Glucan	β-1,3- and β-1,6-glucan	Glucose	Galactose
SEY6210	KRE9	pRS424	138.13 ± 5.15	354.55 ± 1.54	1.53 ± 0.06	1.25 ± 0.05
HAB813	kre9Δ::HIS3	pRS424	32.33 ± 2.00	301.51 ± 17.20	No zone	No zone
HAB813	kre9Δ::HIS3	pSB2-1 (CgKRE9/pRS424)	85.29 ± 2.24	315.28 ± 19.66	1.20 ± 0.09	1.02 ± 0.03
HAB813	kre9Δ::HIS3	pSBG9-1 (CgKNH1/pRS424)	32.86 ± 1.31	267.45 ± 10.53	No zone	0.70 ± 0.00
SEY6210	KRE9	pRS416	NDc	ND	1.48 ± 0.08	1.23 ± 0.08
HAB813	kre9Δ::HIS3	pRS416	ND	ND	No zone	No zone
HAB813	kre9Δ::HIS3	CgKRE9-pRS416	ND	ND	1.10 ± 0.05	1.02 ± 0.10

^aβ-Glucan levels are expressed as micrograms of glucan per milligram (dry weight) of cell wall. 

^bKiller zone size (diameter) was determined by seeded-plate assays as previously described (8). 

^cND, not determined. 

^dAll values are the means of at least three determinations ± 1 standard deviation. 

Disruption of CgKRE9 and CgKNH1 genes and construction of tetracycline-sensitive mutants of CgKRE9 (Tet^s CgKRE9).

To explore the physiological essentialness of CgKRE9 and CgKNH1, each gene was disrupted with the C. glabrata TRP1 (CgTRP1) and HIS3 (CgHIS3) genes, respectively (Fig. 1). Transformation for disruption of CgKRE9 was performed on plates containing either glucose or galactose as a carbon source. cgkre9Δ mutants were obtained from only galactose plates, whereas cgknh1Δ mutants were obtained from glucose plates. This carbon source dependency on the growth of cgkre9Δ mutant was confirmed by spotting cells precultured on galactose medium onto plates containing either 2% galactose, 2% glucose, or 2% glucose and galactose. Although cgknh1Δ cells on all plates and cgkre9Δ cells on the galactose containing plate grew as well as wild-type cells, the growth of cgkre9Δ cells was severely impaired on plates containing glucose as a carbon source (data not shown). These results suggest that the presence of glucose is involved in the slow-growth phenotype of the cgkre9Δ mutant. As shown in Fig. 1D, microscopic examination of cgkre9Δ cells transferred from galactose to glucose medium revealed the presence of aggregates of cells with abnormal morphology, which are also observed in the S. cerevisiae kre9Δ null mutant (6). However, cgknh1Δ cells showed no morphological change compared to the wild type (Fig. 1C and E).

To test for a possible synthetic lethality between cgkre9 and cgknh1 mutations, a C. glabrata tetracycline-controllable gene expression system (40) was applied to control the expression of CgKRE9. This system uses the same tetracycline-responsive promoters and tetR fusion activator as the system for S. cerevisiae. As shown in Fig. 2A, a tetracycline-sensitive mutant (Tet^s CgKRE9) was generated by replacing the cognate CgKRE9 promoter region with the tetracycline-responsive promoter in C. glabrata ACG22, harboring the tetR-GAL4AD fusion activator gene (40). Consistent with the growth phenotype of a cgkre9Δ mutant, tetracycline (50 μg/ml) inhibited the growth of Tet^s CgKRE9 cells specifically on glucose medium (Fig. 2B). This glucose-specific tetracycline sensitivity was complemented by introducing an extragenic copy of CgKRE9 on pCgACH-3 (29), a centromeric plasmid for C. glabrata (data not shown). When CgKNH1 was disrupted in a Tet^s CgKRE9 mutant, cells failed to grow on glucose or galactose media in the presence of tetracycline (Fig. 2B). This result indicates that the disruption of both CgKRE9 and CgKNH1 is synthetically lethal in C. glabrata.

Killer phenotypes and β-1,6-glucan levels of cgkre9Δ and cgknh1Δ mutants.

