1 Department of Microbiology, Columbia University, 701 West 168th Street, NY 10032, USA
2 Biological Sciences Program, Department of Biological Sciences, Columbia University, NY 10027, USA
3 Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, NY 10032, USA
Correspondence
Aaron P. Mitchell
apm4{at}columbia.edu
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ABSTRACT |
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Present address: Department of Microbiology, University of Minnesota, MN 55455, USA.
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INTRODUCTION |
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One distinct property of C. albicans is its ability to produce chlamydospores, an ability shared only with the closely related species Candida dubliniensis. Chlamydospores are large, highly refractile cells with thick cell walls that form on the ends of elongated suspensor cells attached to hyphae and, occasionally, to pseudohyphae (Odds, 1988). They are rich in RNA (Vidotto et al., 1996
) and can germinate in some cases (Jansons & Nickerson, 1970
; McGinnis, 1980
; Raudonis & Smith, 1982
).
Chlamydospores can be induced to form under nutrient-poor oxygen-limited conditions at low temperatures (Calderone, 2002). In cornmeal agar, which is the typical inducing medium in vitro, both light and glucose inhibit chlamydospore formation while nitrogen has no effect (Dujardin et al., 1980a
, b
). Chlamydospores have been found in the lung of an AIDS patient (Chabasse et al., 1988
), and thus may be relevant to infection. The ability to form chlamydospores is nearly universal among C. albicans clinical isolates (Al-Hedaithy & Fotedar, 2002
), thus arguing that chlamydospores have a functional role in C. albicans biology.
The genetic requirements for chlamydospore formation are of interest for four reasons. First, chlamydospores are the output of a developmental process, so the system may be viewed as a model for development in other organisms. Second, it is unclear why the C. albicans genome has so many genes without close homologues in other organisms, and the possibility that they play a role in a C. albicans-specific process like chlamydospore formation may explain their presence. Third, some conserved regulatory pathways respond to well-defined signals, so identification of chlamydospore regulators may reveal the specific external signals that govern their formation. Finally, because chlamydospores form under growth conditions that are not routinely employed for C. albicans cultivation, their genetic requirements may reveal unique functional relationships, biological roles or regulatory signals that govern activity of known gene products.
The latter point has been most clearly reflected in the few known examples of chlamydospore regulators. Transcription factor Efg1p was first characterized as a positive regulator of hyphae formation (Liu, 2001). Efg1p is required for chlamydospore formation but not for hyphal development in cornmeal agar upon oxygen limitation (Sonneborn et al., 1999
); in fact, the efg1/efg1 mutant is hyperfilamentous. This observation was the first indication that Efg1p may function as a negative regulator of hyphae formation under some circumstances, a finding that was echoed in analysis of matrix-induced hyphae formation (Giusani et al., 2002
). A second case is the MAP kinase Hog1p, which is activated by oxidative stress and promotes resistance to oxidants (Alonso-Monge et al., 1999
). Like Efg1p, Hog1p is required for chlamydospore formation but not for hyphal development (Alonso-Monge et al., 2003
). It is possible that chlamydospores are thus formed in response to oxidative stress. While the mechanistic roles in chlamydospore formation of Efg1p and Hog1p are not clearly understood, these findings have expanded our view of these key regulators and may ultimately prove relevant to understanding their roles as virulence factors.
C. albicans is a diploid organism that lacks a complete sexual cycle, and it has been difficult to apply genetic screens to the organism. Identification of C. albicans morphogenetic regulators has relied to a great extent on the use of Saccharomyces cerevisiae as both a biological model and surrogate host for identification of candidate genes (Liu, 2001; Berman & Sudbery, 2002
). These benefits do not seem applicable to chlamydospore formation, which does not occur in S. cerevisiae. Recently, we developed a forward genetic strategy for gene discovery in C. albicans itself (Davis et al., 2002
). We created a panel of 217 defined insertion mutants and screened them for phenotypic alterations. Our initial study reported identification of Mds3p as a new regulator of alkaline pH-induced hyphae formation. Here, we report an analysis of this mutant collection for defects in chlamydospore formation. Our results define a role for alkaline pH-response regulators in acidic conditions, suggest a functional connection between the Isw2p-dependent chromatin remodelling and Efg1p or Hog1p, and define a new phenotypic defect the inability to form either hyphae or chlamydospores. Our results illustrate the utility of this mutant collection for phenotypic testing.
