Compilation of Candida annotation data.

As detailed in Materials and Methods, we used assembly version 19 of the C. albicans genome [6] to identify 11,615 putative ORFs. These included genes encoding proteins greater than 150 aa as well as genes encoding smaller proteins of 50–149 aa that have a coding function greater than 0.5 as determined with a GeneMark matrix [11]. These ORFs were then compared to the set of 7,680 C. albicans ORFs defined by the Stanford Genome Technology Center (SGTC), thus permitting their classification using the same systematic identifiers of the format orf19.n [6]. The 3,936 novel ORFs without an orf19.n counterpart were assigned a new reference number of the format orf19.n.i where orf19.n is the five-prime closest (contig-wise) ORF defined by the SGTC and i is an integer that varies between one and the number of novel ORFs found in the orf19.n to orf19.(n + 1) interval. To simplify correlation with previously published data that use the orf6.n or earlier nomenclatures, we have produced a Web-accessible translation tool (http://candida.bri.nrc.ca).

Positional information for each ORF was merged with data from a variety of different sources, including the SGTC (http://www-sequence.stanford.edu/group/candida/index.html), CandidaDB (http://genolist.pasteur.fr/CandidaDB; [12]), the Agabian laboratory (http://agabian.ucsf.edu/canoDB/anno.php), and the Johnson/Fink laboratories [13], whose annotation data had been updated with a Magpie annotation [7]. This large dataset was then reformatted into EMBL-style files, thus allowing for input in the Artemis annotation software [14]. Volunteer annotators accessed a custom-made database to reserve and download EMBL files containing sequence and annotation data for each of the 266 DNA sequence contigs. To help in validating various and sometimes conflicting sources of information, translated protein sequences from putative C. albicans ORFs were compared to putative protein sequences extracted from five fungal genomes—Saccharomyces cerevisiae [15,16], Schizosaccharomyces pombe [17], Neurospora crassa [18], Aspergillus nidulans (Aspergillus nidulans Database, http://www-genome.wi.mit.edu/annotation/fungi/aspergillus/), and Magnaporthe grisea (Magnaporthe grisea Database, http://www-genome.wi.mit.edu/annotation/fungi/magnaporthe/)—as well as to the genomes of five other eukaryotes—Arabidopsis thaliana [19], Drosophila melanogaster [20], Caenorhadbitis elegans [21], Mus musculus [22], and Homo sapiens [23]—and the GenBank non-redundant (NR) protein database. Comparisons against the translated C. albicans genome were also performed to help identify overlapping genes and putative gene families.

To help interpret such a large number of sequence comparisons, we organized sequence similarity data in a Web-accessible database using a novel visualization concept whereby we used a colorimetric display to indicate BLAST similarity, which was easy and rapid to scan visually (Figure 1). The annotators could thus rapidly determine which genes are potentially unique to C. albicans (e.g., orf19.4741 and orf19.4786), those that are members of gene families (e.g., orf19.4736 and orf19.4779), genes that only have homologs in fungal genomes (e.g., orf19.4756 and orf19.4778), or those with homologs in all eukaryotic genomes (e.g., orf19.4732 and orf19.4784). Finally, a strong hit against the complete NR database, but not in the other genomes (orf19.4772 and orf19.4800), allowed us to identify C. albicans genes that had already been described and submitted to the sequence databases prior to the publication of assembly orf19. Clicking on the relevant boxes opened an additional window containing the precompiled sequence alignments, thus permitting the validation of interesting observations. These visualization tools and the results of sequence comparisons are available at http://candida.bri.nrc.ca/candida/index.cfm?page=blast.

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Figure 1. Visualization of Protein Sequence Similarities

Sample from a Web page used by annotators of the C. albicans genome to visualize the significance of the best hit from whole-proteome BLASTP searches. Each putative ORF was compared to the NR database, the Candida ORF list itself (Ca19; showing results from the four top hits), and amino acid sequences from the proteomes of S. cerevisiae (Sac), S. pombe (S.p), M. grisea (Mag), N. crassa (Neu), H. sapiens (H.S), M. musculus (M.m), D. melanogaster (Dro), C. elegans (C.e), and A. thaliana (A.t). The BLASTP e-value from the top hit was converted to a color scale as indicated. Examples of C. albicans genes with interesting similarity patterns are indicated.

https://doi.org/10.1371/journal.pgen.0010001.g001

The coordinates and annotations for all 11,615 putative ORFs were thus verified, corrected, and (if necessary) rewritten by the annotators. We removed ORFs smaller than 300 bp with no significant sequence similarity to other genes, either within the C. albicans genome or in the sequence databases. In cases where two ORFs overlapped by more than 50%, the smallest gene was removed unless it showed even a slight sequence similarity to another gene in the sequence databases. In other cases, we encountered two, or more, contiguous ORFs that obviously were part of the same gene. These interruptions were usually due to unidentified introns or presumed sequencing errors. In these cases, we decided to merge the relevant gene fragments into a single entry. A total of 5,262 ORF entries were thus removed from the database, or merged with neighboring ORFs, leaving 6,354 confirmed genes. Sequence and/or annotation data can be obtained in Dataset S1 or at http://candida.bri.nrc.ca.