Although cgkre9Δ cells showed severe growth defects on glucose medium, spontaneous second-site suppressor mutations partially restoring growth arose when the cells were cultured by serial passage on glucose medium. Since it is known in S. cerevisiae that those second-site suppressors have no effects on the killer phenotypes except for enhanced growth of the original mutants (4, 8, 34, 48), we used such growth-suppressed cgkre9Δ mutants for further analysis as described below.

To address the killer phenotypes of cgkre9Δ and cgknh1Δ mutants, we asked whether C. glabrata was sensitive to the K1 killer toxin. C. glabrata wild-type strain 2001HTU (Table 1) was found to be sensitive to the toxin on plates containing glucose or galactose as carbon sources, as measured by killer zones formed in a seeded-plate assay (Table 3). When mutant cells were assayed, growth-suppressed cgkre9Δ cells clearly formed smaller killer zones than those of wild-type cells, whereas cgknh1Δ cells formed slightly larger killer zones than those of wild-type cells (Table 3). We also examined the killer sensitivity of cgkre9Δ cells which had been stored on galactose medium to prevent second-site suppressor mutations. Although such mutant cells grew extremely slowly on glucose plates, sizes of killer zones of the cells were the same as those of growth-suppressed cgkre9Δ cells (data not shown).

TABLE 3.

Alkali-insoluble glucan and cellular chitin levels in C. glabrata cells grown on either glucose or galactose^e

Medium	Strain	Genotype	Killer zone sizeb (cm)	Alkali-insoluble glucan(s)a		CFW sensitivityc	Chitind
				β-1,6-Glucan	β-1,3- and β-1,6-glucan
YPD	2001HTU	WT	1.35 ± 0.00	52.48 ± 0.54	178.44 ± 4.54	R	0.88 ± 0.07
	SNBG5	cgkre9Δ::CgTRP1	0.73 ± 0.08	20.14 ± 1.34	233.56 ± 5.75	S	3.87 ± 1.10
	SNBG2-26	cgknh1Δ::CgHIS3	1.55 ± 0.00	52.57 ± 1.40	179.46 ± 4.29	R	0.90 ± 0.04
YPGal	2001HTU	WT	1.17 ± 0.02	76.14 ± 1.07	243.82 ± 9.06	R	1.02 ± 0.02
	SNBG5	cgkre9Δ::CgTRP1	0.63 ± 0.03	38.66 ± 1.62	260.74 ± 1.92	R	1.12 ± 0.05
	SNBG2-26	cgknh1Δ::CgHIS3	1.53 ± 0.02	84.11 ± 2.20	242.65 ± 5.46	R	1.08 ± 0.03

^aβ-Glucan levels are expressed as micrograms of glucan per milligram (dry weight) of cell wall. 

^bKiller zone size (diameter) was determined by seeded-plate assays as previously described (8). 

^cCFW sensitivity was scored by growth of 10⁴ cells on plates containing CFW (25 μg/ml). R, resistant; S, sensitive. 

^dChitin levels are expressed as micrograms of N-acetylglucosamine per milligram of dry cells. 

^eAll values are the means of at least three determinations ± 1 standard deviation. 

To establish that the killer toxin resistance seen in the growth-suppressed cgkre9Δ cells was directly due to decreased levels in β-1,6-glucan, we attempted to determine β-1,6-glucan levels in C. glabrata cells. Following the method used in S. cerevisiae, alkali-insoluble cell wall fractions were digested with Zymolyase, a commercial β-1,3-glucanase preparation, and residual polymers were measured as hexose. As shown in Table 3, in growth-suppressed cgkre9Δ cells, hexose levels in the alkali-insoluble Zymolyase-resistant fraction were reduced to 40 and 50% of wild-type levels in cells grown on glucose and galactose medium, respectively. To verify the presence of β-1,6-linkage in these fractions, alkali-soluble and alkali-insoluble Zymolyase-resistant fractions from all three strains grown on glucose medium were subjected to a dot blot analysis using affinity-purified anti-β-1,6-glucan polyclonal antibody (33). In cgkre9Δ cells, the amount of material recognized by the antibody in both fractions was estimated at less than 50% of those of wild-type by comparing signals from serially diluted spotted samples (data not shown). These results strongly suggest that disruption of CgKRE9 results in a more than 50% reduction of cell wall β-1,6-glucan independent of the carbon source used for growth.

