Dosage-dependent functions of fatty acid desaturase Ole1p in growth and morphogenesis of Candida albicans

Shankarling Krishnamurthy1,{dagger}, Armêl Plaine1,{dagger}, Juliane Albert1, Tulika Prasad2, Rajendra Prasad2 and Joachim F. Ernst1

1 Institut für Mikrobiologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
2 Jawaharlal Nehru University, New Delhi 110067, India

Correspondence
Joachim F. Ernst
Joachim.ernst{at}uni-duesseldorf.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Conditions in the infected human host trigger virulence attributes of the fungal pathogen Candida albicans. Specific inducers and elevated temperatures lead to hyphal development or regulate chlamydospore development. To explore if these processes are affected by membrane lipids, an investigation of the functions of the Ole1 fatty acid desaturase (stearoyl-CoA desaturase) in C. albicans, which synthesizes oleic acid, was undertaken. A conditional strain expressing OLE1 from the regulatable MET3 promoter was unable to grow in repressing conditions, indicating that OLE1 is an essential gene. In contrast, a mutant lacking both alleles of OLE2, encoding a Ole1p homologue, was viable and had no apparent phenotypes. Partial repression of MET3p–OLE1 slightly lowered oleic acid levels and decreased membrane fluidity; these conditions permitted growth in the yeast form, but prevented hyphal development in aerobic conditions and blocked the formation of chlamydospores. In contrast, in hypoxic conditions, which trigger an alternative morphogenetic pathway, hyphal morphogenesis was unaffected. Because aerobic morphogenetic signalling and oleic acid biosynthesis require oxygen, it is proposed that oleic acid may function as a sensor activating specific morphogenetic pathways in normoxic conditions.


Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; ER, endoplasmic reticulum

{dagger}Both authors contributed equally to this work.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The fluidity of cellular membranes is determined to a large extent by their lipid composition and by ambient temperatures. High levels of unsaturated fatty acids, low amounts of sterols in eukaryotic membranes and high temperatures increase fluidity (Carratu et al., 1996; Chatterjee et al., 1997; Horvath et al., 1998). It has been proposed that changes in the physical state of membranes are directly sensed and transmitted by specific signalling pathways triggering protective stress responses (Moskvina et al., 1999). During a heat shock such membrane-induced events could contribute to the complete set of stress responses, which alternatively are activated by protein unfolding (Ananthan et al., 1986; Torok et al., 1997). In yeast an increase in levels of saturated fatty acids within membrane lipids lowered the response to heat shock (Carratu et al., 1996). Similarly, artificially increasing or lowering membrane fluidity in cyanobacteria lowered and, respectively, increased the set point of the heat-shock response (Horvath et al., 1998; Wada et al., 1990; Vigh et al., 1993). Several organisms adapt to low temperatures by increasing the degree of fatty acid desaturation, which leads to an increase in membrane fluidity and simultaneously increases responses to elevated temperatures (Cossins, 1994; Vigh et al., 1998).

In the yeast Saccharomyces cerevisiae the OLE1 gene product encodes a stearoyl-CoA desaturase (EC 1.14.99.5) located in membranes of the endoplasmic reticulum (ER), which transforms the CoA derivatives of palmitic and stearic acid into the corresponding {Delta}9 monounsaturated palmitoleic (C16 : 1) and oleic acid (C18 : 1) derivatives, respectively (Stukey et al., 1989). The C16 : 1 derivative is the predominant unsaturated fatty acid in S. cerevisiae, while other fungi including Candida species also produce C18 : 2 (linoleic) and C18 : 3 (linolenic) fatty acids, which are not present in S. cerevisiae (reviewed by Mishra et al., 1992). Fatty acid desaturases use electrons in cytochrome b5 (derived from NADH by NADH-dependent cytochrome b5 reductase) and molecular oxygen to form a double bond between C-atoms 9 and 10 of fatty acids. Remarkably, fungal fatty acid desaturases contain a cytochrome b5 domain as integral parts of their enzyme structures, while vertebrate enzymes use separate cytochrome b5 molecules (Mitchell & Martin, 1995). The active site in the native protein comprises three histidine-rich sequences, which fold to form two iron-binding sites. Yeast Ole1p contains four putative transmembrane regions, which by forming two pairs of membrane-traversing regions could attach the desaturase to the ER membrane and leave most of the Ole1p sequences within the cytoplasm (Stukey et al., 1990). OLE1 expression is regulated by fatty acids, oxygen and temperature. Saturated fatty acids induce a 1·6-fold increase in transcription, while unsaturated fatty acids repress OLE1 transcription up to 60-fold (McDonough et al., 1992; Bossie & Martin, 1989). At low temperatures and during oxygen limitation, OLE1 expression is induced (Kwast et al., 1998; Nakagawa et al., 2002). A deletion analysis of the OLE1 promoter identified a 111 bp fatty acid-regulated region (FAR) which is essential for transcription activation and repression by unsaturated fatty acids (Choi et al., 1996). In addition, the low oxygen response promoter element (LORE) mediates oxygen repression of OLE1 (Nakagawa et al., 2001; Vasconcelles et al., 2001). Two genes encoding components of fatty acid transporters, FAA1 and FAA4, were found to be essential for unsaturated fatty acid-repression of OLE1 via FAR sequences (Faergeman et al., 2001). Also, the acyl-CoA binding protein and the Ssn6–Tup1 complex were shown to be involved in repression of OLE1 (Fujimori et al., 1997). On the other hand, the Hap1 transcriptional activator (Choi et al., 1996) and two transcription factors, Spt23 and Mga2p, which are initially synthesized as inactive ER-bound precursors, positively regulate OLE1 expression (Zhang et al., 1999). Ole1p is a naturally short-lived enzyme and is degraded by ubiquitin/proteasome-dependent ER-associated degradation (Braun et al., 2002). Lowering of Ole1p activity leads to a loss of mitochondrial inheritance (Stewart & Yaffe, 1991) and disturbs the integrity of the nuclear membrane (Zhang et al., 1999), although growth is not affected even if unsaturated fatty acid levels are reduced down to one-eighth of the normal level (Stukey et al., 1989).