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METHODS |
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C. albicans strains were grown in YPD plus uridine (2 % dextrose, 2 % Bacto Peptone, 1 % yeast extract and 80 µg uridine ml-1) at 30 °C. Following transformations, selection was done on synthetic medium (2 % dextrose, 6·7 % yeast nitrogen base plus ammonium sulfate and the necessary auxotrophic requirements).
Screening for chlamydospore formation.
The GKO library of 217 insertion mutants was screened as follows. Strains were grown overnight in YPD plus uridine at 30 °C, diluted with YPD to an OD600 value of 1·0, and consecutively serially diluted 1 : 10 three times. Then, 100 µl of the third serial dilution was plated onto cornmeal agar (17 g cornmeal agar l-1 plus 80 µg histidine ml-1 plus 0·33 % Tween 80) under a glass coverslip to maintain a semi-anaerobic condition (Dalmau inoculation technique), and grown in the dark for 7 days at 25 °C. Plates were examined over the following 21 days for chlamydospores. Four independent isolates from each disruption mutant were screened.
Screening for filament formation in the embedded cell condition.
The screening of the chlamydospore defective mutants for filamentation ability was done using the embedded cells technique (Giusani et al., 2002) because this more closely resembles the burrowing of the filaments that occurs during chlamydospore formation. Strains were grown overnight in YPD plus uridine at 30 °C, diluted with YPD plus uridine to 4x104 c.f.u. ml-1, cultured for 4 h at 30 °C, and diluted to 400 c.f.u. ml-1 in YPS plus uridine agar (1 % sucrose, 2 % Bacto Peptone, 1 % yeast extract, 2 % Bacto Agar and 80 µg uridine ml-1); plates were poured with the cells mixed inside. Plates were incubated for 4 days at 37 °C.
Gene complementation.
The open reading frame (ORF) affected by each insertion was identified through the Stanford Candida albicans genome database (http://www-sequence.stanford.edu/group/candida). Complementing SUV3 (orf19.4519), SCH9 (orf19.829) and ISW2 (orf19.7401) plasmids were made as follows. PCR was used to produce a fragment for SUV3, SCH9 and ISW2 from approximately 900 bps upstream of the ATG to approximately 400 bps downstream of the stop codon of the ORF for each gene template. (Primer sequences are given in Table 2.) These fragments were inserted into the pGEMT-Easy vector (Promega), which contains NotI sites flanking the insertion. The inserts were then released with NotI and ligated into NotI-digested dephosphorylated pDDB78, a HIS1 vector (Spreghini et al., 2003
), to generate plasmids pMLR101 (containing the SUV3 insert), pCJN103 (containing the SCH9 insert) and pCJN101 (containing the ISW2 insert).
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For comparison to the complemented strains, the mutant strains were made His+ by transforming each mutant with NruI-digested plasmid pDDB78 to histidine prototrophy. Strain CJN223 was derived from mutant GKO443; strain CJN19 was derived from mutant GKO781; strain CJN16 was derived from mutant GKO585.
Suppression studies.
Plasmids pDDB61, containing the full-length RIM101, and pDDB71, containing an activated truncation of RIM101 with a stop codon following amino acid N405 (RIM101-405), were used. The construction of these plasmids was described previously (Davis et al., 2000b). To target integration to the RIM101 locus, pDDB61 and pDDB71 were digested with PpuMI. All strains constructed for the RIM101 suppression study were generated by transforming the appropriate transposon insertion mutant with either the digested pDDB61 or the digested pDDB71 to create full-length or truncated RIM101 at the RIM101 locus, respectively.
