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Corresponding author: Richard S. Zitomer, Department of Biological Sciences, University at Albany/SUNY, Albany, NY 12222., rz144{at}csc.albany.edu (E-mail)
Communicating editor: A. P. MITCHELL
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.
MANY yeast species have the capacity to grow under strongly hypoxic conditions. Cells adapt to hypoxia by inducing genes that encode functions that enable them to use limiting oxygen more efficiently. We have studied the regulation of these hypoxic genes extensively in one yeast, the model organism Saccharomyces cerevisiae (for review see 
Rox1 consists of 368 amino acids (
In our genetic analysis of the Rox1 protein, we have isolated a large number of mutations in the HMG domain and characterized their effects on DNA binding and bending in vitro and repression in vivo (
We report here our attempt to clone the ROX1 homologue from Candida albicans. We began with this yeast for several reasons. First, it can grow under hypoxic conditions and, therefore, is likely to have a hypoxic regulon. Second, this yeast is phylogenetically close enough to S. cerevisiae to expect conservation of general regulatory mechanisms. Indeed, this conservation has been extensively exploited in the study of filamentous growth in C. albicans (for reviews see
We identified a C. albicans gene with extensive similarity to the S. cerevisiae ROX1 gene. We demonstrated that it bound specifically to the hypoxic target DNA sequence and that it served as a hypoxic repressor in S. cerevisiae. However, to our surprise, we found that deletion of the gene caused constitutive hyphal growth and that it did not regulate the HEM13 hypoxic gene of C. albicans. Therefore, this gene appears to encode a repressor of hyphal growth rather than a hypoxic repressor.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Yeast strains and cell growth:
The C. albicans strain RM1000 was used in this study (
r1::gK contains a deletion of the ROX1 gene and an integrated ANB1-lacZ fusion (r1m3 is a derivative of MZ22-4r1::gK containing a deletion of the MOT3 gene (r1::gK containing the tup1::TRP1 allele.
Cells were maintained and grown without selection on rich YPD medium (
Plasmids:
All plasmids were maintained in Escherichia coli strain HB101 as described (
The C. albicans library in pEMBLy23 was provided by P. T. Magee (University of Minnesota) and a second library was provided by G. R. Fink (MIT). The C. albicans transforming plasmid pABSK2 carrying the C. albicans URA3 selectable marker and the ARS2 replication origin was provided by H. Chibana (University of Minnesota). Plasmids pGEM-URA3 and pGEM-HIS1 containing the C. albicans URA3 and HIS1 genes, respectively, were obtained from Dr. A. P. Mitchell (Columbia) (
The plasmids below were constructed for this study. The sequences are numbered with the A of the ATG translational initiation codon as +1, and sequences 3' are numbered consecutively in positive integers and sequenced 5' in negative integers.
100/245 (
Sequence analysis:
The sequence of both strands of the C. albicans ROX1 gene was determined using the Sequenase kit (United States Biochemical, Cleveland) and synthetic primers.
Construction of deletion strains:
The deletions of the ROX1 gene in C. albicans were generated by transforming cells with PCR fragments. The URA3 deletion allele fragment was generated using one synthetic primer that contained the ROX1 sequences from +90 to +141 followed by URA3 sequences from -404 to -382 and a second primer containing the ROX1 sequences from +1314 to +1366 followed by URA3 sequences from +920 to +942. These primers were used to PCR amplify the URA3 gene from pGEM-URA3, resulting in a fragment containing the URA3 gene flanked by the ROX1 sequences of the primers. C. albicans strain RM1000 was transformed with this fragment as described (
The HIS1 deletion allele was generated in a similar fashion except that the primers contained HIS1 sequences (-337 to -317 for one primer and 900 to 916 for the other) at the 3' end following the ROX1 sequences. The HIS1 sequences were amplified from pGEM-HIS1.
The correct constructs were confirmed by PCR analyses of genomic DNA. The strategy involved the use of four sets of primers. The first set hybridized to sequences internal to the deleted ROX1 sequences such that only the wild-type allele would generate a PCR product. For the second set of primers, one hybridized outside the deleted ROX1 sequences and the other hybridized to one end of the URA3 sequences such that a PCR product would be generated only from the rox1::URA3 allele. The third set was similar to the second, except a HIS1 primer was used instead of the URA3 primer; this set was specific for the rox1::HIS1 allele. Finally, a set of primers that amplified the actin gene was used as a control for the quality of the DNA preparations. The data generated with these primers have been reviewed by the Communicating Editor.