The human fungal pathogen Candida albicans is able to assume different growth forms, which appear to have different roles for host–cell interaction and virulence (reviewed by Ernst, 2000). At body temperatures and in the presence of inducing agents, a true hyphal form is induced, which may be involved in anchoring within and penetrating tissues, while at lower temperatures, in the absence of inducers, a unicellular yeast form is favoured (Joshi et al., 1993; Sonneborn et al., 1999). The yeast form, in special genetic backgrounds at low temperatures, may spontaneously form a rod-like appearance (opaque). Thick-walled chlamydospores appear in certain media, preferentially at lower temperatures. The striking dependence of temperature on the generation of morphological forms suggests a role of membrane fluidity in morphogenesis of C. albicans. Although some signalling pathways mediating the yeast–hypha transition have been defined, no membrane sensors are yet known which mediate environmental cues and specifically could act as cellular ‘thermometers’. In this report, we characterize the OLE1 gene of C. albicans and show that it is essential for viability. We show for the first time in a fungal developmental system that levels of oleic acid in cellular lipids are critical for morphogenetic competence. Thus, a modest reduction in oleic acid does not affect growth in the yeast form, but effectively prevents the formation of hyphal filaments and chlamydospores in aerobic conditions. Our results indicate, however, that overall membrane fluidity is not directly responsible for the morphogenetic potential of C. albicans, but that oleic acid has a specific role to activate specific morphogenetic pathways.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
C. albicans strains used in this study are listed in Table 1. Transformed strains were generated as described by Wilson et al. (1999). Strains were routinely grown in YPD or SD medium (Sherman et al., 1986). A 1 % solution of the non-ionic detergent Igepal CA-630 (Sigma) was used for solubilization of 0·5 mM oleic acid in media (Stukey et al., 1989). Strains were incubated in microaerophilic conditions by using a CampyGen bag in an anaerobic jar (Oxoid). The PCK1 promoter (PCK1p) was induced in SCAA medium (0·67 % yeast nitrogen base without amino acids and 2 % Casamino acids) or SL medium (0·67 % yeast nitrogen base, 2 % sodium lactate) and repressed in SD medium (Leuker et al., 1997). To repress the MET3 promoter (MET3p), SD medium supplemented with different concentrations of methionine and/or cysteine was used (Care et al., 1999).


View this table:
[in this window]
[in a new window]
 
Table 1. Strains and plasmids used in this study

 
Strains were grown for 3–4 days at 37 °C on Lee's medium (Lee et al., 1975), or on Spider-Plates (Liu et al., 1994) or on 5 % horse serum solidified by 2 % agar to induce hyphae. Corn meal agar (CMA) (Difco) containing 0·33 % Tween 80 was used for chlamydospore induction (Joshi et al., 1993). Strains were streaked lightly on the agar surface and covered by coverslips. Following incubation at room temperature for 5 days, photographs of chlamydospores and filaments were taken with a Zeiss Axioscop microscope across the coverslips.

S. cerevisiae Y0779 ({Delta}ole1 : : LEU2 leu2,3-112 lys2-801 trp1-1 ura3-52), kindly provided by S. Jentsch, was cultured at 30 °C in SD medium supplemented with 0·2 % oleic acid in 0·2 % NP40 (Braun et al., 2002). It was transformed by expression plasmids containing OLE1 (pSKM24) or OLE1-GFP (pSKM62) under transcriptional control of the C. albicans PCK1 promoter.

Chromosomal deletions of OLE1 and OLE2.
C. albicans sequence data were obtained from the Stanford Genome Technology Center website (http://www-sequence.stanford.edu/group/candida). The genomic region of OLE1 was isolated by PCR using DNA of strain CAI4. The entire OLE1 coding region was disrupted by the Ura-blaster method (Fonzi & Irwin, 1993). A cassette for disruption of OLE1 was constructed in several steps. First, sequences 5' and 3' of the OLE1 ORF were amplified by PCR and subcloned. A fragment (0·98 kb) of the 5' sequences flanking the start of the CaOLE1 ORF was amplified using primers Ole1disA and Ole1disB (5'-CTAGAGCTCGGATCCCACGGAACTAAAC-3'/5'-CTAGAGCTCGGATCCAGCAGCAATGGCATTC-3'; bold, regions of homology; italics, BamHI); similarly, 728 bp of the 3' untranslated sequences were amplified using primers Ole1disC and Ole1disD (5'-CTAGAGCTCTGCAGAAGGAAAAGCAATC-3'/5'-CTAGAGCTCTGCAGGCGACTACATACATAC-3'; bold, regions of homology; italics, PstI). PCR fragments were subcloned into pUC18, which resulted in plasmids pSKM8 and pSKM7, respectively. The BamHI fragment of pSKM8 was cloned into the BglII site of p5921. The PstI fragment of pSKM7 was inserted into the PstI site of the resulting plasmid pSKM55. A plasmid with OLE1 flanking sequences in the correct orientation was obtained (pSKM56). Its HindIII fragment containing the OLE1 disruption cassette was used to transform strain CAI4.

OLE2 was disrupted similarly by subcloning the regions flanking and partially including the ORF. The 876 bp 5' region was amplified by genomic PCR using primers 709 and 710 (5'-TATGGATCCAAAACTCCTGTAGATGG-3'/5'-TATGGATCCACATACAAGACTGC-3'; bold, regions of homology; italics, BamHI); the 870 bp 3' region was amplified by genomic PCR using primers 711 and 712 (5'-TATAGATCTGACTGCTGCGGTG-3'/5'-TATAGATCTGCCAACTTTTCTAATGC-3'; bold, regions of homology; italics, BglII). The BamHI fragment carrying the 5' region was subcloned into the BglII site of p5921, while the BglII fragment carrying the 3' region was subcloned into the BamHI site of the resulting vector, to construct pAP9a. The SacI–SphI fragment of pAP9a was used for sequential disruption of both OLE2 alleles (Fonzi & Irwin, 1993).

To place OLE1 under control of the MET3 promoter we followed a previously described strategy (Care et al., 1999). First, a fragment of 624 bp corresponding to the 5' end of the OLE1 ORF was amplified by PCR using the primers OleBHI-Nterm (5'-CTTAAGCTTGGATCCCATGTGAGAAAACC-3') and OleStpBHI-HindIII (5'-CTTAAGCTTGGATCCTAAGATTGCTTTTCC-3'; bold, regions of homology; underlining, HindIII, BamHI) and the PCR fragment was cloned into pUC18, resulting in plasmid pSKM20. The BamHI fragment of pSKM20 was inserted into the single BamHI site of pCaDis, downstream of the MET3 promoter (Care et al., 1999). The resulting plasmid pSKM25 was linearized by NcoI (which cuts within the OLE1 fragment) and was used to transform the heterozygous strain {Delta}O8.2 (OLE1/ole1{Delta} : : hisG).

Overexpression of OLE1 and OLE2.
To overexpress OLE1 in C. albicans we used pBI-1, a derivative of pRC2312 (Stoldt et al., 1997) containing the PCK1 promoter. The entire OLE1 coding region was amplified by genomic PCR using primers OLEHindIII-BHIATG (5'-CTTAAGCTTGGATCCACAATGACTACAGTTG-3') and OLEStpBHI-HindIII (5'-CTTAAGCTTGGATCCTAAGATTGCTTTTCC-3') (italics: HindIII, BamHI sites) and cloned into pUC18 to generate pSKM19. The 1·072 kb BamHI fragment of pSKM19 was inserted downstream of PCK1p into the BglII site of pBI-1, thereby generating OLE1-overexpression plasmid pSKM24. Similarly, the entire OLE2 ORF was amplified by genomic PCR using primers 707 and 708 (5'-TATGGATCCAGGCAAATAATATATCC-3'/5'-TATGGATCCATTAATCTGTTAAAGTAG-3'; italics, BamHI) and inserted, as a 1·58 kb fragment, into the BglII site of pBI-1 to generate the OLE2-overexpression vector pAP5.