A nomenclature for C. albicans genes.

Following consultations with the C. albicans research community during the fifth and sixth American Society for Microbiology Conferences on Candida and Candidiasis, it was agreed that C. albicans gene names should follow the format established for S. cerevisiae [24]. Gene names consist of three letters (the gene symbol) followed by an integer (e.g., ADE12); the gene symbol should be an acronym for, or relate to, the gene function, gene product, or mutant phenotype. It is preferable that a given gene symbol have only one meaning, so that all genes using that symbol are related in some way, for instance, by sharing a function, participating in a shared pathway, or belonging to the same gene family. In addition, gene symbols that are used in S. cerevisiae gene names should retain the same meaning when used for C. albicans genes. The prefix ‘Ca' has sometimes been used on gene names to denote that a gene is derived from C. albicans; however, while the use of prefixes adds clarity to discussions of genes from different species that share a name (e.g., comparing CaURA3 to ScURA3), the prefix is not considered part of the gene name proper. Finally, allele designations and deletion symbols should come after the gene name (ICG1–8 and icg1Δ for example). For more details on genetic nomenclature, see the Candida Genome Database (CGD; [25]) Web page on this topic (http://www.candidagenome. org/Nomenclature.html).

Wherever possible, genes that are orthologous between C. albicans and S. cerevisiae should share the same name. We have provided 3,409 suggested names (in the SuggGene field of the EMBL files) for many C. albicans ORFs based on their orthology to S. cerevisiae genes; these are not yet considered the standard C. albicans gene names, but rather provide guidance for investigators wishing to name these genes. CGD assigns standard names to C. albicans genes for which there are published data (the PubGene field). The annotation contains 355 such entries. Generally, CGD considers the first published name in the correct format to be the standard name; common usage and uniqueness are also considered. All names that have been used for a gene are collected in CGD, regardless of their format, so that information from the literature can be traced to the correct gene. In the current annotation, additional published gene names have been placed in the Synonym field.

Public access to the data.

The complete annotation dataset, results of BLAST sequence similarity searches, and the identification of conserved protein domains can be obtained from our Web page (http://candida.bri.nrc.ca/). Furthermore, CGD (http://www.candidagenome.org), funded by the National Institute for Dental and Craniofacial Research of the National Institutes of Health, will curate the scientific literature and provide tools for accessing and analyzing the C. albicans genome sequence. In addition, CGD will act as a central repository for gene names and modifications, as approved by the C. albicans research community at the American Society for Microbiology Candida and Candidiasis meeting in Austin, Texas, in March 2004. CGD itself will not name C. albicans genes, but instead will act as a clearinghouse for the standard gene names and aliases, as the Saccharomyces Genome Database (SGD) does for the S. cerevisiae community. CGD hopes that researchers will follow CGD's gene nomenclature guidelines (see above) and keep CGD informed of any new gene names. Prior to publication, researchers may reserve a gene name, which will then become the standard name upon publication. Finally, the CandidaDB database (http://genolist.pasteur.fr/CandidaDB) [12], which has provided an annotation of the C. albicans genome sequence since January 2001, will be updated to take into account the complete annotation dataset and will continue to provide tools for accessing and analyzing the C. albicans genome sequence complementary to those available at the CGD and the Biotechnology Research Institute.

Intron analysis.

There are 215 ORFs containing at least one intron, four of which have two introns, one gene (encoding the Hxt4p transporter) has three, and the SIN3 gene has four. A total of 43 (20.2%) of these genes encode ribosomal proteins, 63 (29.6%) encode products with enzymatic activity, and 26 (12.2%) encode trans-membrane proteins involved in small molecule transport. We measured the relative position of introns in their host ORFs and observed that a significant proportion of them are located in the 5′ end of ORFs, with 32% of introns being located within the first 10% of the coding sequences. A survey of the distribution of introns in 18 eukaryotic genomes, including S. cerevisiae and H. sapiens, also indicated a similar bias in intron-poor genomes. It has been argued that this 5′ bias is an indication that introns are particularly difficult to remove by cDNA recombination, because of the high activity of these genes and paucity of full-length cDNA, and that this finding lends some support to the idea that introns are being lost more frequently than they are being gained in these lineages [26], although a more recent study of four fungal genomes suggests the presence of additional mechanisms [27].

We surveyed the intron phase distribution and found that C. albicans has 50.5%, 20.4%, and 29.1% of phase zero, one, and two introns, respectively. A similar result was observed in fungal, plant, and animal genomes [27,28], suggesting that a similar intron phase distribution may be present in ancient introns and that the intron loss has no preference selection on intron phases. Seventy out of 215 intron-containing ORFs have reciprocal best matches with S. cerevisiae genes that also contain introns. Among these 70 ORFs, 25 introns (35.7%) share the same position and the same phase. This suggests that these commonly positioned introns descended from a common ancestor, as suggested previously [29].