Sensitivity to CFW and cellular chitin levels in cgkre9 and cgknh1 mutants.

CFW, a negatively charged fluorescent dye that preferentially binds to nascent chains of chitin and interferes with cell wall assembly (16, 50), is a useful compound for surveying a broad range of cell wall defects in S. cerevisiae (32, 46). To test for cell wall defects in cgkre9Δ and cgknh1Δ mutants, CFW sensitivities of both growth-suppressed cgkre9Δ and cgknh1Δ cells were determined by a spotting assay (31) on plates containing glucose or galactose as a carbon source. Although cgknh1Δ cells grew as well as wild-type cells even in the presence of 25-μg/ml CFW, growth-suppressed cgkre9Δ failed to grow at this concentration of CFW when glucose was used as a carbon source (Table 3).

In S. cerevisiae, kre9Δ mutant cells gave strong fluorescence when stained by CFW (6). This evidence and glucose-specific CFW sensitivity of growth-suppressed cgkre9Δ cells led us to determine cellular chitin levels in C. glabrata cells. As shown in Table 3, on glucose medium, more than fourfold more cellular chitin was detected in growth-suppressed cgkre9Δ cells than in wild-type cells, while cgknh1Δ cells had almost the same amount of chitin as wild-type cells. On galactose medium, no significant difference was seen in chitin levels among these three strains.

To assess a possible correlation between this chitin increase and the second-site mutations suppressing the growth defect on glucose medium, we measured cellular chitin levels in cgkre9Δ cells without such suppressor mutations. For this purpose, two different strategies were taken. In one, a Tet^s CgKRE9 mutant was used. In the other, cgkre9Δ cells, which had been stored on galactose medium, were switched from galactose to glucose medium. As shown in Fig. 7A, although the repression of CgKRE9 expression is expected to be partial since the inoculum for the tetracycline assay was increased to permit sufficient cells to be obtained for the chitin measurement, addition of tetracycline resulted in an ∼17-fold increase of chitin levels in the Tet^s CgKRE9 mutant cells while there was no obvious change in cells of the parent strain, ACG22. When cgkre9Δ cells were transferred from galactose to glucose medium, cellular chitin levels increased by >15-fold (Fig. 7B). These results suggest that a considerable amount of chitin is present in cgkre9Δ cells grown in the presence of glucose and that such levels are unrelated to second-site mutations leading to growth suppression.

FIG. 7.

Cellular chitin levels in cgkre9 mutants of C. glabrata. (A) Effects of addition of tetracycline on cellular chitin levels in Tet^s CgKRE9 mutants. About 10⁶ cells were cultured on YPD with (solid bars) or without (open bars) tetracycline (50 μg/ml) at 30°C for 20 h, and the cellular chitin levels were measured. As the wild type (WT), strain ACG22 (Table 1) was used. (B) Effect of switching the carbon source on cellular chitin levels in cgkre9Δ mutant. Cells precultured on YPGal were inoculated onto either YPD (solid bars) or YPGal (hatched bars) and cultured at 30°C for 20 h, and the cellular chitin levels were measured. As the wild type (WT), strain 2001HTU (Table 1) was used. Error bars, standard deviations.

Overexpression of CgKNH1 and S. cerevisiae KRE9 in cgkre9Δ cells.

We asked if multiple copies of either CgKNH1 or S. cerevisiae KRE9 could complement the phenotypes of a cgkre9Δ mutant. CgKNH1 was cloned into pRS316 (56), which is known to be a multicopy plasmid for C. glabrata (60). CgKNH1-pRS316 and KRE9-pRS316 (6) were transformed into growth-suppressed cgkre9Δ cells. As summarized in Table 4, the killer sensitivities and β-1,6-glucan levels of the mutant cells were partially restored by multiple copies of S. cerevisiae KRE9 whereas multiple copies of CgKNH1 showed no effect. Further, multiple copies of either CgKNH1 or S. cerevisiae KRE9 allowed growth-suppressed cgkre9Δ cells to grow as well as wild-type cells on plates containing glucose and CFW (25 μg/ml). In the cells harboring CgKNH1-pRS316, the chitin increase was slightly suppressed (Table 4).