For C-terminal tagging of OLE1 with green fluorescent protein (GFP), we generated a PCR fragment containing PCK1p-OLE1 with primers PCKp-HIII (5'-AGAAGCTTGGCTGCAGGTCGAC-3') and Ole1-GFP (5'-AATAAGCTTCCAGATTGCTTTTCCTTCTCC-3') (bold, regions of homology; underlined, HindIII), using pSKM24 as a template DNA and the resulting PCR fragment was cloned into pUC18 (pSKM58). The HindIII fragment of pSKM58 containing PCK1p-OLE1 without the stop codon was cloned into the single HindIII site in-frame with GFP into vector pRCGFP3 to generate pSKM62. To C-terminally tag OLE1 with a myc epitope, we used primers PCKp-HIII-BHI-SalI (5'-GTCGACGGATCCAAGCTTGGCTGCAGGTCGAC-3'; bold, regions of homology; underlined, SalI, BamHI, HindIII) and Ole1-Myc (5'-AATGGATCCTCACAAGTCTTCCTCGGAGATTAGCTTTTGTTCACCAGATTGCTTTTCCTTCTCC-3'; bold, regions of homology; underlined, BamHI; italics, myc epitope) to generate a PCR fragment, which was cloned into pUC18. The BamHI fragment of the resulting plasmid pSKM59 was cloned into the BglII site of pBI-1 to generate plasmid pSKM63.

Blotting procedures.
Total RNA was isolated from liquid cultures as described (Stoldt et al., 1997). The conditional strain {Delta}O7.2/25-2 was grown in SD medium without or with 0·25 mM methionine/cysteine. Following denaturing gel electrophoresis, RNA blots were probed with the 966 bp HindIII–EcoRI fragment of pSKM19, which corresponds with the 5' region of the OLE1 ORF, or with the 938 bp EcoRI fragment derived from the OLE2 ORF.

Immunoblottings for detection of Ole1p-myc fusions in crude extracts of transformants carrying pSKM63 were carried out as described (Weber et al., 2001).

Fatty acid analyses.
Lipids were extracted from cells disrupted by glass beads, as described (Daum et al., 1999), and subjected to methanolysis using BF3/methanol. Fatty acyl methyl esters were separated by GC on a Shimadzu GC-17A (version 3) gas chromatograph using a FS-CW-20M-0·25 polarity capillary column (0·25 mmx25 m; film thickness 0·25 mm) with a temperature gradient (160–200 °C at 1 °C min–1; hold at 200 °C for 20 min). Fatty acids were identified by comparisons to a commercial standard (RM-6; Supelco).

Determination of membrane fluidity.
Cells grown to an OD600 value of 1 (or OD600 0·7 for the conditional strain) were washed and converted to spheroplasts by zymolase 20T treatment. Spheroplasts were washed thrice in 20 mM Tris/HCl pH 7·5/10 mM MgSO4/0·6 M sorbitol and resuspended in this buffer. An aqueous solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) was prepared by diluting a 2 mM solution in tetrahydrofuran into 50 ml of 20 mM Tris/HCl pH 7·5 and removing traces of tetrahydrofuran by flushing with nitrogen. DPH (2 µM) was added to spheroplasts (4x108 cells ml–1) and incubated at 30 °C in a water bath shaker for 1 h. Fluorescence polarization was determined by excitation with vertically polarized monochromatic light (360 nm) and measurements of emission intensities at 426 nm using an analyser oriented parallel or perpendicular to the excitation light (Smriti et al., 1999; Kaur & Bachhawat, 1999).

The degree of fluorescence polarization (P) was calculated according to the following formula

where IV is the corrected fluorescence intensity and subscripts V and H indicate the values obtained with vertical or horizontal orientation, respectively, of the polarizer and analyser (in that order). The corrected fluorescence was determined by subtracting the intensity of light measured with unlabelled control spheroplasts from the intensity observed with labelled cells. The optical components used in the instruments have particular polarizing properties causing interferences which are corrected by calculating factor G (called grating factor). G is calculated as IHV/IHH.

GFP fluorescence microscopy.
Cells were used for fluorescence microscopy directly without fixation. Nuclei were stained by the addition of 10 µg 4',6-diamidino-2-phenylindole (DAPI) ml–1 to the cell suspension. All cells were viewed using a Zeiss Axioplan 2 fluorescence microscope. Images were taken with a Quantix Digital CCD camera using METAMORPH software and processed in corel PHOTOPAINT 11.0.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the C. albicans OLE1 and OLE2 genes
Inspection of C. albicans genomic sequences (http://www-sequence.stanford.edu/group/candida) revealed two genes with homology to the S. cerevisiae OLE1 gene encoding {Delta}9 stearoyl-CoA desaturase, which were designated OLE1 (orf6.6333; CA3921) and OLE2 (orf6.5882; CA3576). The respective gene products share 33 % identity among themselves and have 57 and 32 % identity, respectively, to Ole1p of S. cerevisiae (Stukey et al., 1989). The conceptual C. albicans Ole1 protein contains 486 residues, with a predicted molecular mass of 55·3 kDa, while Ole2p contains 526 residues (61 kDa). The molecular mass of Ole1p was confirmed by a myc-tagged derivative of Ole1p, which in immunoblottings of extracts of pSKM63 transformants revealed a single protein of about 55 kDa using an anti-myc antibody (data not shown). Calculation of a phylogenetic tree of fatty acid desaturases revealed close homologies among most fungal Ole1 proteins, while the C. albicans Ole2 protein, as well as the Aspergillus fumigatus Ole1 protein, were more distantly related, being situated on a common branch with mammalian desaturases (Fig. 1A). C. albicans Ole1p contains blocks of homology compared to desaturase family 1 members, such as signature I sequences between residues 93–110 and 139–159, as well as signature V sequences between residues 309 and 323 (Fig. 1B). As with other fungal, but not mammalian, desaturases, Ole1p and Ole2p contain integral cytochrome b5 domains between residues 407–440 and residues 438–467, respectively. Computer analysis using the TMPRED program (http://www.ch.embnet.org/software/TMPRED_form.html) predicts at least four transmembrane regions able to form two pairs of transmembrane regions, consistent with the current model for the topology of ScOle1p (Stukey et al., 1990).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1. Comparisons of {Delta}9 fatty acid desaturases. (A) A phylogenetic tree was calculated from a CLUSTAL_X alignment using the TREEVIEW software (version 1.6.5). Bar, 0·1 amino acid substitutions per site. Ole1-type proteins of Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Histoplasma capsulatum (Hc), Aspergillus fumigatus (Af), Homo sapiens (Hs) and Rattus norvegicus (Rn) were compared. (B) Homology among fatty acid desaturase signature regions. The fatty acid desaturase signature V sequence is underlined. Identical residues are shaded grey and highly conserved residues are marked with an asterisk.