Analysis of protein domains.

Table 3 shows the most abundant protein domains that were identified in the C. albicans proteome. As a comparison, we also performed this analysis on the same ten eukaryotic proteomes that were used in the BLASTP sequence comparisons. Compared to the S. pombe and S. cerevisiae proteomes, the C. albicans proteome shows a slight increase in the abundance of leucine-rich repeats (IPR001611), some zinc finger transcription factors (IPR001138), esterases/lipases (IPR000379), and trans-membrane transporters for polyamines (IPR002293) and for amino acids (IPR004841). If the analysis is expanded to the other fungal proteomes, only the increased abundance in leucine-rich repeats appears to be unique to C. albicans.

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Table 3.

Number, Abundance Ranking, and Proportion of Gene Products Containing the Indicated Interpro Protein Domain in C. albicans and Other Eukaryotes

https://doi.org/10.1371/journal.pgen.0010001.t003

The ATP-binding cassette transporter superfamily.

The ATP-binding cassette (ABC) protein superfamily represents one of the largest protein families known to date among available genome sequences. These proteins share similar molecular architecture with the presence of at least one conserved ABC domain and the presence of membrane-spanning segments (transmembrane segments [TMSs]). The ABC domain typically contains Walker A and Walker B motifs and an ABC signature motif. The ABC domain and TMSs can be arranged in a duplicated forward (TMS₆-ABC)₂ or reverse (ABC-TMS₆)₂ topology, however “half size” ABC proteins also exist. As indicated in Table 6, the C. albicans genome contains at least 27 genes with ABC domains that include these topologies. These genes have been categorized, according to a classification established in S. cerevisiae, into six subfamilies (the MDR, PDR, MRP/CFTR, ALD, YEF3, and RLI subfamilies) [42]. The MDR, PDR, MRP/CFTR, and ALD subfamilies likely all encode transporter proteins, while the other subfamilies, YEF3 and RLI, generally lack TMSs and are considered as non-transporter ABC proteins. The C. albicans ABC proteins fall neatly into the categories developed for S. cerevisiae, and they are also present in approximately the same numbers (with the exception of the MRP/CFTR subfamily; see below). The predicted topology of each protein detailed in Table 6 is also largely comparable between the two yeast species. Among the 27 ABC proteins so far identified in C. albicans, the functions of only nine have been previously characterized. The largest group of known ABC transporters belongs to the CDR gene family, among which are CDR1 and CDR2, two genes upregulated in azole-resistant clinical isolates that function in multidrug resistance [43–45]. CDR3 and CDR4 have been shown to function as phospholipid flippases and their expression is controlled by the white–opaque switching system [46,47]. Four MRP/CFTR-like transporters are present in C. albicans, and among them three show the NH₂-terminal extension with additional transmembrane segments that is typical for many MRP-like transporters (see Table 6). For unknown reasons, homologs of additional members of this family, such as the S. cerevisiae genes ScYBT1, ScNFT1, and ScVMR1, are lacking in C. albicans [42,48]. Interestingly, the vacuolar MRP-like transporter encoded by MLT1 has been implicated in virulence [49]. Since MRP/CFTR transporters are often involved in detoxification of heavy metals or xenobiotics, the presence or absence of discontinuous alleles of some ABC transporter genes (e.g., orf19.6383) may indicate strain differences in ABC transporter function and resulting susceptibility to environmental stresses. Most of the ABC transporter genes listed in Table 6 were given names through their closest homologs in S. cerevisiae; however, the functional assignments of these genes awaits further investigation.

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Table 6.

Genes Encoding Members of the ABC Transporter Family

https://doi.org/10.1371/journal.pgen.0010001.t006

The ALS family.

The ALS genes encode large cell-surface glycoproteins that function in host–pathogen interactions [50,51]. The ALS genes are composed of three domains: a 5′ domain that is approximately 1,300 bp in length and relatively conserved in sequence across the family, a central domain composed entirely of tandemly repeated copies of a 108-bp sequence, and a 3′ domain of variable length and sequence that encodes a serine/threonine-rich portion of the protein [50]. Efforts to characterize the ALS genes started independently of the C. albicans genome project [38,52–54]) and were aided greatly by information that emerged as the genome sequencing effort progressed [55–57]. Table 7 lists the current ORFs that correspond to genes in the ALS family. The ALS family includes eight different genes [55], each with an extensive degree of allelic variability, sometimes within a given strain (Table 7) or across the wider population of C. albicans isolates [58–60]. Because of sequence assembly difficulties, mainly attributable to the length and repetitive nature of sequences within the ALS central domain, only three of ALS ORFs in this project are in agreement with ALS gene sequences derived independently of the genome project and reported in the literature (Table 7). The annotation effort described here did not edit the underlying assembly 19 sequence. However, gap sequencing that is presently being carried out and the production of a final genome assembly will correct these errors. Published ALS gene sequences can be found on the CGD Web site.