TABLE 4.

Effects of multiple copies of either CgKNH1 or S. cerevisiae KRE9 on the phenotypes of growth-suppressed cgkre9Δ cells^f

Strain	Allele at CgKRE9 locus	Plasmid	Alkali-insoluble glucan(s)a		Killer zone sizeb (cm)	CFW sensitivityc	Chitind
			β-1,6-Glucan	β-1,3- and β-1,6-glucan
2001HTU	CgKRE9	pRS316	76.05 ± 3.40	228.22 ± 6.11	1.46 ± 0.02	R	0.94 ± 0.02
SNBG5	cgkre9Δ::CgTRP1	pRS316	35.14 ± 1.42	246.14 ± 3.79	1.10 ± 0.02	S	3.71 ± 0.38
SNBG5	cgkre9Δ::CgTRP1	CgKNH1-pRS316	32.72 ± 0.78	218.78 ± 10.09	0.89 ± 0.01	R	2.95 ± 0.52
SNBG5	cgkre9Δ::CgTRP1	KRE9-pRS316	44.97 ± 3.01	204.59 ± 4.96	1.56 ± 0.02	R	NDe

^aβ-Glucan levels are expressed as micrograms of glucan per milligram (dry weight) of cell wall. 

^bKiller zone size was determined by seeded-plate assays as previously described (8). 

^cCFW sensitivity was scored by growth of 10³ cells on plates containing CFW (25 μg/ml). R, resistant; S, sensitive. 

^dChitin levels were expressed as micrograms of N-acetylglucosamine per milligram of dry cells. 

^eND, not determined. 

^fAll values are the means of at least three determinations ± 1 standard deviation. 

DISCUSSION

The CgKRE9 and CgKNH1 genes have been identified by functional screening using an S. cerevisiae Tet^s KRE9 knh1Δ mutant. Both C. glabrata gene products have significant overall identity with their S. cerevisiae counterparts (Fig. 5B). Partial restoration of the killer sensitivity and β-1,6-glucan levels of kre9Δ mutant cells harboring multiple copies of CgKRE9 (Table 2) clearly indicates that CgKRE9 is an ortholog of S. cerevisiae KRE9. Furthermore, a single copy of CgKRE9 was sufficient to partially complement the killer phenotype of the kre9Δ mutant (Table 2). This result also supports the argument for the functional similarity between Kre9p and CgKre9p and implies that the promoter activity of CgKRE9 and the N-terminal signal for secretion of CgKre9p are active in S. cerevisiae.

Disruption of CgKRE9 resulted in cells with phenotypes similar to that of the S. cerevisiae kre9Δ null mutant (6): a severe growth defect on glucose medium, resistance to the K1 killer toxin, a reduction of β-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium (Table 3; Fig. 1D). Some of these phenotypes were partially complemented by multiple copies of S. cerevisiae KRE9 (Table 4). Recent cloning of the C. albicans KRE9 (CaKRE9) gene has demonstrated that CaKre9p is also required for β-1,6-glucan synthesis in C. albicans (33). These lines of evidence indicate that the function of Kre9p as an essential component for β-1,6-glucan biosynthesis is conserved at least among S. cerevisiae, C. albicans, and C. glabrata.

cgknh1Δ mutants, however, had no phenotype beyond a slightly increased sensitivity to the K1 killer toxin. Further, multiple copies of CgKNH1 failed to restore the killer sensitivity and alkali-insoluble β-1,6-glucan levels in cgkre9Δ cells grown on glucose medium (Table 4). However, in addition to the synthetic lethality suggested by the tetracycline sensitivity of Tet^s CgKRE9 cgknh1Δ mutant (Fig. 6B), its ability to complement a range of kre9 defects in S. cerevisiae and C. glabrata implies that CgKnh1p is related to Kre9p/CgKre9p and is an ortholog of S. cerevisiae Knh1p. These complementation abilities include S. cerevisiae kre9 mutant phenotypes (Fig. 4 and Table 2), CFW sensitivity, and chitin increase of growth-suppressed cgkre9Δ cells (Table 4).