 
Disruption of OLE1 and OLE2 alleles
Derivatives of the wild-type strain CAI4 were constructed which contained different disrupted OLE1 alleles (Fig. 2A). Heterozygous OLE1/ole1 strains such as strain {Delta}O8 were generated without difficulty, while the construction of a homozygous ole1/ole1 strain failed repeatedly. This result suggested that OLE1 in C. albicans is essential, as is ScOLE1 in S. cerevisiae (Stukey et al., 1989). To confirm this hypothesis we modified the remaining intact copy of OLE1 in the heterozygous strain {Delta}O8-2 by placing its ORF under transcriptional control of the MET3 promoter, which is repressed by methionine and/or cysteine (resulting strains {Delta}O7.2/25-2 and {Delta}O8.2/25-2). The genomic configuration of two independently constructed lineages of homozygous, heterozygous and conditional strains was verified by Southern blottings as exemplified in Fig. 2(A). Pairs of isogenic mutant strains were identical in all phenotypes, as described below.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. OLE1 alleles and transcripts. (A) Top, genomic configuration of the C. albicans OLE1 wild-type locus and its deleted derivatives. H, HindIII; E, EcoRV; asterisks indicate the probe used in the corresponding Southern blot. Genomic DNA of strains was cut by HindIII and analysed by Southern blotting (bottom). Strains tested were: lane 1, CAI4 (OLE1/OLE1); lane 2, {Delta}O8 (OLE1/ole1{Delta} : : hisG-URA3-hisG); lane 3, {Delta}O8-2 (OLE1/ole1{Delta} : : hisG); and lanes 4 and 5, {Delta}O8.2/25-2 (MET3p : : OLE1/ole1{Delta} : : hisG). (B) OLE1 transcripts. Total RNA of strain CAF2-1 (lanes 1 and 4) and of the conditional strain {Delta}O7.2/25-2 (lanes 2, 3 and 5, 6) was analysed by Northern blotting, using a probe homologous to the OLE1 ORF. Strains were grown in the absence (lanes 1–3) and in the presence (lanes 4–6) of 0·25 mM methionine/cysteine. The positions of the MET3p-OLE1 and OLE1 transcripts, as well as of the truncated {Delta}ole1 transcript, are indicated. rRNA stained by ethidium bromide was used as loading control.

 
To confirm that OLE1 in strain {Delta}O7.2/25-2 was under transcriptional control of the MET3 promoter, we performed a Northern analysis of total RNA of this strain, grown without or with limiting amounts (0·25 mM) of methionine (see below). In the absence of methionine an OLE1 transcript was detected (Fig. 2B, lanes 2, 3), which was missing in the presence of methionine (lanes 5, 6). The size of this transcript of 2 kb corresponded to the OLE1 transcript in a control strain (lanes 1, 4). Growth in the presence of methionine did not influence the authentic OLE1 transcript and, in agreement, also did not regulate the expected shortened ole1{Delta} transcript of about 1·8 kb in the conditional strain, which arose by chromosomal integration of the MET3p-OLE1 plasmid (Care et al., 1999).

Using the above-mentioned procedure we also generated two independent lines of CAI4 derivatives, in which one or both OLE2 alleles were disrupted (data not shown). At the OLE2 locus strains cII and CIV are heterozygous (ole2/OLE2), while strains JA1 and JA2 are homozygous mutants (ole2/ole2). URA3-minus derivatives of strain JA1 were isolated by FOA selection (resulting strains JA1x and JA2x), which subsequently were transformed by the expression vector pAP5 to reconstitute OLE2. In Northern blottings using an OLE2 probe on total RNA of strain CAF2-1, we observed that OLE2 was expressed, generating a transcript of about 2 kb, but signal intensity was much lower compared to the OLE1 transcript (data not shown). Thus, OLE2 appears to be expressed at low levels, at least in the conditions used here.

Growth depends on OLE1 expression levels
The conditional strain {Delta}O8.2/25-2 (MET3p-OLE1/ole1{Delta} : : hisG) was streaked on SD medium containing cysteine and/or methionine to repress OLE1 (Fig. 3A). Growth was blocked completely at 2·5 mM cysteine, 0·25 mM methionine and 0·05 mM of a cysteine/methionine mixture, while the heterozygous strains {Delta}O7 and {Delta}O8 were not affected. Likewise, in liquid SD medium containing 2·5 mM methionine/cysteine, growth of the conditional strains was blocked completely, with a terminal phenotype of mostly unbudded cells that tended to aggregate (data not shown), suggesting a block in the G1 or G0 phase of the cell cycle. The threshold level, at which methionine/cysteine blocked growth, strongly depended on the type of media and growth conditions used: whereas cysteine/methionine at a concentration of 0·05 mM prevented growth on solid SD medium, it did not block growth in liquid SD medium up to 0·25 mM cysteine/methionine. Also, on Lee's medium, growth of the conditional strain was inhibited significantly only at methionine concentrations above 5 mM. This medium dependence may be due to different efficiencies of MET3p repression in different conditions. The addition of 0·5 mM oleic acid in 1 % Igepal CA-630 as solubilizer was able to restore growth of the conditional strain {Delta}O8.2/25-2 in OLE1-repressing conditions (Fig. 3B). In contrast, the conditional control strain CA2d1m, which contains the essential SEC20 gene under control of MET3p (Weber et al., 2001), could not be rescued by oleic acid. Thus, these results indicate that OLE1 is essential in C. albicans and suggest that it is involved in oleic acid biosynthesis.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3. Growth of the C. albicans ole1 conditional mutants. (A) Six independent isolates of conditional ole1 mutants (MET3p : : OLE1/ole1{Delta} : : hisG) were tested for growth at 30 °C on SD medium containing the indicated concentrations of cysteine and methionine (sectors 1–6); the heterozygous strains {Delta}O7 and {Delta}O8 (OLE1/ole1{Delta} : : hisG-URA3-hisG) were used as controls. (B) Oleic acid complementation of the conditional ole1 mutant. Isolates of mutant {Delta}O8.2/25-2 (sectors 1–3) were grown on SD medium containing 1 % Igepal, 2·5 mM methionine/cysteine (Met/Cys) and 0·5 mM oleic acid as indicated. As controls the wild-type CAF2-1 (OLE1/OLE1), the heterozygous strain {Delta}O8 (OLE1/ole1{Delta} : : hisG-URA3-hisG) and the conditional mutant CA2d1m (MET3p-SEC20/ole1{Delta} : : hisG) were tested. (C) Complementation of the ole1 mutation of S. cerevisiae. Transformants of the S. cerevisiae strain Y779 (ole1) carrying an empty vector (pBI-1), a vector expressing PCK1p-OLE1 (pSKM24) or PCK1p-OLE1-GFP (pSKM62) were streaked out on SD medium lacking uracil with or without 0·2 % oleic acid (OA) and incubated for 3 days at 30 °C.

 
To further confirm the role of Ole1p as a fatty acid desaturase, we attempted to complement the S. cerevisiae ole1 mutant Y0779, which lacks this activity (Braun et al., 2002), by the C. albicans OLE1 gene. Expression vector pSKM24 contains CaOLE1 under transcriptional control of the C. albicans PCK1 promoter, the CARS1 replicator and the CaURA3 gene, which we found to be functional in the heterologous host S. cerevisiae. Transformants of strain Y0779 containing pSKM24 were able to grow on SD medium in the absence of added oleic acid, while transformants with the control vector pBI-1 did not grow (Fig. 3C). A similar result was obtained using medium containing 2 % galactose as the carbon source, indicating that the heterologous C. albicans PCK1 promoter is active in S. cerevisiae in the presence of glucose. Taken together, these results strongly suggest that the C. albicans OLE1 gene encodes {Delta}9 stearoyl desaturase activity.