Protein purification and DNA binding analysis:
The HMG domain of the C. albicans Rox1 protein was expressed from pMAL-CaROX1(HMG) in the E. coli strain PR745 (New England Biolabs, Beverly, MA) as a fusion to the maltose binding protein (MBP). The protein purification procedure was identical to that described for the S. cerevisiae MBP-Rox1(HMG) fusion (
ß-Galactosidase assays:
The assays for ß-galactosidase expression in yeast were carried out as described (
Preparation of RNA, reverse transcription, and RT-PCR:
RNA was prepared from yeast cells as described (
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cloning and sequence of the C. albicans ROX1 gene:
The cloning of the C. albicans ROX1 gene was based upon the appearance of a 320-bp DNA sequence with similarity to the HMG-domain coding region of the S. cerevisiae ROX1 gene and annotated as a ROX1 homologue in the C. albicans database maintained at the University of Minnesota. PCR primers were generated to the ends of this sequence, and it was amplified, subcloned, and used as a probe in colony hybridization to two different libraries of C. albicans genomic DNA. Positive clones were sequenced and contained only the HMG domain and upstream sequences; the C-terminal coding sequence could not be found in either library. To obtain this region, RNA was prepared from C. albicans cells, and cDNA was generated to total poly(A) containing RNA. The ROX1 C-terminal coding region and the 3'-untranslated region was amplified from this cDNA pool using oligo(dT) and a ROX1-HMG specific primer. The PCR products were cloned and sequenced, and those containing the continuation of the ROX1 coding sequence were identified from the overlap with the previously sequenced regions. Finally, the genomic sequences from -547 through the 3'-untranslated region were cloned by PCR. For the purposes of this study, the C. albicans gene is referred to as ROX1 and the S. cerevisiae gene is referred to as ScROX1.
The complete DNA sequence from -547 to +1366, where the A of the first ATG initiation codon in the open reading frame is +1, has been deposited in the EMBL database. The protein coding sequence for the HMG domain from codon 1 to 189 is presented in Fig 1. The core amino acid sequence of the HMG domain from residues 30 to 119 is aligned with that of the ScRox1 HMG domain from residues 1 to 94. The two sequences are 50% identical and 74% conserved over this region. The most conserved region lies between the C. albicans residues 38 to 60, where 17 of the 23 residues are identical and the remainder are highly conserved substitutions. This region represents the first helix of the HMG domain that makes the majority of the specific contacts with DNA (
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The C-terminal region, which we presume contains the repression domain based upon complementation studies below, has little homology to the ScRox1 repression domain. This observation was not surprising because our previous genetic studies with the S. cerevisiae repression domain suggested that it did not require a highly conserved sequence. Rather, the region is redundant in function but not in sequence. Nonetheless, there are small pockets of sequence similarity between this region in the two Rox1 sequences, although their significance is unclear at this time.
The C. albicans ROX1 complements a rox1 deletion in S. cerevisiae:
To determine whether the cloned C. albicans gene functions as a DNA binding repressor protein, we determined its ability to complement a rox1 deletion allele in S. cerevisiae. Complementation was measured by the ability to repress the ANB1-lacZ fusion, a reporter hypoxic gene. We generated a number of constructs that tested the ability of the HMG domain, the repression domain, and the full-length protein to function in the heterologous species. As can be seen in Table 1, a construct consisting of the HMG domain from residues 38 to 199 fused to the ScRox1 repression domain and, driven by the ScROX1 promoter, was capable of repression of the reporter (line 3). However, surprisingly, a shorter region of the HMG domain, lacking residues 120 to 199 that are not present in the ScRox1 and not part of the basic HMG domain, was not capable of complementation (line 2), indicating that this region plays some important but unknown role in Rox1 DNA binding or protein stability that is not required in the ScRox1.
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Also evident from Table 1, the C-terminal sequences of the C. albicans Rox1 contain a repression domain. The fusion protein containing this region joined to the ScRox1 HMG domain was able to repress the reporter gene (line 4). Therefore, despite the extensive sequence divergence, this region is still recognized as a functional repression domain in S. cerevisiae.