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Table 7.

Assembly 19 ORFs That Correspond to ALS Genes

https://doi.org/10.1371/journal.pgen.0010001.t007

Assembly of the C. albicans genome sequence revealed the contiguous positions of ALS5, ALS1, and ALS9 on Chromosome 6, which was verified by independent studies [57]. Additional testing revealed that, in SC5314, the large alleles of ALS5, ALS1, and ALS9 occupy the same chromosome while the small alleles of each gene are found on the homologous chromosome [57]. But allelic variability and arrangement on homologous chromosomes will vary for each C. albicans strain. Allelic variation can be extreme for ALS genes, and is most commonly associated with the tandem repeat domain, although it is also present within other domains of the coding region [56,57,59]. Presenting the sequence of a single ALS allele, as done in these annotation data, loses the sense of allelic diversity that can have a significant effect on evaluation of ALS protein function. For example, testing the two ALS3 alleles from strain SC5314 in a common adhesion assay format showed that the allele with more tandem repeat copies produced a protein with greater adhesive capability than the smaller allele [60]. Table 7 notes GenBank entries for ALS alleles from strain SC5314 that aid understanding of allelic diversity for the various ALS genes.

The MEP family.

Members of the MEP gene family encode ammonium permeases and, along with the OPT family described below, feature prominently in our list of fungal-specific genes. They thus represent potentially interesting targets for the development of antifungal drugs. Experimental evidence suggests that MEP1 and MEP2 encode the only specific ammonium permeases in C. albicans, since Δmep1 Δmep2 double mutants exhibited no detectable ammonium uptake and were unable to grow at ammonium concentrations below 5 mM [61], a phenotype that is similar to that of S. cerevisiae mutants deleted for all three ammonium permeases [62,63]. The third C. albicans gene, represented by orf19.4446, encodes a protein with much lower similarity to the other ammonium permeases of C. albicans and S. cerevisiae (approximately 44% to all proteins) but might encode an ammonium permease that is not expressed under the growth conditions used in these assays.

In addition to its role in ammonium transport, Mep2p also controls nitrogen-starvation-induced filamentous growth of C. albicans. Mutants in which only the MEP2 gene was deleted grew as well as the wild-type strain at low ammonium concentrations but failed to filament under these conditions. This role of MEP2 in filamentous growth of C. albicans at low ammonium concentrations is similar to the function of its counterpart ScMEP2 in pseudohyphal growth of S. cerevisiae under limiting ammonium conditions [63]. However, in contrast to the latter, MEP2 seems to have a much broader role in filamentous growth of C. albicans since Δmep2 mutants also had a filamentation defect when amino acids or urea instead of ammonium served as the limiting nitrogen source (J. Morschhäuser, personal communication).

The OPT family.

Oligopeptide transporters represent another group of fungal-specific surface proteins that transport peptides of four or five amino acids in length into the cell and together with the di- and tripetide transporters allow growth when peptides are the only available nitrogen source. This is presumably the position of C. albicans cells when they have invaded host tissues and are secreting their battery of peptidases and other catabolic enzymes. The founding member of the oligopeptide transporter gene family was OPT1 from C. albicans [64]. Analysis of the C. albicans genome sequence as well as cloning of the corresponding genes demonstrated that C. albicans in fact possesses a large gene family encoding putative oligopeptide transporters. The OPT genes were annotated according to their decreasing similarity to OPT1. The OPT2, OPT3, and OPT4 genes are highly similar to each other. The similarity of the remaining members of the family then drops considerably, but we have detected genes now named OPT6, OPT7, and OPT8. Deletion of the OPT1 alleles in the C. albicans wild-type strain SC5314 resulted in increased resistance of the mutants to a toxic tetrapeptide, providing experimental evidence that Opt1p indeed functions as an oligopeptide transporter in C. albicans [65]. Preliminary observations indicate that at least the OPT2 to OPT5 genes also encode functional oligopeptide transporters (O. Reuß and J. Morschhäuser, unpublished data).

Zinc cluster transcription factors.