In contrast to the results obtained for OLE1, both alleles of OLE2 could be deleted without any difficulty and the resulting mutants (e.g. strain JA2) grew as well as the wild-type in all media, in the absence of oleic acid. Furthermore, the CaOLE2 expression vector pAP5 was unable to reconstitute the S. cerevisiae ole1 mutant Y0779. Thus, these experiments provided no evidence for a function of the OLE2 gene product as a {Delta}9 stearoyl desaturase.

Hyphal morphogenesis depends on OLE1 expression levels
We examined, next, if partial repression of OLE1 expression in the conditional strain would still allow growth, but prevent hyphal morphogenesis. Common media used for induction of hypha formation already contain methionine and/or cysteine: Lee's medium contains 0·075 mM methionine (Lee et al., 1975), which in SD medium blocks growth of the conditional strain {Delta}O8.2/25-2 completely (Fig. 3A); Spider and serum media contain complex sources of nitrogen likely to include methionine or cysteine. As stated above, growth of the conditional strain was not affected on Spider medium or on Lee's medium compared to the wild-type strain. In contrast, hypha formation on both media was completely blocked (Fig. 4) and microscopy revealed that colonies consisted entirely of yeast-form cells. The importance of a sufficient dosage of wild-type OLE1 expression was confirmed by the OLE1/ole1 heterozygous strain, which had a reduced ability to form hyphae. A more complex phenotype was observed on serum medium, on which the conditional and the heterozygous strains produced hyphae, although their growth was retarded (presumably because of methionine/cysteine as well as traces of oleic acid in serum). The effect of lowered OLE1 expression on hyphal morphogenesis was confirmed in liquid SD medium containing 5 % serum and methionine/cysteine, although these experiments were hampered by the fact that both amino acids impaired hypha formation partially even in the control strain CAF2-1 (data not shown).



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 4. Filamentation defect of ole1 conditional mutants. Strains were grown on Spider or Lee's medium or on agar containing 5 % horse serum. Strains CAF2-1 (OLE1/OLE1), the heterozygous strain {Delta}O8 (OLE1/ole1{Delta} : : hisG-URA3-hisG) and the conditional mutant {Delta}O8.2/25-2 (MET3p : : OLE1/ole1{Delta} : : hisG) were tested. Following 5 days growth at 37 °C, colony phenotypes were recorded microscopically (magnification 2·5-fold). Restoration of the filamentation defect was obtained on Lee's medium in the presence of 0·5 mM oleic acid (OA) or in hypoxic conditions generated in an anaerobic jar.

 
The addition of oleic acid to Spider medium restored hyphal morphogenesis of the conditional strain (Fig. 4), suggesting that the lack of oleic acid was the reason for the defective morphogenetic phenotype. Surprisingly, hyphal morphogenesis was also partially restored in microaerophilic (hypoxic) conditions, in which an alternative signalling pathway leading to hyphal morphogenesis is activated (Brown et al., 1999; Sonneborn et al., 1999). This result suggested that lowered oleic acid biosynthesis does not change the ability to form hypha per se, but rather affects the induction of hypha formation.

Since these experiments had shown that low oleic acid levels could prevent morphogenesis, we also asked if increased levels would enhance hypha formation. Therefore, we pre-grew strain CAI4(pSKM24) in inducing SCAA medium to overexpress OLE1 by the PCK1 promoter. Although in this medium a small fraction of the transformant cells (about 5 %) formed elongated abnormal filaments resembling pseudohyphae, no typical true hyphae were detected. When 10 % horse serum was added and cells were incubated at 37 °C, they began to form true hyphae with similar kinetics as transformant cells which had been grown in PCK1p-repressing S4D medium and as control cells carrying empty vector pBI-1 (data not shown). Germ tubes were of identical lengths in all strains, suggesting that different Ole1p enzyme levels were not correlated with induction and elongation of hyphae. To confirm that the growth conditions had indeed led to an overproduction of Ole1p, we grew transformant CAI4(pSKM63), producing a myc-tagged version of Ole1p, in identical conditions, and by immunoblottings could verify that Ole1p production was increased in SCAA-grown cells five- to tenfold, as compared to S4D-grown cells (data not shown).

Thus, these results indicated that a minimal dosage of Ole1p and of oleic acid is crucial to allow efficient hypha formation in several but not all conditions known to induce hyphae in C. albicans. In contrast to these results, an ole2 deletion strain (JA2) was able to form true hyphae as was the wild-type in all conditions tested, suggesting that Ole2p does not have an essential role in hyphal morphogenesis.

Chlamydospore formation requires wild-type levels of OLE1 expression
Because of the requirement for OLE1 in hypha formation, we also considered the possibility that another morphogenetic event in C. albicans, chlamydospore formation, would be affected by OLE1 expression. Therefore, we streaked the conditional MET3p-OLE1 strain onto CMA containing a low level of methionine/cysteine and covered cells by a coverslip to generate microaerophilic conditions (Joshi et al., 1993; Sonneborn et al., 1999). Following 5 days incubation at 25 °C, chlamydospores of all strains were visible in medium lacking methionine/cysteine, while only the wild-type and the heterozygous strain produced chlamydospores in the presence of methionine/cysteine (Fig. 5). The conditional strain grew equally as well in this condition as the wild-type and heterozygous strains, but failed to form chlamydospores and this defect remained even after prolonged incubations. In addition to this defect, the conditional strain formed pseudohyphae that aggregated strongly, which was a phenotype not seen in the heterozygous and wild-type strains. Thus, wild-type expression levels of OLE1 and corresponding levels of oleic acid are necessary to allow chlamydospore formation in C. albicans. In contrast to these results, strains JA1 and JA2 were able to form chlamydospores at normal levels, indicating that Ole2p is not involved in chlamydospore formation.



View larger version (87K):
[in this window]
[in a new window]
 
Fig. 5. Defective chlamydospore formation of the conditional ole1 mutant. Strains were streaked out lightly on chlamydospore induction medium (CMA) without or with 0·25 mM methionine/cysteine, covered by a coverslip and incubated for 4–5 days at 25 °C. Strains used were CAF2-1 (OLE1/OLE1), {Delta}O8 (OLE1/ole1{Delta} : : hisG-URA3-hisG) and {Delta}O8.2/25-2 (MET3p : : OLE1/ole1{Delta} : : hisG). Photographs of chlamydospores and filaments were magnified 100-fold (across coverslips on plates).

 
Intracellular localization of Ole1p
To examine the intracellular location of Ole1p, we examined a transformant carrying pSKM62, which expresses a fusion of OLE1 to the GFP gene under transcriptional control of the PCK1 promoter. Complementation of a S. cerevisiae ole1 mutant by pSKM62-encoded Ole1-GFP demonstrated that this fusion is a functional stearoyl CoA desaturase (Fig. 3C). In transformants grown in PCK1p-inducing medium, green fluorescence was observed throughout the cell, but it was especially seen at a location surrounding the nucleus marked by DAPI staining (Fig. 6). This staining pattern is consistent with the localization of Ole1p in the ER membrane and agrees with the localization of the homologous protein in S. cerevisiae (Stukey et al., 1990). However, because at low expression levels (i.e. in S4D medium) the transformants did not show any fluorescence, we cannot exclude the possibility that high levels of Ole1-GFP biosynthesis had an influence on its intracellular distribution.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6. Intracellular localization of an Ole1p-GFP fusion. A transformant of strain CAI4 carrying pSKM62 (PCK1p-OLE1-GFP) was grown in PCK1p-inducing SCAA medium. Cells were stained with DAPI and analysed by fluorescence microscopy in a Zeiss Axioplan 2 microscope. The differential interference contrast (DIC) image is shown along with the DAPI and GFP labellings.