Finally, the intact ROX1 gene, including the regulatory sequences, complemented the rox1 deletion, demonstrating that the intact gene encodes an authentic DNA binding repressor protein (Table 1). Furthermore, the ROX1 gene did not repress anaerobic ANB1-lacZ expression. ScROX1 expression is regulated by oxygen, and constitutive expression results in both aerobic and anaerobic repression of the hypoxic genes (
The HMG domain binds to the Rox1 consensus sequence:
The cross-species complementation experiments indicated that the Rox1 HMG domain bound to the ScRox1 target site. To confirm this conclusion directly, the ability of the Rox1 HMG domain to bind the ScRox1 consensus sequence in vitro was determined. The ROX1 HMG-domain coding sequence from codons 38 to 174 was fused to the E. coli malE gene, encoding the MBP, and the fusion protein was expressed in and purified from bacterial cells. Gel retardation studies using this fusion protein indicated that the protein bound to the ScRox1 binding site (Fig 2, lanes 2, 3, and 4). Binding was specific for sequence containing the ScRox1 binding site as determined by competition with either a sequence containing two Rox1 sites of the ANB1 OpA (lane 5 and 6), which decreased the amount of labeled complex visible, or a similar sequence with a deletion of the ScRox1 binding sites (lane 7), which did not reduce the amount of labeled complex.
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Deletion of ROX1 results in constitutive filamentous growth:
To determine whether the ROX1 gene regulates the hypoxic genes in C. albicans, it was necessary to generate a deletion strain. Since C. albicans is diploid with no known meiotic division, the deletion had to be constructed in each homologue. This was achieved by successive gene replacements using a ura3/ura3 his1/his1 strain. One copy of the gene was precisely deleted and replaced with the URA3 gene to generate the heterozygote, and then the second copy was precisely deleted and replaced with the HIS1 gene. Two independent homozygous deletion clones were obtained and, surprisingly, the colony morphology of both was quite irregular. Microscopic inspection revealed that the cells grew as hyphae in rich medium as opposed to the budding growth of the wild-type and heterozygote strains (Fig 4). Hyphal growth is normally triggered only under specific conditions, most strongly in serum (
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Given that we isolated only two homozygous deletion strains and that it had an unexpected phenotype, we attempted to reisolate the double deletion, but this time first generating the HIS1 replacement. By extraordinary luck, often better than careful experimental design, one His+ transformant had the irregular colony morphology and, when analyzed by PCR, was found to be homozygous for the HIS1 replacement. Again, these cells grew as hyphae (Fig 3). Since these cells were still ura3 auxotrophs, we transformed them with a URA3 vector carrying the wild-type ROX1 gene. These transformants reverted back to a budding growth pattern (Fig 3), although there were many cells that appeared to be initiating hyphal growth, probably due to the unstable nature of the plasmid. (Since cell growth was maintained under selective conditions, growth of those cells that lost the plasmid could not continue and give rise to hyphae.) The double deletant transformed with the vector alone remained filamentous (Fig 3). Given that independent isolates of the homozygous deletion yielded the same hyphal growth phenotype and that this phenotype could be suppressed by transforming cells with a wild-type copy of the gene, we conclude that ROX1 encodes a repressor of hyphal growth.
To determine whether the homozygous rox1 deletant formed pseudo- or true hyphae, we stained cells with calcofluor, a chitin-specific fluorescent dye that reveals the septum between true hyphal cells. As can be seen in Fig 4, the deletant culture grown on rich medium contained a mixture of cells; single budded cells, pseudohyphae (Fig 4A), and true hyphae (Fig 4A and Fig B) were all visible. The (morphological index) value for the filamentous cells shown in Fig 4B averages 3.4–3.9 with an average of 3.7, close to the value expected for true hyphae (
The hypoxic HEM13 gene is not regulated by Rox1, and the ROX1 gene is not regulated by oxygen or serum:
The repression of hyphal growth by Rox1 could be direct, through repression of specific genes controlling filamentation, or indirect through repression of one or more hypoxic genes that regulate hyphal growth. To test whether Rox1 represses hypoxic genes in C. albicans, we analyzed the regulation of the HEM13 gene by RT-PCR. HEM13 encodes coproporphyrinogen III oxidase and its S. cerevisiae homologue is strongly repressed by the ScRox1 (
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The hypoxic response in S. cerevisiae cells is regulated by the transcriptional regulation of the ScROX1 gene; the gene is transcriptionally activated in the presence of oxygen and repressed in its absence. Although the C. albicans gene appeared not to be the repressor of the hypoxic genes, we determined whether its transcription was regulated by oxygen using ROX1-specific probes for RT-PCR analyses. As seen in Fig 5B, the levels of the ROX1 product were similar in the samples prepared from aerobic and anaerobic RNA indicating that ROX1 expression is not regulated by oxygen.