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RESULTS AND DISCUSSION |
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The C. albicans sch9/sch9 insertion mutant (CJN19) was also defective in producing both chlamydospores and embedded hyphae under C-I conditions (Fig. 1E). In S. cerevisiae, Sch9p is a protein kinase that has multiple roles in stress resistance, longevity and nutrient sensing (Fabrizio et al., 2001
; Thevelein & de Winde, 1999
). It is impossible to interpret the role that a global regulator like Sch9p may play in chlamydospore formation with confidence. However, considering that chlamydospores are thought to be a storage structure and that Sch9p is implicated in glycogen accumulation, it is possible that Sch9p is a component of a storage pathway required for chlamydospore formation.
The C. albicans isw2/isw2 insertion mutant (CJN16) was defective in chlamydospore formation but did produce embedded hyphae (Fig. 1G). In S. cerevisiae, Isw2p is part of a chromatin remodelling complex that is recruited to promoter regions by the repressor Ume6p (Goldmark et al., 2000
; Fazzio et al., 2001
). The similarity of the isw2/isw2, efg1/efg1 and hog1/hog1 mutant phenotypes is consistent with the simple hypothesis that C. albicans Isw2p may act in conjunction with either Efg1p or Hog1p. This hypothesis suggests that the isw2/isw2 mutant may have other phenotypes in common with efg1/efg1 or hog1/hog1mutants. However, the isw2/isw2 mutant is not hyperfilamentous in a matrix-induced hyphae formation assay and is not hypersensitive to oxidative stresses (data not shown). Therefore, if Isw2p acts in conjunction with either Efg1p or Hog1p, its role may be limited to C-I conditions.
Relationship of pH-response regulators and chlamydospore formation
The rim13/rim13 and mds3/mds3 insertion mutant strains produced fewer chlamydospores and embedded hyphae than the reference strain (data not shown). In addition, chlamydospores were not produced by the mutants until 12 days, compared to 57 days for the reference strain (data not shown). We verified this defect with independently constructed deletion mutants (Fig. 2A, B, D, E) and corresponding strains with reconstituted wild-type alleles (Fig. 2C, F
). Rim13p and Mds3p act in alkaline pH-response pathways in C. albicans and S. cerevisiae, yet C-I medium pH is 5·5. We determined that the medium remains approximately at pH 5·5 in the region of growth under the coverslip after chlamydospores are produced. Preparation of C-I medium at pH 8·0 abolished chlamydospore production by reference strains. These observations argue that Rim13p and Mds3p promote chlamydospore formation, not because of a metabolic alkalinization of C-I medium. Instead, Rim13p and Mds3p function in this context in acidic conditions.
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Utility of the C. albicans insertion mutant library
We found that there were 197 different genes represented by the insertions through comparison of insertion site sequences with the Stanford C. albicans genome from Assembly 19. These genes are listed as follows: AAH1 (orf19.9791), AAT22 (orf19.4669), ABZ1 (orf19.1291), ACH1 (orf19.3171), AFG1 (orf19.4743), ALS7 (orf19.7400), APG7 (orf19.8326), APL5 (orf19.248), ARO80 (orf19.3012), ARR3 (orf19.3122), AXL2 (orf19.5292), BMH1 (orf19.3014), BNI4 (orf19.4457), BPH1 (orf19.6261), BPT1 (orf19.5100), BUD4 (orf19.4257), BUL1 (orf19.5094), CAT8 (orf19.2808), CKA2 (orf19.3530), CRD1 (orf19.6100), CUP2 (orf19.5001), DAL52 (orf19.3208), DIE2 (orf19.6971), DOA4 (orf19.7207), ECM21.3 (orf19.12351), EXG1 (orf19.2990), FTH1 (orf19.12265), FUN31 (orf19.7451), GLO3 (orf19.5445), GSG1 (orf19.3764), GCN1 (orf19.5328), HAL9 (orf19.3190), HAP1 (orf19.10169), HCH1 (orf19.3396), HIT1 (orf19.2723), HOF1 (orf19.5664), HSM3 (orf19.1331), IFA21 (orf19.12603), IFE1 (orf19.769), IFF4 (orf19.7472), IFK2 (orf19.856), IPF243 (orf19.3254), IPF635 (orf19.7614), IPF721.3f (orf19.5365), IPF745 (orf19.5352), IPF918 (orf19.7576), IPF1292 (orf19.7381), IPF2029 (orf19.5251), IPF2125 (orf19.4116), IPF2127.3 (orf19.11598), IPF2147 (orf19.7194), IPF2542 (orf19.13950), IPF2653 (orf19.7410), IPF2971 (orf19.4284), IPF3032 (orf19.5023), IPF3189 (orf19.3563), IPF3448 (orf19.6185), IPF3514 (orf19.6850), IPF3647 (orf19.6729), IPF4004 (orf19.658), IPF4123 (orf19.2033), IPF4305 (orf19.530), IPF4500 (orf19.4846), IPF4684 (orf19.1857), IPF4876 (orf19.6267), IPF4905 (orf19.411), IPF5363 (orf19.3009), IPF5546 (orf19.4428), IPF5795 (orf19.811), IPF5846 (orf19.5495), IPF5912 (orf19.6950), IPF6050 (orf19.6737), IPF6396 (orf19.580), IPF6493 (orf19.9115), IPF6631 (orf19.2763), IPF6787 (orf19.998), IPF6845 (orf19.9331), IPF6954 (orf19.3818), IPF7393 (orf19.9364), IPF7409 (orf19.4668), IPF7525 (orf19.4529), IPF7559 (orf19.6970), IPF8075 (orf19.3701), IPF8113 (orf19.2938), IPF8470 (orf19.14148), IPF8642 (orf19.11450), IPF8666 (orf19.6952), IPF8726 (orf19.1795), IPF8730 (orf19.1793), IPF9171 (orf19.10248), IPF9206 (orf19.3678), IPF9282 (orf19.1510), IPF9538 (orf19.271), IPF9846 (orf19.3202), IPF10001 (orf19.6194), IPF10482.exon2 (orf19.425), IPF10916 (orf19.4763), IPF11006 (orf19.4658), IPF11424 (orf19.4518), IPF11492 (orf19.2392), IPF11644 (orf19.2660), IPF12141 (orf19.9508), IPF12282 (orf19.4893), IPF12368 (orf19.4369), IPF12412 (orf19.3125), IPF12473 (orf19.5571), IPF12584 (orf19.12706), IPF13353 (orf19.4872), IPF13377 (orf19.2350), IPF13689 (orf19.9081), IPF13810 (orf19.1825), IPF13943 (orf19.5235), IPF14019 (orf19.4746), IPF14379 (orf19.4474), IPF14485 (orf19.13704), IPF14468 (orf19.1907), IPF14652 (orf19.2653), IPF15081 (orf19.4409), IPF16755 (orf19.4966), IPF17037 (orf19.4214), IPF17054 (orf19.5037), IPF18512 (orf19.695), IPF19808 (orf19.5016), ISW2 (orf19.7401), JEN1 (orf19.7447), KAR3 (orf19.564), KAR9 (orf19.5011), KEM1.3 (orf19.4969), KNS1 (orf19.4979), LAP41 (orf19.1628), LIP6 (orf19.4823), LPG20 (orf19.771), MDS3 (orf19.6760), MED8 (orf19.11973), MEP3 (orf19.1614), MKK2 (orf19.14178), MRP8 (orf19.3844), MUB1 (orf19.7412), MUC1 (orf19.11659), NMD5 (orf19.4188), NIT2 (orf19.7291), NTH1 (orf19.7479), NUP60 (orf19.2901), NUP85 (orf19.5887), OAF1 (orf19.7583), PAN3 (orf19.4010), PEX14 (orf19.1805), PGM2 (orf19.10359), PHM5 (orf19.7016), PHO23 (orf19.1759), POX18 (orf19.10841), PRM9 (orf19.2508), PTP1 (orf19.6365), PTP3 (orf19.7610), PTR3 (orf19.4535), PUF3 (orf19.1795), RBK1 (orf19.6344), RBT1 (orf19.8907), REV3.5f (orf19.7389), RIC1 (orf19.6036), RIM11 (orf19.1593), RIM13 (orf19.3995), ROD1 (orf19.1509), SCH9 (orf19.829), SEC34 (orf19.4440), SLA2 (orf19.7201), SLK19 (orf19.6763), SMF12 (orf19.2270), SMK1 (orf19.7208), SPE1 (orf19.6032), SPR1 (orf19.2237), SSN8 (orf19.7355), SSY1 (orf19.814), STR2 (orf19.8635), SUV3 (orf19.4519), SYG1 (orf19.768), THI13 (orf19.7324), UGA2 (orf19.4543), VAC7.3 (orf19.1409), YAL061 (orf19.769), YBL053 (orf19.4139), YCR090 (orf19.1394), YHB1 (orf19.3707), YIL130 (orf19.4244), YKL090 (orf19.4243), YLR106 (orf19.813), YME1 (orf19.1252), YMR317 (orf19.5761) and YOR054 (orf19.1276); and ORFs lacking assigned gene names 19.1005, 19.2061, 19.2348, 19.3906, 19.4285, 19.4513, 19.5866 and 19.8837.