 
Fatty acid analyses
To prove the effects of altered OLE1 or OLE2 expression, we determined fatty acid compositions of the conditional MET3p-OLE2 strain, the ole2 mutant and of transformants carrying overexpression vectors. In pre-tests we observed that fatty acid compositions were strongly dependent on growth media used, in agreement with a previous study on S. cerevisiae (Chatterjee et al., 2001). For example, growth in SD medium led to a strong increase in C16 : 0 and a strong decrease in C18 : 2 and C18 : 3 fatty acids compared to growth in SCAA or SD/methionine/cysteine medium. Therefore, to exclude medium effects, we compared mutant or overexpression strains following growth in the same medium.

The conditional mutant {Delta}O7.2/25-2 and the control strain CAF2-1 were pre-grown in SD medium and then inoculated into SD medium containing low amounts of methionine and cysteine (0·25 mM). At these levels, the conditional strain downregulated OLE1 to an extent to prevent hypha formation, but to still allow growth. Following about three cell doublings, we isolated lipids and generated fatty acid methyl esters, which were analysed by GC. Representative results indicate that levels of C18 : 1 (oleic) acids were reduced in the conditional strain, while the C18 : 0 precursor was strongly increased relative to amounts in the control strain (Table 2). On the other hand, levels of C18 : 2 and C18 : 3 acids were identical in the control and conditional strains, suggesting that lowering of OLE1 expression primarily affects the C18 : 0 to C18 : 1 conversion. We also note that the levels of C16 : 0 and C16 : 1 acids were diminished in the conditional strain. In contrast, fatty acid composition in the ole2 deletion strain JA2 appeared similar to the control strain CAF2-1 (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Fatty acid composition and membrane fluidity

 
We also checked if strains carrying overexpression vectors for OLE1 or OLE2 would show altered fatty acid compositions compared to a control strain transformed with an empty vector. A transformant with the OLE1-overexpression vector pSKM24 showed only a slight increase in C18 : 1 levels compared to the control, while contents of C18 : 2 and C18 : 3 fatty acids were not altered (Table 2). A similar pattern including an increase in C18 : 1 was obtained in a strain carrying the OLE2-overexpression vector pAP5. The latter result is the only evidence that OLE2, at least at elevated expression levels, may function as a {Delta}9 stearoyl desaturase.

Membrane fluidity measurements
It appeared possible that lowering of membrane oleic acid would lead to decreased membrane fluidity. Alternatively, we speculated that cells would cope with alterations in oleic acid levels by compensatory alterations in membrane lipids and/or proteins, which would maintain membrane fluidity at a relatively constant level. To decide between these alternatives, we measured membrane fluidity in strains with altered OLE1 expression levels, by detection of the mobility of the fluorochrome 1,6-diphenyl-1,3,5-hexatriene, using fluorescence polarization measurements. Results were expressed as P-values, which at low values indicate high fluorochrome mobility, i.e. high fluidity, whereas elevated values indicate decreased membrane fluidity.

Partial repression of MET3p-OLE1 in the conditional strain {Delta}O7.2/25-2 led to increased P-values, indicating a decrease in fluidity, as expected for a decrease in oleic acid (Table 2). On the other hand, deletion of OLE2 also decreased fluidity, although levels of unsaturated fatty acids were very similar in the ole2 mutant and the control strain. Furthermore, membrane fluidity was unaltered in a strain with an OLE2-overexpression vector and fluidity was lowered rather than increased in a transformant carrying an OLE1-overexpression plasmid relative to the control. Thus, we did not detect any correlation between fatty acid composition and membrane fluidity. A complicating factor in these studies was the fact that membrane fluidity depended on the type of growth medium, because CAF2-1 control cells grown in SCAA and SD medium had different fluidities (P-values of 0·145 and 0·131, respectively); furthermore, membrane fluidity in the control CAI4-transformant was unusually low (P=0·184).

Since strains with different levels of overall membrane fluidity were obtained, we tested a possible correlation between membrane fluidity and the ability to form hyphae. In spite of the significantly enhanced membrane rigidity of the OLE1-overexpression strain, it was able, upon addition of 10 % serum, to form hyphae with about equal kinetics compared to control cells. The different rigidities of the control strains grown in SCAA medium or SD medium also did not alter the ability of cells to form hyphae. Furthermore, the addition of the fluidizer benzyl alcohol (Horvath et al., 1998) to wild-type cells did not change the response to hypha-inducing agents (data not shown). Thus, overall membrane fluidity per se does not appear to be a crucial factor to establish morphogenetic competence, whereas levels of individual membrane components such as oleic acid, which may occur in membrane subdomains or ‘rafts’, may be relevant.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we characterized a first gene determining fatty acid metabolism in the human fungal pathogen C. albicans. The conclusion that OLE1 encodes a stearoyl desaturase required for the biosynthesis of oleic acid (C18 : 1) is based on (a) its high homology to such desaturases in other organisms and especially in S. cerevisiae, (b) its ability to complement an ole1 mutation in S. cerevisiae, (c) rescue of growth and morphogenetic phenotypes occurring during low OLE1 expression by external oleic acid, and (d) higher levels of oleic acid in an OLE1 overexpression strain and lowered levels in a conditional strain, in which OLE1 was downregulated. In the latter experiment, levels of C18 : 2 and C18 : 3 fatty acids, which do not occur in S. cerevisiae, were not downregulated, suggesting that C18 : 1 produced by OLE1 is not the direct precursor of C18 : 2 or C18 : 3 acids, but that a separate desaturase is involved. We suspected that the OLE2 gene described here would fulfil this function. The OLE2 gene product shares relatively low homology with OLE1 of yeast-like fungi, but has greater homology to genes encoding desaturases of filamentous fungi and mammals. However, in an ole2 deletion strain the pattern of fatty acids was similar to a wild-type strain, ruling out a function of Ole2p in the generation of C18 : 1, C18 : 2 and C18 : 3 fatty acids. Interestingly, a strain overexpressing OLE2 showed elevated oleic acid levels, which we take as a hint of a possible desaturase function of Ole2p, which is detectable at high production levels. It is possible that OLE2 is involved in the synthesis of other fatty acid derivatives, such as leukotrienes and prostaglandins, in C. albicans (Noverr et al., 2002). The task of generation of C18 : 2 and C18 : 3 fatty acids may be assumed by the gene products of two ORFs (orf6.1443; orf6.5913) in the C. albicans genome, which encode proteins that are highly homologous to {Delta}12 fatty acid desaturases of Aspergillus nidulans (Calvo et al., 2001).