Proteins of the zinc finger superfamily represent one of the largest classes of DNA-binding proteins in eukaryotes. Several different classes of zinc finger domains exist that differ in the arrangement of their zinc-binding residues [66]. One of these domains, which appears to be restricted to fungi, consists of the Zn(II)₂Cys₆ binuclear cluster motif in which six cysteines coordinate two zinc atoms [67,68]. S. cerevisiae possesses 54 zinc cluster factors defined by the presence of the zinc cluster signature motif CX₂CX₆CX_5–16CX₂CX_6–8C, which is generally located at the N-terminus of the protein. These proteins function as transcriptional regulators involved in various cellular processes including primary and secondary metabolism (e.g., Gal4p, Ppr1p, Hap1p, Cha4p, Leu3p, Lys14p, and Cat8p), pleiotropic drug resistance (e.g., Pdr1p, Pdr3p, and Yrr1p), and meiosis (Ume6p) [68,69]. Quite often, they bind as homo- or heterodimers to two CGG triplets organized as direct, indirect, or inverted repeats and separated by sequences of variable length [68,70]. A large proportion of these factors (50%) also contain a middle homology region (Fungal_trans in the Pfam Protein Families Database) located in the central portion of the protein that has been proposed to participate in DNA binding and to assist in DNA target discrimination [67].

Analysis of the C. albicans proteome using a combination of sequence analyses tools (SMART, Pfam, and PHI-BLAST) allowed us to identify 77 binuclear cluster proteins. These factors are characterized by the presence of the zinc cluster signature motif CX₂CX₆CX_5–24CX₂CX_6–9C generally located at the N-terminus of the protein (72 out of 77) and with a spacing between cysteines 3–4 and 5–6 slightly different from the S. cerevisiae motif. As observed in S. cerevisiae, a large proportion of the C. albicans factors also contain a middle homology region (29 out of 77). To our knowledge, only six of the C. albicans zinc cluster genes have been characterized in detail, including SUC1, involved in sucrose utilization [71], FCR1, implicated in pleiotropic drug resistance [72], CWT1, required for cell wall integrity [73], and CZF1, FGR17, and FGR27, involved in filamentous growth [9,74]. The functions of many uncharacterized C. albicans zinc cluster factors (approximately 20%) can be inferred from the fact that they display high levels of sequence similarity (top BLASTP e-value ≤ 1e⁻²⁰) with the products of S. cerevisiae genes with a known function. In the case of GAL4, however, the C. albicans homologous ORF identified (orf19.5338) encodes a significantly smaller protein (261 aa) than S. cerevisiae Gal4p (881 aa), lacking the C-terminal two-thirds of the protein that contains one of two transcriptional activating domains, and must therefore have a somewhat different function. Approximately half of the C. albicans zinc cluster genes do not appear to have homologs in S. cerevisiae (using a BLAST cutoff of < 1e⁻²⁰) and are therefore likely to participate in processes specific to C. albicans. Finally, it is noteworthy that many of the zinc cluster factors known to be involved in pleiotropic drug resistance in S. cerevisiae, such as Pdr1p, Pdr3p, Yrr1p, Yrm1p, Rds1p, and Rdr1p, do not appear to possess close structural homologs in C. albicans. Since pleiotropic drug resistance is frequently observed in C. albicans, it is likely that this organism possesses functional homologs of these genes or other novel processes that remain to be identified.

Phospholipases.

Depending on the site of attack, phospholipases are classified as phospholipase A, B, C, or D. Phospholipase A enzymes hydrolyze the 1-acyl ester (PLA₁) or the 2-acyl ester (PLA₂) of phospholipids. In fungi, phospholipase B enzymes hydrolyze both acyl groups and often also have lysophospholipase activity, removing the remaining acyl moiety on lysophospholipids [77]. Phospholipase C and phospholipase D enzymes are phosphodiesterases that cleave the glycerophosphate bond and remove the base group of phospholipids, respectively. While a major role of phospholipase function is membrane homeostasis, additional functions comprise nutrient digestion and generation of signaling molecules. Some phospholipases are toxins or components of venoms. Bacterial phospholipases have been shown to be involved in pathogenesis by promoting hemolysis, cytolysis, and tissue destruction, as well as interfering with host signal transduction [78].

As indicated in Table 8, the largest and best-characterized group of phospholipases in C. albicans is the five-member phospholipase B gene family. A related gene family is present in S. cerevisiae, albeit with three members, again reflecting the general increase in gene numbers for enzymes involved in lipid metabolism in C. albicans. All PLB proteins harbor NH₂-terminal signal peptides for secretion; Plb3p, Plb4p, and Plb5p additionally contain hydrophobic COOH termini with putative GPI anchor attachment sites for localization to the plasma membrane or further processing for tethering to the cell wall [79,80]. To date, PLB1 and PLB2 are the best-characterized members of the gene family [81–84]. Inactivation of PLB1 [82,83] and PLB5 (S. Theiss, G. Ishdorj, M. Kretschman, C. Y. Lan, T. Nichterlein, et al., unpublished data) reduced virulence in animal models.

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Table 8.