We have demonstrated that cellular chitin levels were significantly increased in cgkre9 mutants on glucose medium (Table 3 and Fig. 7). It is known that chitin levels are also increased in several cell wall mutants of S. cerevisiae such as gas1Δ, fks1Δ, and knr4Δ mutants (22, 27, 45, 47). Based on genetic interaction between gas1Δ and chs3Δ mutations and the sensitivity to nikkomycin Z (a competitive inhibitor of chitin synthases) of a gas1Δ mutant, it has been hypothesized that such a chitin increase is essential for growth as a compensation mechanism to support the impaired cell wall integrity of these mutants (27, 45, 47). However, the increase of chitin in cgkre9 cannot simply be concluded to be the result of such a compensation mechanism, since it is correlated with a severe growth defect on glucose medium and is independent of the reduction of β-1,6-glucan. This idea that increased chitin levels slow the growth of cgkre9 mutants is supported by several observations in the present study. First, considerable amounts of cellular chitin were detected in both tetracycline-treated Tet^s CgKRE9 cells grown on glucose medium (Fig. 7A) and cgkre9Δ cells transferred from galactose to glucose medium (Fig. 7B). Second, there was no obvious increase in chitin levels in cgkre9Δ cells grown on galactose medium (Table 3 and Fig. 7B), on which they grew as well as the wild type did, in spite of a 50% reduction of alkali-insoluble β-1,6-glucan (Tables 3 and 4).

The mechanism and physiological relevance of the chitin increase in cgkre9 mutants and its apparent glucose dependence remain to be elucidated. In S. cerevisiae, at least five genes have been known to be involved in the chitin synthase activity (11, 14). Cloning of these homologs and an enzymatic analysis of chitin synthesis in C. glabrata will be helpful in addressing this question. It will be useful to see if a chitin increase is common to S. cerevisiae kre9 and other kre mutants, since second-site mutations suppressing growth defects have been isolated in many kre mutants and act without restoration of killer sensitivity or β-1,6-glucan levels (4, 8, 34, 48). Glucose-specific cross-linking changes in the cell wall of cgkre9Δ cells may result in elevated chitin levels and a severe growth defect on glucose medium.

Extensive sequencing of regions around both the CgKRE9 and CgKNH1 loci show that genomic organization in the 3′ regions of both homologs is conserved between C. glabrata and S. cerevisiae (Fig. 3). This synteny in regions of two chromosomes further indicates a close evolutionary relationship between C. glabrata and S. cerevisiae, consistent with the phylogenetic trees deduced from comparison of 5S (2) and 18S (43) rRNA genes. Further, CgKre9p and CgKnh1p have lower overall identity between themselves than to their orthologous S. cerevisiae counterparts (Fig. 5B). This observation implies that the duplication of the KRE9 and KNH1 genes took place before the divergence of these two fungi from a common ancestor. In contrast, no chromosomal conservation between S. cerevisiae and C. albicans was found in the 8-kbp fragment containing the CaKRE9 locus (data not shown). This result supports the idea of a more distant relationship of C. albicans and S. cerevisiae based on phylogenetic trees deduced from the distribution of the serine-tRNA gene (42, 43) and comparison of rRNA genes (2, 43). Although the presence of a KNH1 homolog in C. albicans still remains a possibility, this result suggests that extensive genomic reorganization around the CaKRE9 locus has occurred since its divergence from a common ancestor with S. cerevisiae. For example, it is possible that the duplication event leading to the KRE9 and KNH1 pair in S. cerevisiae and C. glabrata occurred after the divergence of these yeast lineages from that of C. albicans.

In summary, although the molecular functions of the Kre9p/Knh1p proteins still remain to be characterized, the evolutionary conservation of the essentiality of these proteins supports the idea that compounds that interfere with their functions would be new antifungal drugs affecting a broad spectrum of pathogenic fungi. Our data also indicate that C. glabrata is a useful model pathogenic fungus for understanding biological processes, including cell wall biosynthesis.