Complete repression of OLE1 prevented growth, indicating that Ole1p and its product oleic acid provide essential functions. We presume that oleic acid is also needed for specific functions, such as for oleic acid-dependent membrane sensors, and that it has a role in regulating overall and localized membrane fluidity. Low levels of OLE1 expression did not impair growth of the yeast form, but significantly blocked hyphal morphogenesis on solid and in liquid induction media. A specific set of signalling pathways leading to hyphal morphogenesis is known to be required in most induction conditions in the presence of oxygen (Ernst, 2000), while in embedded hypoxic conditions an alternative signalling pathway, which is downregulated in wild-type cells, is operative (Brown et al., 1999; Sonneborn et al., 1999). Because low OLE1 expression permitted hypha formation in embedded/hypoxic conditions but prevented filamentation in aerobic conditions, it appears likely that threshold levels of oleic acid are required specifically for the function of the aerobic pathways. Thus, oleic acid does not appear to have a general role in filament formation, for example for late events in hyphal development, but it is implicated in early events of activation of specific signalling pathways. Conceivably, because the biosynthesis of oleic acid requires oxygen, it could be a signalling molecule activating (yet unknown) membrane sensors transmitting external cues to internal pathways operative in aerobic conditions.

It is known that elevated temperatures increase membrane fluidity, while lowered temperatures decrease fluidity. Because elevated temperatures are a decisive environmental cue to trigger hyphal morphogenesis in C. albicans, we speculated that the state of its membrane fluidity could act as a cellular ‘thermometer’ signalling directly to morphogenetic pathways. It has indeed been reported that stress responses and in particular the heat-shock responses in yeasts are activated by membrane perturbations (Carratu et al., 1996; Moskvina et al., 1999). Lowering OLE1 expression in C. albicans indeed led to decreased membrane fluidity, as expected for a direct role of membrane fluidity in adjusting the set point of temperature induction of hyphal growth. However, further findings provide arguments against a direct correlation between membrane fluidity and morphogenesis: (1) elevated temperatures did not restore hypha formation in the MET3p-OLE1 conditional strain, (2) no correlation between membrane fluidity and hyphal induction was detected, (3) benzyl alcohol, a membrane fluidizer, did not increase hypha formation, and (4) overexpression of OLE1 led to a slightly increased level of oleic acid, but strongly increased membrane rigidity, which nevertheless did not interfere with hypha formation. Increased rigidity in the latter experiments may be due to compensatory increases in other membrane components, for example an increase in ergosterol levels, which may maintain the physical state of membranes constant. According to a similar principle, it has been described that the composition of the C. albicans cell wall is subject to compensatory alterations (Kapteyn et al., 2000). Furthermore, a genome-wide transcriptional profiling of genes induced during hyphae induction recently revealed that a heat shock alone is not able to induce hyphae-specific genes and hyphal morphogenesis (Nantel et al., 2002). Thus, we favour a model in which stress responses and induction of morphogenesis do not share the same dependence on membrane fluidity. We rather postulate that levels of oleic acid have a direct effect on specific components of the hyphal induction machinery.

In addition to the defect in hyphal morphogenesis, the development of chlamydospores was blocked at low OLE1 expression levels. Although the functions of the thick-walled chlamydospores in the biology and virulence of C. albicans are currently unclear, it is evident that their development requires a specific morphogenetic pathway, which depends on oleic acid. The finding of a threshold level of oleic acid for morphogenetic events in C. albicans suggests that the reason for defective phenotypes in several morphogenetic mutants of C. albicans may be an impaired lipid and/or fatty acid and/or oleic acid metabolism. In agreement with this notion, in S. cerevisiae the Tup1 regulator represses transcription of OLE1 (Fujimori et al., 1997) and C. albicans tup1 mutants grow in a pseudohyphal form (Braun & Johnson, 1997), while we observed that OLE1 overexpression favours an abnormal pseudohyphal growth form, although only in a small fraction of cells. Our results further suggest that Ole1p may be a suitable target for future antifungal agents. Because OLE1 is essential, it is likely that potential Ole1 inhibitors will prevent cell growth and lead to a rapid loss of viability. This would be an advantage compared to azole inhibitors of ergosterol biosynthesis, which do not kill fungal pathogens. Even at low doses of inhibitors, which reduce but do not eliminate Ole1 function, hyphal morphogenesis and consequently virulence of C. albicans would be blocked. A major structural difference between mammalian and fungal Ole1 proteins is the presence of an integral cytochrome b5 domain in fungal desaturases, which may allow the development of selective inhibitors.


   ACKNOWLEDGEMENTS
 
We are very grateful to G. Daum for helpful advice on lipid analyses. We thank S. Jentsch for contributing strains. Nucleotide sequence data for C. albicans were obtained from the Stanford Genome Technology Center website (http://www-sequence.stanford.edu/group/candida). Sequencing of C. albicans was accomplished with the support of the NIDCR and the Burroughs Wellcome Fund. We thank the Alexander von Humbodt Foundation for the support of S. K. and gratefully acknowledge the support by a grant of the Volkswagen-Stiftung to R. P. and J. F. E.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ananthan, J., Goldberg, A. L. & Voellmy, R. (1986). Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232, 522–524.[Medline]

Bossie, M. A. & Martin, C. E. (1989). Nutritional regulation of yeast delta-9 fatty acid desaturase activity. J Bacteriol 171, 6409–6413.[Medline]

Braun, B. R. & Johnson, A. D. (1997). Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science 277, 105–109.[Abstract/Free Full Text]

Braun, S., Matuschewski, K., Rape, M., Thoms, S. & Jentsch, S. (2002). Role of the ubiquitin-selective CDC48(UFD1/NLP4) chaperone (segregase) in ERAD of OLE1 and other substrates. EMBO J 21, 615–621.[Abstract/Free Full Text]

Brown, D. H., Jr, Giusani, A. D., Chen, X. & Kumamoto, C. A. (1999). Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene. Mol Microbiol 34, 651–662.[CrossRef][Medline]

Calvo, A. M., Gardner, H. W. & Keller, N. P. (2001). Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans. J Biol Chem 276, 25766–25774.[Abstract/Free Full Text]

Care, R. S., Trevethick, J., Binley, K. M. & Sudbery, P. E. (1999). The MET3 promoter: a new tool for Candida albicans molecular genetics. Mol Microbiol 34, 792–798.[CrossRef][Medline]

Carratu, L., Franceschelli, S., Pardini, C. L., Kobayashi, G. S., Horvath, I., Vigh, L. & Maresca, B. (1996). Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast. Proc Natl Acad Sci U S A 93, 3870–3875.[Abstract/Free Full Text]

Chatterjee, M. T., Khalawan, S. A. & Curran, B. P. (1997). Alterations in cellular lipids may be responsible for the transient nature of the yeast heat shock response. Microbiology 143, 3063–3068.[Abstract]

Chatterjee, M. T., Khalawan, S. A. & Curran, B. P. G. (2001). Subtle alterations in growth medium composition can dramatically alter the percentage of unsaturated fatty acids in the yeast Saccharomyces cerevisiae. Yeast 18, 81–88.[CrossRef][Medline]

Choi, J. Y., Stukey, J., Hwang, S. Y. & Martin, C. E. (1996). Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 271, 3581–3589.[Abstract/Free Full Text]

Cossins, A. R. (1994). Temperature Adaptation of Biological Membranes. London: Portland Press.