If Rox1 were directly involved in repressing hyphal growth, its expression might be regulated by the signals that activate filamentation. One of the most potent activators is serum as seen in Fig 3. Therefore, we analyzed the accumulation of ROX1 mRNA in cells grown with and without fetal calf serum by RT-PCR. As is evident from the results presented in Fig 5B, there was no effect of serum on ROX1 RNA levels, suggesting that repressor function may be regulated at the level of translational or protein stability, activity, or compartmentalization.
The oxygen regulation of Rox1 hypoxic gene repression in S. cerevisiae is mediated by Mot3 and is Tup1dependent:
The results above presented the paradox that the C. albicans Rox1 expressed from its own promoter appeared to regulate the hypoxic genes in S. cerevisiae, implying that the gene was regulated by oxygen as is the ScROX1, but it was not regulated by oxygen in its native cell. It seemed unlikely that the whole heme-dependent regulatory scheme that operates in S. cerevisiae was bent to an alternative use in C. albicans, especially since the C. albicans HEM13 gene was regulated by oxygen. Therefore, we sought an alternative explanation.
Operator A, which is responsible for the bulk of the repression of the hypoxic S. cerevisiae ANB1 gene, contains two binding sites for Rox1 and a site for a second protein, Mot3 (r1m3). In this strain transformed with the S. cerevisiae ROX1 plasmid (YCp(22)ROX1H) the aerobic ANB1-lacZ expression resulted in 10.8 units of ß-galactosidase compared to 126 units in the same strain transformed with the vector (YCplac22). However, when transformed with the C. albicans gene (YCp(22)Ca-ROX1), the double deletant expressed only 103 units of ß-galactosidase. Thus, unlike the ScRox1, the C. albicans Rox1 required Mot3 for repression. Furthermore, RT-PCR analysis indicated that the C. albicans ROX1 mRNA levels were not regulated by oxygen in S. cerevisiae (results not shown). Thus results demonstrate that oxygen regulation of ANB1-lacZ by the heterologous ROX1 gene does not require oxygen regulation of the C. albicans gene in S. cerevisiae.
Finally, to determine whether repression by the C. albicans Rox1 was dependent on the general repressor Tup1 in S. cerevisiae, we measured the expression of the ANB1-lacZ fusion in the rox1 tup1 strain MZ22-4PC transformed with YCp(33)CaROX1 or the empty vector. The presence of CaROX1 resulted in only a 1.7-fold repression in this strain compared to the 5.5-fold repression in the congenic TUP1 wild-type strain (Table 1). Therefore, repression by the C. albicans Rox1 is Tup1 dependent.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We report here the identification of a gene encoding an HMG-containing DNA binding repressor of hyphal growth from C. albicans. The evidence for these conclusions is as follows. The protein sequence is very similar within its HMG domain to that of the Rox1 repressor of S. cerevisiae and, when expressed in and purified from bacterial cells, this C. albicans HMG domain was capable of specific binding to DNA. The gene complemented a S. cerevisiae ROX1 deletion to repress the expression of an hypoxic reporter gene, indicating that the protein has repressor activity. Finally, the deletion of the gene in C. albicans results in constitutive hyphal growth. Given this last finding, combined with the lack of effect of the deletion on the regulation of the C. albicans HEM13 hypoxic gene, it appears inappropriate to continue to refer to this gene as ROX1, the homologue of the S. cerevisiae hypoxic repressor gene. We propose the name RFG1, Repressor of Filamentous Growth.