Phospholipases in C. albicans

https://doi.org/10.1371/journal.pgen.0010001.t008

Putative PLC and PLD phosphodiesterases are also represented in the C. albicans genome. Orf19.6629 is a likely homolog to S. cerevisiae ScISC1, which encodes a PLC with neutral sphingomyelinase activity. Besides the recently published PLC1 gene [85], two almost identical genes encode phosphatidylinositol phospholipase C proteins (PI-PLC). The latter lack homologs in S. cerevisiae, but are similar to bacterial PI-PLCs. PLD1 was shown to be involved in the morphological transition from yeast to hyphae and required for full virulence in animal models [86]. Interestingly, the PLD1 gene product and another phospholipase-like protein (encoded by orf19.4151) show significant sequence similarity to S. cerevisiae proteins that are involved in meiosis and sporulation (ScSpo1p, ScSpo14p, and ScSpo22p). As already shown for PLD1, the ScSPO1 homolog, the functional roles of these proteins are likely to differ from their counterparts in S. cerevisiae since C. albicans has not been shown to undergo meiosis [10].

Another intriguing group of phospholipase genes in C. albicans are patatin-like phospholipases encoded by orf19.1504, orf19.5426, and orf19.6396. These proteins might account for phospholipase A activities in C. albicans that could be involved in intracellular storage or mobilization of lipids.

Sphingolipid metabolism.

C. albicans also displays differences from S. cerevisiae with respect to sphingolipid metabolism. Pathways leading to and from fungal-type sphingomyelins have been studied extensively in S. cerevisiae, where by-products mediate many important structural and signaling functions that affect cell proliferation, the definition of cell membrane domains and polarity, apoptosis, and stress responses [87,88]. Many of the associated enzymes are essential and are targets of fungal toxins, and thus are candidates for anti-fungal drug development [88]. C. albicans shares the same fundamental pathways in sphingolipid biosynthesis/degradation plus four additional enzymes. Two of these, a glucosyl transferase (CGT1) and a delta-4 sphingolipid desaturase (DES1), have been previously studied. The presence of glycosyl ceramides in C. albicans has been known for some time [89,90], and the gene responsible for their synthesis has been cloned and expressed in Pichia [91]. The molecules play a common role in differentiation in dimorphic fungi [92]. Homologs of the delta-4 sphingolipid desaturase enzyme include the mouse, human, and Drosophila degenerative spermatocyte proteins, which play a role in meiosis [93]; its function in C. albicans may relate to membrane structure, or the production of signaling molecules, as is the case in plants. An interesting component of sphingolipid metabolism in C. albicans is a sphingomyelin transfer protein (Het1p) similar to the Podospora anserina HET-C2 protein. The P. anserina protein is involved in self/non-self discrimination, a fungal version of the vertebrate major histocompatibility locus [94,95]. It is possible that the protein is involved in regulating the sphingomyelin composition of C. albicans membranes, a factor that may relate to acquisition of resistance to amphotericin B and azoles [96]. Finally, C. albicans encodes four acid sphingomyelinases, two of which may be secreted, that have not been studied in fungi. Based on the actions of metazoan secreted acid sphingomyelinases, these enzymes may be involved in regulation of membrane raft formation and generation of ceramide, a second messenger that is known to regulate apoptosis in higher eukaryotes. Secreted sphingomyelinases of pathogenic bacteria, which are enzymatically similar but structurally unrelated to those of C. albicans, have been shown to lyse phagosomal membranes [97], facilitate entry into both phagocytic and nonphagocytic cells [98,99], act as hemolysins that abet piracy of iron from the host [100,101], and induce host cell apoptosis [102,103].

Identification of C. albicans ORFs and merging of preliminary annotations.

Nucleotide sequence data for assembly 19 were retrieved from the SGTC Web site (http://www-sequence.stanford.edu//group/candida/). Assembly 19 is composed of a haploid supercontig set (contigs 19–831 to 19–10262), here referred to as the haploid set, and a allelic supercontig set (contigs 19–20001 to 19–20161), here referred to as the allelic set [6].

The CAAT-Box software package [109] was used to identify annotation-relevant ORFs in assembly orf19. A set including ORFs longer than 300 codons and a set with all intergenic regions obtained after subtraction of ORFs larger than 80 codons were created. These sets were used to build a GeneMark matrix [11] that was subsequently used to evaluate the coding probability of all ORFs in assembly 19. ORFs longer than 150 codons, and ORFs longer than 40 codons and with a GeneMark coding function greater than 0.5 [11] over their whole length, were selected and assigned a reference number of the format IPFn.i where IPF stands for individual protein file, n is an integer specific to the IPF, and i corresponds to the number of times the IPF has been modified between assembly 5, 6, and 19 of the C. albicans genome sequence. In total, 11,025 and 9,089 IPFs were selected in the haploid and allelic sets, respectively. IPFs shorter than 150 codons in the haploid set were further inspected for (1) overlaps with larger IPFs on a different frame and (2) homology to proteins in the NR database of non-redundant proteins from GenBank. IPFs that overlapped with a larger IPF or did not show a significant homolog (BLASTP e-value < 1e⁻³) [110] were designated FALSORF. Of the 11,025 IPFs identified in the haploid set, 3,505 were FALSORFs.