Daum, G., Tuller, G., Nemec, T. & 14 other authors (1999). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 15, 601–604.[CrossRef][Medline]

Ernst, J. F. (2000). Transcription factors in Candida albicans – environmental control of morphogenesis. Microbiology 146, 1763–1774.[Free Full Text]

Faergeman, N. J., Black, P. N., Zhao, X. D., Knudsen, J. & DiRusso, C. C. (2001). The acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular utilization. J Biol Chem 276, 37051–37059.[Abstract/Free Full Text]

Fonzi, W. & Irwin, Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.[Abstract/Free Full Text]

Fujimori, K., Anamnart, S., Nakagawa, Y., Sugioka, S., Ohta, D., Oshima, Y., Yamada, Y. & Harashima, S. (1997). Isolation and characterization of mutations affecting expression of the delta9-fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae. FEBS Lett 413, 226–230.[CrossRef][Medline]

Horvath, I., Glatz, A., Varvasovszki, V. & 8 other authors (1998). Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a "fluidity gene". Proc Natl Acad Sci U S A 95, 3513–3518.[Abstract/Free Full Text]

Joshi, K. R., Solanki, A. & Prakash, P. (1993). Morphological identification of Candida species on glucose agar, rice extract agar and corn meal agar with and without Tween-80. Indian J Pathol Microbiol 36, 48–52.[Medline]

Kapteyn, J. C., Hoyer, L. L., Hecht, J. E. & 6 other authors (2000). The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 35, 601–611.[CrossRef][Medline]

Kaur, R. & Bachhawat, A. K. (1999). The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae. Microbiology 145, 809–818.[Abstract]

Kwast, K. E., Burke, P. V. & Poyton, R. O. (1998). Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast. J Exp Biol 201, 1177–1195.[Abstract/Free Full Text]

Lee, K. L., Buckley, H. R. & Campbell, C. C. (1975). An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148–153.[Medline]

Leuker, C. E., Sonneborn, A., Delbrück, S. & Ernst, J. F. (1997). Sequence and regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. Gene 192, 235–240.[CrossRef][Medline]

Liu, H., Köhler, J. & Fink, G. R. (1994). Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266, 1723–1726.[Medline]

McDonough, V. M., Stukey, J. E. & Martin, C. E. (1992). Specificity of unsaturated fatty acid-regulated expression for the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 267, 5931–5936.[Abstract/Free Full Text]

Mishra, P., Bolard, J. & Prasad, R. (1992). Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast. Biochim Biophys Acta 1127, 1–14.[Medline]

Mitchell, A. G. & Martin, C. E. (1995). A novel cytochrome b5-like domain is linked to the carboxyl terminus of the Saccharomyces cerevisiae delta-9 fatty acid desaturase. J Biol Chem 270, 29766–29772.[Abstract/Free Full Text]

Moskvina, E., Imre, E.-M. & Ruis, H. (1999). Stress factors acting at the level of the plasma membrane induce transcription via the stress response element (STRE) of the yeast Saccharomyces cerevisiae. Mol Microbiol 32, 1263–1272.[CrossRef][Medline]

Nakagawa, Y., Sugioka, S., Kaneko, Y. & Harashima, S. (2001). O2R, a novel regulatory element mediating Rox1p-independent O2 and unsaturated fatty acid repression of OLE1 in Saccharomyces cerevisiae. J Bacteriol 183, 745–751.[Abstract/Free Full Text]

Nakagawa, Y., Skumoto, N., Kaneko, Y. & Harashima, S. (2002). Mga2p is a putative sensor for low temperature and oxygen to induce OLE1 transcription in Saccharomyces cerevisiae. Biochem Biophys Res Commun 291, 707–713.[CrossRef][Medline]

Nantel, A., Dignard, D., Bachewich, C. & 12 other authors (2002). Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13, 3452–3465.[Abstract/Free Full Text]

Noverr, M. C., Toews, G. B. & Huffnagle, G. B. (2002). Production of prostaglandins and leuktrienes by pathogenic fungi. Infect Immun 70, 400–402.[Abstract/Free Full Text]

Sherman, F., Fink, G. & Hicks, J. (1986). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Smriti, Krishnamurthy, S. & Prasad, R. (1999). Membrane fluidity affects functions of Cdr1p, a multidrug ABC transporter of Candida albicans. FEMS Microbiol Lett 173, 475–481.[CrossRef][Medline]

Sonneborn, A., Bockmühl, D. P. & Ernst, J. F. (1999). Chlamydospore formation in Candida albicans requires the Efg1p morphogenetic regulator. Infect Immun 67, 5514–5517.[Abstract/Free Full Text]

Stewart, L. C. & Yaffe, M. P. (1991). A role for unsaturated fatty acids in mitochondrial movement and inheritance. J Cell Biol 115, 1249–1257.[Abstract]

Stoldt, V. R., Sonneborn, A., Leuker, C. & Ernst, J. F. (1997). Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J 16, 1982–1991.[Abstract/Free Full Text]

Stukey, J. E., McDonough, V. M. & Martin, C. E. (1989). Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae. J Biol Chem 264, 16537–16544.[Abstract/Free Full Text]

Stukey, J. E., McDonough, V. M. & Martin, C. E. (1990). The OLE1 gene of Saccharomyces cerevisiae encodes the {Delta}9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J Biol Chem 265, 20144–20149.[Abstract/Free Full Text]

Torok, Z., Horvath, I., Goloubinoff, P., Kovacs, E., Glatz, A., Balogh, G. & Vigh, L. (1997). Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions. Proc Natl Acad Sci U S A 94, 2192–2197.[Abstract/Free Full Text]

Vasconcelles, M. J., Jiang, Y., McDaid, K., Gilooly, L., Wretzel, S., Porter, D. L., Martin, C. E. & Goldberg, M. A. (2001). Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. J Biol Chem 276, 14374–14384.[Abstract/Free Full Text]

Vigh, L., Los, A. D., Horváth, I. & Murata, N. (1993). The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipid stimulated the expression of the desA gene in Synechocystis PCC6803. Proc Natl Acad Sci U S A 90, 9090–9094.[Abstract]

Vigh, L., Maresca, B. & Harwood, J. L. (1998). Does the membrane's physical state control the expression of heat shock and other genes? Trends Biochem Sci 23, 369–374.[CrossRef][Medline]

Wada, H., Gombos, Z. & Murata, N. (1990). Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347, 200–203.[CrossRef][Medline]

Weber, Y., Santore, U. J., Ernst, J. F. & Swoboda, R. K. (2001). Divergence of eukaryotic secretory components: the Candida albicans homolog of the Saccharomyces cerevisiae Sec20 protein is N-terminally truncated, and its levels determine antifungal drug resistance and growth. J Bacteriol 183, 46–54.[Abstract/Free Full Text]

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, 1868–1874.[Abstract/Free Full Text]

Zhang, S., Skalsky, Y. & Garfinkel, D. J. (1999). MGA2 or SPT3 is required for transcription of the {Delta}9 desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae. Genetics 151, 473–483.[Abstract/Free Full Text]

Received 13 January 2004; revised 16 February 2004; accepted 23 February 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Krishnamurthy, S.
Articles by Ernst, J. F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Krishnamurthy, S.
Articles by Ernst, J. F.
Agricola
Articles by Krishnamurthy, S.
Articles by Ernst, J. F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.