Our results in this study illustrate the utility of the C. albicans insertion mutant library for analysis of C. albicans biological attributes. First, we have identified a role for alkaline pH-response regulators in an acidic growth condition, thus suggesting that a novel signal may activate these pathways. Second, phenotypic similarity suggested at first that Isw2p might have been a participant in either the Efg1p or the Hog1p pathways; this hypothesis was not supported by further phenotypic assays, but such observations may be useful with other genes in the future. Third, some of our negative results are informative; for example, the sla2/sla2 insertion mutant in the library is defective in hyphae formation under many growth conditions (Davis et al., 2002) as expected (Asleson et al., 2001
), but not under C-I conditions. Fourth, we note that all of the chlamydospore-defective insertion mutants could be complemented, thus providing a further indication that secondary mutations do not pose an overwhelming problem for the UAU1 mutagenesis strategy (Enloe et al., 2000
; Davis et al., 2002
). Finally, extrapolation of our results to the entire C. albicans genome suggests that roughly 200 genes may be required in total for chlamydospore formation, an estimate that seems reasonable in comparison to S. cerevisiae sporulation (Vershon & Pierce, 2000
), for example.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alonso-Monge, R., Navarro-Garcia, F., Molero, G., Diez-Orejas, R., Gustin, M., Pla, J., Sanchez, M. & Nombela, C. (1999). Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181, 30583068.
Alonso-Monge, R., Navarro-Garcia, F., Roman, E., Negredo, A., Eisman, B., Nombela, C. & Pla, J. (2003). The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans. Eukaryotic Cell 2, 351361.
Asleson, C. M., Bensen, E. S., Gale, C. A., Melms, A.-S., Kurischko, C. & Berman, J. (2001). Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p. Mol Cell Biol 21, 12721284.
Berman, J. & Sudbery, P. E. (2002). Candida albicans: a molecular revolution built on lessons from budding yeast. Nat Rev Genet 3, 918930.[CrossRef][Medline]
Boveris, A. & Cadenas, E. (1982). Production of superoxide radicals and hydrogen peroxide in mitochondria. In Superoxide Dismutase, vol. II, pp. 1530. Edited by L. Oberley. Boca Raton, FL: CRC Press.
Calderone, R. A. (2002). Candida and Candidiasis. Washington, DC: American Society for Microbiology.
Chabasse, D., Bouchara, J. P., de Gentile, L. & Chennebault, J. M. (1988). Chlamydospores de Candida albicans observees in vivo chez un patient attaint de SIDA. Ann Biol Clin (Paris) 46, 817818.[Medline]
Davis, D., Edwards, J. E., Jr, Mitchell, A. P. & Ibrahim, A. S. (2000a). Candida albicans RIM101 pH response pathway is required for hostpathogen interactions. Infect Immun 68, 59535959.
Davis, D., Wilson, R. B. & Mitchell, A. P. (2000b). RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20, 971978.
Davis, D. A., Bruno, V. M., Loza, L., Filler, S. G. & Mitchell, A. P. (2002). Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis. Genetics 162, 15731581.
Dujardin, L., Walbaum, S. & Biguet, J. (1980a). Chlamydosporulation de Candida albicans: deroulement de la morphogenese, influence de la lumiere et de la densite densemencement. Ann Microbiol (Paris) 131A, 141149.