All IPFs identified in the haploid set were compared through reciprocal BLASTP to the set of 7,680 C. albicans ORFs defined at the SGTC that uses the systematic designation orf19.n. BLASTP results were parsed using Readblast [111]. IPFs without an orf19.n counterpart were assigned a new reference number of the format orf19.n.i, where orf19.n is the closest upstream (using SGTC contig coordinates) ORF defined by the SGTC and i is an integer that varies between 1 and the number of IPFs located between orf19.n and orf19.(n + 1). For instance, if three ORFs were found between orf19.1234 and orf19.1235, these would be referred to as orf19.1234.1, orf19.1234.2, and orf19.1234.3. Taken together, 11,616 orf19 ORFs were identified in the haploid set, of which 3,936 were not present in the SGTC orf19 set.

A similar procedure was applied to the allelic set of sequences, and a total of 9,552 orf19 ORFs were identified, of which 3,012 were not present in the SGTC orf19 set. The haploid and allelic sets of orf19 ORFs were compared by reciprocal BLASTP in order to define allelic and unique sequences in the allelic set (see below).

The 11,615 orf19 ORFs identified in the haploid set were compared by reciprocal BLASTP to the 9,168 ORFs identified by the SGTC using assembly 6 of the C. albicans genome sequence (designated orf6.n). A similar reciprocal comparison was run using the set of 6,165 C. albicans proteins available in the CandidaDB database that have been defined by applying a procedure similar to that outlined above on assembly 6 and through a manual curation aiming to reach a non-redundant protein set (http://genolist.pasteur.fr/CandidaDB; [12]). Furthermore, orf19 ORFs were reciprocally compared to the S. cerevisiae proteome using data available at the SGD [112]. All data were parsed using Readblast [111], and a matrix was generated that correlated ORFs from each dataset.

We used the genome annotation tool Artemis [14], which provides very detailed annotation capability, visually mapping desired features onto the target sequence. In preparation, we loaded our heterogeneous data into the required EMBL-style files: (1) the orf19 reference (field: Assembly_ID); (2) the orf6 reference (field: old_Assembly_ID); (3) the CandidaDB entry number (field: db_xref); (4) the entry number for the Comprehensive Yeast Genome Database, which provides a detailed analysis of protein features of all entries available in CandidaDB [113] (field: db_xref); (5) the GenBank entry number for C. albicans proteins previously characterized and annotated (field: db_xref); (6) the IPF reference (field: db_xref); (7) annotation data available from CandidaDB (proposed gene name and proposed function; field: Annotator); (8) annotation data of the Agabian's laboratory based on the orf6 protein set (http://agabian.ucsf.edu/canoDB/anno.php) (field: Annotator); (9) annotation data of the Fink's and Johnson's laboratories based on the orf6 protein set (unpublished) (field: note_AJ); (10) annotation comments available from CandidaDB (field: note_GF); (11) Pfam matches [114] obtained using the orf19 protein set (field: pfam_match); (12) Clusters of Orthologous Groups matches [115] obtained from the Comprehensive Yeast Genome Database using the CandidaDB protein set (field: COGs_MIPS); (13) EC number matches obtained from the Comprehensive Yeast Genome Database using the CandidaDB protein set (field: EC_number_MIPS); (14) S. cerevisiae closest protein including e-value, putative orthology, protein function, gene name, and alternate gene names, genome reference number obtained from SGD (field: Note); (15) GO annotation of the S. cerevisiae protein obtained from SGD (field: GO); (16) the reference of the allelic orf19 in the allelic set of sequences including supercontig number and location (field: Allele); and (17) chromosome assignment data available from the Magee's and Whiteway's laboratories (http://206.167.190.233/candida/index.cfm?page=CaChrom) (field: Chromosome).

Furthermore all ORFs were assigned a color code in order to facilitate annotation using the annotation tool Artemis [14]. All orf19 ORFs corresponding to an IPF classified as FALSORF and orf19 ORFs identified at the SGTC and not found among IPFs were color-coded in grey. orf19 ORFs with an unambiguous allele (90% identical amino acids over the whole length of the longest ORF) were color-coded in red (>150 codons) or pink (<150 codons). orf19 ORFs with a questionable allele (90% identical amino acids over the whole length of the shortest ORF) were color-coded in green (>150 codons) or pale green (<150 codons). orf19 ORFs without a clear allele (less than 90% identical amino acids over the whole length of the shortest ORF or no reciprocal match) were color-coded in blue (>150 codons) or light blue (<150 codons).

From this “master” file of sequences and their corresponding preliminary annotation, groups of contigs were selected and saved as partially annotated subsequences that were reserved and retrieved by members of the annotation consortium, and once fully annotated, were returned to a central Web site. A version of Artemis was distributed to the consortium that included a modified “options” file [116] allowing project-specific qualifiers to be used, and also featuring the C. albicans–specific translation Table 12 [117].

Whole genome BLAST searches and visualization of sequence homologies.

