Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.     

Dual-colour fluorescence microscopy using yEmCherry-/GFP-tagging of eisosome components Pil1 and Lsp1 in Candida albicans

PCR-based gene targeting technologies have previously been developed for Candida albicans molecular genetic manipulation. Modular marker plasmids for the functional analysis of C. albicans genes have been generated to delete genes, exchange promoters and tag genes with GFP. Here, we have embedded two fluorescent proteins encoded by Venus and yEmCherry into the pFA-plasmid series and demonstrate their usefulness in dual colour microscopy. To this end we analysed the localization of C. albicans homologues of Pil1 and Lsp1, which in S. cerevisiae are components of eisosomes. We find that Pil1/Lsp1-containing eisosomes are cortical protein complexes in C. albicans.Pil1 and Lsp1, tagged with either GFP or yEmCherry, strictly co-localized during all growth stages. Eisosomes, however, localized at distinct positions not overlapping with either cortical actin patches or the endocytosis marker protein Abp1 in yeast or the Spitzenk?rper in hyphal cells. To demonstrate the use of Venus yellow fluorescent protein we performed time lapse microscopy of yeast and hyphal stages using a histone H4-Venus tag. As demonstrated, these additions to the toolbox enable a wide range of in vivo applications in C. albicans.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5⁺ or Escherichia coli kan^r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.     

Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins

We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).     

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.     

Molecular cloning of the RPS0 gene from Candida tropicalis.

The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalis RPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both RPS0 genes in S. cerevisiae. Southern blot analysis suggests that CtRPS0 is present at a single locus within the C. tropicalis genome.     

New tools for phenotypic analysis in Candida albicans: the WAR1 gene confers resistance to sorbate

Availability of the complete sequence of the Candida albicans genome allows for global gene analysis. We designed a gene deletion method to facilitate such studies. First, we constructed C. albicans strains that are both Deltaura3 and Deltatrp1. Second, we designed a system that relies on in vitro recombination, using the Gateway((R)) technology, for efficient generation of deletion cassettes. They are generated in two steps: (a) upstream and downstream DNA fragments of the chromosomal region to be deleted are amplified by PCR and introduced into two separate entry vectors; (b) the second step involves a quadruple recombination event including the two entry vectors, a plasmid bearing a marker of interest and a destination vector, in order to generate a plasmid containing the deletion cassette. The deletion plasmid contains very rare restriction sites for convenient excision of the knockout cassette. Selection in C. albicans can be performed with one of the following markers: the C. albicans URA3 gene, a modified S. cerevisiae TRP1 gene or the mycophenolic acid resistance (MPA(R)) gene. Upon integration into the genome, these markers can be removed by the use of 5-fluoroorotic acid (URA3), 5-fluoroanthranilic acid (TRP1) or the FLP recombinase (MPA(R)). Using this approach, we show that removal of the C. albicans orf19.1035 gene results in sensitivity to the weak acid sorbate, while its overexpression increases resistance to this compound. We named it WAR1, in analogy to its S. cerevisiae orthologue.     

Efficient gene targeting in Kluyveromyces lactis

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.     

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4⁺ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.     

One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha.

Previous evidence based on the experience of our laboratory showed that one-step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one-step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR-mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed.     

The Candida albicans 14-3-3 gene, BMH1, is essential for growth.

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.     

A single-copy suppressor of the Saccharomyces cerevisae late-mitotic mutants cdc15 and dbf2 is encoded by the Candida albicans CDC14 gene.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.     

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.     

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers

Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces.     

Identification of a Candida albicans homologue of the PHO85 gene, a negative regulator of the PHO system in Saccharomyces cerevisiae

In a screen for the protein kinase genes of the human pathogenic yeast Candida albicans, a putative homologue (CaPHO85) of PHO85, a negative regulator of the PHO system of Saccharomyces cerevisiae, which is one of the cyclin-dependent protein kinases (CDKs), was isolated. An open reading frame (ORF) of this gene was identified encoding a predicted protein of 326 amino acids with a calculated molecular weight of 37.6 kDa. The amino acid sequence is highly homologous to S. cerevisiae Pho85 (62% identity) and its Aspergillus nidulans homologue (70% identity), but less homologous to Cdc28 (50% identity) of S. cerevisiae and to its C. albicans homologue CaCdc28 (49% identity), both of which are also CDK. The coding region for the C. albicans gene was interrupted by an intron of 81 nucleotides near the sequence encoding the N-terminal region, similarly to the case of the S. cerevisiae PHO85 gene. Alignment of CaPho85 with various yeast CDKs revealed that most of the domains for ATP-binding and protein kinase activity are conserved among fungal species. Southern blot analysis indicated that CaPHO85 is most likely present as a single copy gene. This gene complemented the pho85 mutation of S. cerevisiae by transformation.