Transformation of Candida albicans with a synthetic hygromycin B resistance gene

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2‐containing plasmids or single‐copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT‐1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild‐type C. albicans. We used PCR to fuse CaHygB or SAT‐1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild‐type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.     

Bax-induced cell death in Candida albicans

Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans. Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans. Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans, Bax co-localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region.     

Inter- and intra-species crosses between Candida albicans and Candida guilliermondii

Hybridization was shown to occur both between strains of the imperfect diploid yeast Candida albicans and between C. albicans and the distantly related haploid yeast Candida (Pichia) guilliermondii. Prototrophic hybrids were selected from crosses of multiply marked auxotrophic mutants of the two species. In most cases, mild ultraviolet irradiation of the C. albicans partner was required. Examination of auxotrophic markers in segregants from the hybrids indicated that recombination, rather than heterokaryon formation, had occurred in these crosses. The DNA content of the hybrids varied from diploid or aneuploid (for crosses between C. albicans and C. guilliermondii) to triploid (for C. albicans x C. albicans). It seems possible that genetic exchange analogous to this may occur in nature.     

The SPL1 tRNA splicing gene of Candida maltosa and Candida albicans

The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.     

不同物理诱变方式对休哈塔假丝酵母产乙醇的影响

采用不同物理方式（紫外线、He-Ne激光和微波）对休哈塔假丝酵母（Candida albicans）进行诱变,结合致死率、正突变率、乙醇产量等试验,分析不同诱变因子对该菌株发酵木糖的影响。结果表明,三种物理诱变对菌株诱变能力有差别,具有代表性的紫外线突变株（ZW-6）、He-Ne激光突变株（HN-3）及微波突变株（WB-3）的乙醇产量分别为17.44 g/L、18.49 g/L和18.11 g/L,较原始菌株分别提高13.91%、20.77%和18.29%;乙醇得率分别为0.35 g/g、0.38 g/g和0.37 g/g,较原始菌株分别提高13.80%、23.17%和19.50%。与紫外线及微波各诱变菌株相比,He-Ne激光诱变菌株的正突变率高,激光照射10 min时,正突变率高达48.62%;He-Ne激光诱变菌株的木糖利用率、乙醇产量、乙醇得率均提高最多,提高的范围最大,分别较原始菌株提高1.69%～44.77%、13.12%～40.59%、29.73%～47.75%。因此,确定He-Ne激光为最佳物理诱变因子。     

Construction of FLAG tagging vectors for Candida albicans

We have constructed three new vectors for Candida albicans (pFLAG-Act1, pFLAG-Mal2, and pFLAG-Met3). The proteins can be expressed as C-terminal FLAG-tagged proteins under the control of different promoters (ACT1, MAL2, and MET3). To confirm the protein expression, we used the Renilla reniformis luciferase and the drug efflux pump Cdr1p of Candida albicans as reporters. The luciferase protein expressed by the MET3 promoter was found to have the strongest activity of the three promoters when cultured in a methionine-depleted synthetic medium. Cdr1p was expressed as a C-terminal FLAG-tagged protein using either these vectors or PCR-mediated integration. The fluconazole resistance was increased by the Cdr1p expression in a CDR1 homozygous disruptant. The expressed proteins were detected by Western blotting using the anti-FLAG antibody. We also constructed a Cdr1p-FLAG expressing strain, in which we directly tagged Cdr1p with FLAG on the genome loci, using a PCR-based integrative marker cassette that was amplified using the pFLAG vector. We then confirmed the protein expression by Western blotting. Thus, these new vectors are useful as C. albicans genetic tools.     

Cloning of the Candida albicans nucleoside transporter by complementation of nucleoside transport-deficient Saccharomyces

The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.     

A Candida albicans gene encoding a DNA ligase

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession Number X95001.     

白色念珠菌检测方法的研究进展

白色念珠菌是导致真菌感染的重要致病菌之一,文章对白色念珠菌的生物学特性和检测方法进行了综述。目前我国尚未出台食品中白色念珠菌检测的相关标准。本文通过对白色念珠菌检测技术发展历程的梳理,旨在为广大科研工作者、行业法规制定者提供一些参考依据。     

Vectors designed for efficient molecular manipulation in Candida albicans

Functional studies on genes of Candida albicans have been hampered by the fact that few vectors are available for efficient cloning and expression in C. albicans, in contrast to Saccharomyces cerevisiae. Here we report that six vectors were constructed for molecular manipulation in C. albicans. All of them contained the autonomous replicating sequence ARS2 and the uracil gene as a selective marker. Introduction of multicloning site (MCS) facilitated directional cloning into various convenient restriction sites is discussed. Distal to the MCS, the additions of sequences encoding yeast-enhanced green fluorescent protein 3 (yEGFP3) and the terminator of chitin synthase 2 (TCHS2) enabled us to express an open reading frame (ORF) with its own promoter as a GFP fusion protein, so that its intracellular localization could be easily determined. A vector of 7.4 kb was also constructed to express a cloned ORF as a GFP fusion protein under the control of an inducible MET3 promoter (PMET3) located proximal to the MCS. Since this vector was relatively large in size for expressing ORFs, two additional vectors of 6.7 kb were constructed by inserting PMET3 and TCHS2 proximal and distal to the MCS of the above vector containing MCS only, respectively. These six vectors made it possible to study C. albicans in greater detail. They can be used in identification of a promoter, intracellular localization of a protein, and in the induction of lethal genes.     