ORF sequences were translated to proteins using the translation table for C. albicans [117], and compared using the BLASTP algorithm [118] with the NR database, the C. albicans proteome itself, the putative proteomes of five fungi (S. cerevisiae, S. pombe, N. crassa, A. nidulans, and M. grisea), and the proteomes of five other eukaryotes (A. thaliana, D. melanogaster, C. elegans, M. musculus, and H. sapiens). Sequence data were obtained from the EMBL-EBI Integr8 Browser (http://www.ebi.ac.uk/integr8/EBI-Integr8-HomePage. do) and the Broad Institute for Genome Research (http://www.broad. mit.edu/annotation/). For the most similar proteins by BLAST, negative exponents of the e-values were parsed from the output files, collected in a relational database, and visualized as a color range associated with each reference protein (see Figure 1). For this purpose, a Web-accessible visualization tool was constructed using Macromedia Cold Fusion and an Apache2 Web server. These results can be consulted at For this purpose, a Web-accessible visualization tool was constructed using Maromedia Cold Fusion and an Apache2 Web server. These results can be consulted at http://candida.bri.nrc.ca/candida/index.cfm?page =blast.

Bioinformatic identification of putative introns.

Intron locations were predicted by constructing regular expressions based on consensus data drawn from several known introns in C. albicans (SOD1, EFB1, CMD1, CSK1, and others) as well as the extensive knowledge of the splice site consensus in S. cerevisiae [119] and matching of those expressions to the genomic DNA. Two regular expressions were used: /(.{15,}?TG[AT]A[CT]G)/ and /(.{700}) ([ATC]TACTAAC.{4,24}?[ATC][CTA]AG)(.{90})/. These incorporate several conserved aspects of mRNA splice sites including (1) the splice donor site of G(T/C)A(T/A)GT, where the initial guanine is the first residue within the intron, (2) a gap of between 15 and 700 nucleotides between the donor site and the branch point, (3) the branch point consensus sequence (A/T/C)TACTAAC, (4) a gap of between four and 24 nucleotides between the branch point and the splice acceptor site, (5) the splice acceptor site (T/A/C)(T/A/C)AG, where the final guanine is the final residue within the intron. This procedure provided landmarks that annotators could use to decide whether a gene contained introns, a decision also based on the position of nearby ORFs, on the ability of the intron/exon assembly to extend the size of the reading frame, and, most important, on the ability of the putative intron/exon assembly to improve similarity to sequences in other species. It predicted 1,297 introns in the C. albicans genome, many of which were not near ORFs or were otherwise incorrect. Nevertheless, this procedure was useful in providing markers for confirmation by the annotators.

Identification and classification of STRs.

A STR was defined as a short nucleotide element (1–20 nt) repeated a number of times with a periodicity P, a length L, and a tolerated mutation window W. Since allelic indels are rare in C. albicans, we considered only mutations in STR sequences and not insertion or deletion from the consensus periodic pattern. W indicates the minimun distance between two mutations within a STR. For example, the STR “CTACAACAACAGCAAC” has P = 3, L = 16, and W ≤ 10. When two STRs overlap they are merged together defining a single STR domain. Since short STRs can randomly occur, depending on the G/C content of the genome, we established a significant threshold length L_MIN for every periodicity P and mutation window W to represent the STR length for which the number of STR domains found in a randomized genome is less than 5% of the number found in the real genome. We call STR95 the set of all STR domains that are longer than the threshold value. In the STR95 set, every STR domain has a less than 5% chance of being a random event. With this method of setting the minimal STR length, no significant STR can be found in any randomized genome or in genomes that do not contain STRs in sufficient number to beat the odds 20 to one. More information and data can be found in Dataset S6.

Identification of spurious genes.

For the calculation of gene expression correlation, we used a set of approximately 1,000 S. cerevisiae microarray experiments [127], and 216 genome-wide C. albicans expression profiles [7,13,108,120–126]. The iterative signature algorithm [35,127] was applied to the C. albicans expression data as described. We analyzed ORFs present on the arrays for which an orf19 number could be determined, including some that were subsequently removed from the final set of annotated genes. Pairwise Pearson correlation coefficients were calculated for each ORF with respect to all other ORFs across all of the experiments contained in the dataset. Random subsets of the S. cerevisiae data were generated by randomly selecting 200 experiments from the complete set of approximately 1,000 profiles. ORFs whose correlation coefficient exceeded the threshold value of 3σ with at least one other ORF in the dataset were recorded and excluded from the list of spurious gene candidates. The standard deviation σ of the background correlation of random gene pairs was measured to be σ = 0.16 for S. cerevisiae and σ = 0.21 for C. albicans. Similarly, genes possessing an ortholog in S. cerevisiae were excluded from the list of C. albicans candidates, and vice versa. All remaining ORFs were subsequently ordered by their length, and the 50 shortest ORFs were excluded (many of the shortest 50 genes correspond to real genes in S. cerevisiae).