Dujardin, L., Walbaum, S. & Biguet, J. (1980b). Influence de la concentration du glucose et de l'azote sur la morphologie de Candida albicans et la formation de ses chlamydospores dans un milieu de culture synthetique. Mycopathologia 71, 113118.[Medline]
Enloe, B., Diamond, A. & Mitchell, A. P. (2000). A single-transformation gene function test in diploid Candida albicans. J Bacteriol 182, 57305736.
Fabrizio, P., Pozza, F., Pletcher, S. D., Gendron, C. M. & Longo, V. D. (2001). Regulation of longevity and stress resistance by Sch9 in yeast. Science 292, 288290.
Fazzio, T. G., Kooperberg, C., Goldmark, J. P., Neal, C., Basom, R., Delrow, J. & Tsukiyama, T. (2001). Widespread collaboration of Isw2 and Sin3-Rpd3 chromatin remodeling complexes in transcriptional repression. Mol Cell Biol 21, 64506460.
Giusani, A. D., Vinces, M. & Kumamoto, C. A. (2002). Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of EFg1p-mediated repression. Genetics 160, 17491753.
Goldmark, J. P., Fazzio, T. G., Estep, P. W., Church, G. M. & Tsukiyama, T. (2000). The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p. Cell 103, 423433.[Medline]
Jansons, V. K. & Nickerson, W. J. (1970). Induction, morphogenesis, and germination of the chlamydospore of Candida albicans. J Bacteriol 104, 910921.[Medline]
Lamb, T. M. & Mitchell, A. P. (2003). The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae. Mol Cell Biol 23, 677686.
Lamb, T. M., Xu, W., Diamond, A. & Mitchell, A. P. (2001). Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway. J Biol Chem 276, 18501856.
Liu, H. (2001). Transcriptional control of dimorphism in Candida albicans. Curr Opin Microbiol 4, 728735.[CrossRef][Medline]
McGinnis, M. R. (1980). Laboratory Handbook of Medical Mycology. New York, London, Toronto, Sydney, San Francisco: Academic Press.
Minczuk, M., Dmochowska, A., Palczewska, M. & Stepien, P. P. (2002). Overexpressed yeast mitochondrial putative RNA helicase Mss116 partially restores proper mtRNA metabolism in strains lacking the Suv3 mtRNA helicase. Yeast 19, 12851293.[CrossRef][Medline]
Odds, F. C. (1988). Candida and candidosis, 2nd edn. London, UK: Bailliere Tindall.
Porta, A., Ramon, A. M. & Fonzi, W. A. (1999). PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 181, 75167523.
Ramon, A. M., Porta, A. & Fonzi, W. A. (1999). Effect of environmental pH on morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded by PRR2. J Bacteriol 181, 75247530.
Raudonis, B. M. & Smith, A. G. (1982). Germination of the chlamydospores of Candida albicans. Mycopathologia 78, 8791.[Medline]
Sonneborn, A., Bockmuhl, D. P. & Ernst, J. F. (1999). Chlamydospore formation in Candida albicans requires the Efg1p morphogenetic regulator. Infect Immun 67, 55145517.
Spreghini, E., Davis, D. A., Subaran, R., Kim, M. & Mitchell, A. P. (2003). Roles of Candida albicans Dfg5p and Dcw1p cell surface proteins in growth and hypha formation. Eukaryotic Cell 2, 746755.
Thevelein, J. M. & de Winde, J. H. (1999). Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33, 904918.[CrossRef][Medline]
Vershon, A. K. & Pierce, M. (2000). Transcriptional regulation of meiosis in yeast. Curr Opin Cell Biol 12, 334339.[CrossRef][Medline]
Vidotto, V., Bruatto, M., Accattatis, G. & Caramello, S. (1996). Observation on the nucleic acids in the chlamydospores of Candida albicans. Microbiologica 19, 327334.[Medline]
Wilson, R. B., Davis, D. & Mitchell, A. P. (1999). Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181, 18681874.
Received 10 July 2003;
revised 29 August 2003;
accepted 4 September 2003.