The transition from budding growth to the invasive, hyphal growth in C. albicans is believed to be an important aspect of the organism's pathogenicity, and a great deal of research has focused on how this transition is regulated (reviewed in
By analogy to S. cerevisiae then, we propose that Rfg1 is the regulon-specific DNA binding protein that recruits Tup1 to the filamentation genes for the following reasons. First, deletion of RFG1 results in a similar filamentous growth phenotype as reported for the deletion of TUP1. However, it should be mentioned that the tup1 double deletant grows mostly as pseudohyphal cells on rich media (
It is not clear at this point how Rfg1 activity is regulated. We found that RFG1 mRNA accumulated during hyphal growth in serum, indicating that transcription is not regulated. Regulation might occur at any subsequent step, including translation or protein function or localization. The HMG domain of Rfg1 contains extensive sequences at both the amino and carboxyl ends that are not present in ScRox1, and either of these regions could be targets of control, as could the repression domain. Alternatively, there may be other proteins involved in the repression of the filamentation genes whose synthesis or activity is regulated. Further studies are required to resolve this question.
Finally the question remains as to how the hypoxic genes of C. albicans are regulated. We demonstrated that HEM13 is repressed by oxygen, strongly suggesting that a hypoxic regulon exists in this yeast. However, we have also clearly demonstrated that the Rfg1 protein is not the hypoxic repressor despite its similarity to the S. cerevisiae Rox1 protein, at least in the HMG domain. We cannot rule out the existence of a second gene encoding an HMG-domain repressor protein in the C. albicans genome, although it has not been revealed by the sequence data yet. Alternatively, these genes may be regulated by Mot3 or a novel DNA binding protein.
| ACKNOWLEDGMENTS |
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We thank Drs. G. R. Fink and P. T. Magee for the C. albicans libraries and Drs. H. Chibana and A. Mitchell for the plasmids and strains. Many of the findings reported here have also been discovered by Drs. D. Kadosh and A. Johnson, and their results have been prepared for publication. We thank them for sharing their results with us. They originally proposed the name RFG1, and we have adopted it to avoid needless confusion in the literature. These studies were supported by grant GM-26061 from the National Institutes of Health.
Manuscript received November 6, 2000; Accepted for publication January 8, 2001.
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M. Banerjee, D. S. Thompson, A. Lazzell, P. L. Carlisle, C. Pierce, C. Monteagudo, J. L. Lopez-Ribot, and D. Kadosh UME6, a Novel Filament-specific Regulator of Candida albicans Hyphal Extension and Virulence Mol. Biol. Cell, April 1, 2008; 19(4): 1354 - 1365. [Abstract] [Full Text] [PDF]
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Y. Li, C. Su, X. Mao, F. Cao, and J. Chen Roles of Candida albicans Sfl1 in Hyphal Development Eukaryot. Cell, November 1, 2007; 6(11): 2112 - 2121. [Abstract] [Full Text] [PDF]
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 S. Biswas, P. Van Dijck, and A. Datta Environmental Sensing and Signal Transduction Pathways Regulating Morphopathogenic Determinants of Candida albicans Microbiol. Mol. Biol. Rev., June 1, 2007; 71(2): 348 - 376. [Abstract] [Full Text] [PDF]
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S. M. Mulhern, M. E. Logue, and G. Butler Candida albicans Transcription Factor Ace2 Regulates Metabolism and Is Required for Filamentation in Hypoxic Conditions Eukaryot. Cell, December 1, 2006; 5(12): 2001 - 2013. [Abstract] [Full Text] [PDF]
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D. Kadosh and A. D. Johnson Induction of the Candida albicans Filamentous Growth Program by Relief of Transcriptional Repression: A Genome-wide Analysis Mol. Biol. Cell, June 1, 2005; 16(6): 2903 - 2912. [Abstract] [Full Text] [PDF]
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L. G. Klinkenberg, T. A. Mennella, K. Luetkenhaus, and R. S. Zitomer Combinatorial Repression of the Hypoxic Genes of Saccharomyces cerevisiae by DNA Binding Proteins Rox1 and Mot3 Eukaryot. Cell, April 1, 2005; 4(4): 649 - 660. [Abstract] [Full Text] [PDF]
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M. Bassilana, J. Hopkins, and R. A. Arkowitz Regulation of the Cdc42/Cdc24 GTPase Module during Candida albicans Hyphal Growth Eukaryot. Cell, March 1, 2005; 4(3): 588 - 603. [Abstract] [Full Text] [PDF]
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K. W. Henry, J. T. Nickels, and T. D. Edlind ROX1 and ERG Regulation in Saccharomyces cerevisiae: Implications for Antifungal Susceptibility Eukaryot. Cell, December 1, 2002; 1(6): 1041 - 1044. [Abstract] [Full Text] [PDF]
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