Characterization of glycolytic metabolism and ion transport of Candida albicans

The main energetic pathways, fermentation and respiration, and the general ion transport properties of Candida albicans were studied. Compared to Saccharomyces cerevisiae, we found that in C. albicans: (a) the cell mass yield when grown in YPD was significantly larger; (b) it required longer times to be starved of endogenous substrates; (c) ethanol production was lower but significant; (d) respiration was also lower; (e) it showed a small activity of an alternative oxidase; (f) fermentation and oxidative phosphorylation seemed to compete for both ADP and NADH; and (g) NADH levels were lower. Regarding ion transport and compared to S. cerevisiae: (a) the general mechanism was similar, with a plasma membrane H⁺‐ATPase that generates both a plasma membrane ΔpH and a ΔΨ, the latter being responsible for driving K⁺ inside; (b) its acidification capacity is slightly smaller and less sensitive to activation by high pH; and (c) the presence of K⁺ results in a large activation of both respiration and fermentation, most probably due to the energy required in the process. ADP produced by H⁺‐ATPase stimulation by high pH or the addition of K⁺ at low pH results in the increase of both respiration and fermentation. Copyright © 2012 John Wiley & Sons, Ltd.     

Large circular and linear rDNA plasmids in Candida albicans

Although plasmids containing rRNA genes (rDNA) are commonly found in fungi, they have not been reported in Candida. We discovered that the yeast opportunistic pathogen Candida albicans contains two types of rDNA plasmids which differ in their structure and number of rDNA repeats. A large circular plasmid of unknown size consists of multiple rDNA repeats, each of which includes an associated autonomously replicating sequence (ARS). In contrast, a linear plasmid, which is represented by a series of molecules with a spread of sizes ranging from 50-150 kbp, carries a limited number of rDNA units and associated ARSs, as well as telomeres. The number of linear plasmids per cell is growth cycle-dependent, accumulating in abundance in actively growing cells. We suggest that the total copy number of rDNA is better controlled when a portion of copies are on a linear extrachromosomal plasmid, thus allowing a rapid shift in the number of corresponding genes and, as a result, better adaptation to the environment. This is the first report of a linear rDNA plasmid in yeast, as well as of the coexistence of circular and linear plasmids. In addition, this is a first report of naturally occurring plasmids in C. albicans.     

5-Fluoro-orotic acid induces chromosome alterations in Candida albicans

Treatment of a prototrophic laboratory strain of Candida albicans with 5-fluoro-orotic acid (5-FOA) produced two major types of mutants with chromosomal alterations, 5-FOA-resistant (FoaR) and those remaining sensitive (FoaS). Both major types remained Ura+. FoaR mutants, produced after a long exposure, contained either a duplication of chromosome 4b or an inner enlargement of chromosome 5b. The average mutant frequency was approximately 1.0 x 10(-5). The reverse mutation of FoaR to FoaS also caused the loss of either the extra chromosome 4b or the enlarged chromosome 5b, revealing a causal relationship between the resistance and the specific chromosome constitution. The cells remained sensitive after a relatively short 24 h exposure to 5-FOA medium, but the treatment induced non-specific changes in lengths of various chromosomes. Furthermore, FoaR type mutants acquired a notable chromosomal and phenotypic instability. Our results indicate the necessity of electrokaryotyping of strains that have been exposed to 5-FOA, especially with studies of gene function and with DNA microarray assays.     

Functional analysis of the Candida albicans ALS1 gene product

ALS1 encodes a cell surface protein that mediates adherence of Candida albicans to endothelial cells. The predicted Als1p has an N-terminal region, which contains a signal peptide; a middle region, which contains 20 36-amino acid tandem repeats; and a C-terminal region, which contains a glycosylphosphotidylinositol-anchorage sequence. We used site-directed mutagenesis to delineate the regions in Als1p required for endothelial cell adherence and cell surface expression of the protein. Mutant alleles of ALS1 containing either deletions or insertions were expressed in the normally non-adherent Saccharomyces cerevisiae. These transformants were analysed for endothelial cell adherence and cell surface expression of Als1p. We found that mutations centred around amino acid 285 in the N-terminus completely abolished adherence, but had no effect on cell surface expression of Als1p. Deletion of 15 of the tandem repeats reduced adherence by 50%, whereas deletion of all abolished adherence completely, even though cell surface expression of the N-terminus of Als1p was maintained. Insertions into the C-terminus at amino acids 413 and 254 upstream of the stop codon resulted in a modest loss of adherence, while cell surface expression of Als1p was maintained. An insertion at amino acid 249 in the C-terminus caused complete loss of both adherence and cell surface expression, even though the glycosylphosphotidylinositol-anchorage sequence remained intact. These data suggest a model of Als1p in which the endothelial cell binding region is localized within its N-terminus, the tandem repeats are essential for the proper presentation of the binding site, and the C-terminus is required for localizing Als1p to the cell surface.     

The ALS6 and ALS7 genes of Candida albicans

ALS genes of Candida albicans encode a family of cell-surface glycoproteins that are composed of an N-terminal domain, a central domain of a tandemly repeated motif, and a relatively variable C-terminal domain. Although several ALS genes have been characterized, more ALS-like sequences are present in the C. albicans genome. Two short DNA sequences with similarity to the 5' domains of known ALS genes were detected among data from the C. albicans genome sequencing project. Probes developed from unique regions of these sequences were used to screen a genomic library from which two full-length genes, designated ALS6 and ALS7, were cloned. ALS6 and ALS7 encode features similar to other genes in the ALS family and map to chromosome 3, a chromosome previously not known to encode ALS sequences. ALS6 and ALS7 are present in all C. albicans strains examined. Additional analysis suggested that some C. albicans strains have another ALS gene with a 5' domain similar to that of ALS6. Characterization of ALS7 revealed a novel tandemly repeated sequence within the C-terminal domain. Unlike other ALS family tandem repeats, the newly characterized ALS7 repeat does not appear to define additional genes in the ALS family. However, our data and information from the C. albicans genome sequencing project suggest that there are additional ALS genes remaining to be characterized.