Use of microsatellite markers and gene dosage to quantify gene copy numbers in Candida albicans.

With microsatellite marker typing, the number of alleles must be known for calculation of allelic frequencies in the diploid Candida albicans for a given locus. We describe a gene dosage with a double real-time PCR. Such a dosage should also be useful in exploring the loss of heterozygosity in C. albicans.

Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide.

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.

Are two Cryptococcus neoformans strains epidemiologically linked?

The aim of this study was to standardize a method to determine whether two strains of Cryptococcus neoformans could be considered epidemiologically linked. We hypothesized that strains isolated from the same patient were epidemiologically linked and that those isolated from different patients were unrelated. We used 17 environmental isolates and 97 clinical isolates from 31 patients diagnosed with cryptococcosis (1 to 14 isolates per patient). Using the plasmid pCnTel-1-labeled probe CENTEL, we were able to differentiate some unrelated strains that yielded the same hybridization profile with the C. neoformans middle-repetitive-element CNRE-1 probe. The genetic distances separating the strains isolated from the same patient and those separating the strains isolated from different patients were estimated, and the results obtained with the two probes were compared. Analysis of the results enabled the calculation of two Dice coefficient limits defining the zones containing the pairs of linked strains and the pairs of unrelated strains, as well as an intermediate uncertainty zone for which it was not possible to establish whether the pairs of strains were linked.

UV and X-ray sensitivity of Candida albicans laboratory strains and mutants having chromosomal alterations.

Utilization of L-sorbose, D-arabinose or primary fluconazole resistance in Candida albicans are controlled by copy number of specific chromosomes. On the other hand, spontaneous morphological mutants have a wide range of chromosomal alterations. We have investigated the UV and X-ray sensitivity of these mutants, as well as C. albicans laboratory strains. While L-sorbose utilizing mutants had normal sensitivities, a large subclass of D-arabinose utilizing mutants was abnormally sensitive to UV. Spontaneous morphological mutants responded differently, an expected result because of the heterogeneous nature of their electrophoretic karyotypes. We suggest that the differences in UV and X-ray sensitivity are due to gene imbalance caused by some chromosomal alterations. In this respect, the radiation sensitivity is similar to other features impaired by changes in chromosomes, but is unlike the acquisition of the ability to utilize alternative nutrients or the acquisition of resistance to fluconazole. Our studies also revealed that strains of C. albicans heterozygous for the mating type loci exhibited the same X-ray sensitivity as homozygous or hemizygous strains, a finding which is in contrast to the properties of Saccharomyces cerevisiae, where heterozygous strains are more resistant. This feature of C. albicans strains may be indicative of an inefficient repair system that may be related to inefficiency of mating.

Appearance and properties of L-sorbose-utilizing mutants of Candida albicans obtained on a selective plate.

This is the first report that adaptive mutagenesis can arise by chromosomal nondisjunction, a phenomenon previously associated exclusively with DNA alterations. We previously uncovered a novel regulatory mechanism in Candida albicans in which the assimilation of an alternative sugar, l-sorbose, was determined by copy number of chromosome 5, such that monosomic strains utilized l-sorbose, whereas disomic strains did not. We present evidence that this formation of monosomy of chromosome 5, which is apparently a result of nondisjunction, appeared with increased frequencies after a selective condition was applied, i.e., by adaptive mutagenesis. The rate of formation of l-sorbose-utilizing mutants per viable cell per day ranged from 10(-6) at the initial time of detection to 10(-2) after 4 days of incubation on the selective plate.

Epidemiological evidence for dormant Cryptococcus neoformans infection.

To date, the time of acquisition of a Cryptococcus neoformans infectious strain has never been studied. We selected a primer, (GACA)(4), and a probe, CNRE-1, that by randomly amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP), respectively, regrouped strains from control samples of C. neoformans var. grubii environmental isolates according to their geographical origins. The two typing techniques were then used to analyze 103 isolates from 29 patients diagnosed with cryptococcosis in France. Nine of the 29 patients lived in Africa a median of 110 months prior to moving to France; 17 of the patients originated from Europe. Results showed a statistically significant clustering of isolate subtypes from patients originating from Africa compared to those from Europe. We conclude that the patients had acquired the C. neoformans infectious strain long before their clinical diagnoses were made.

Monosomy of a specific chromosome determines L-sorbose utilization: a novel regulatory mechanism in Candida albicans.

We report the identification of the gene, SOU1, required for L-sorbose assimilation in Candida albicans. The level of the expression of SOU1 is determined by the copy number of chromosome III (also denoted chromosome 5), such that monosomic strains assimilate L-sorbose, whereas disomic strains do not, in spite of the fact that SOU1 is not on this chromosome. We suggest that C. albicans contains a resource of potentially beneficial genes that are activated by changes in chromosome number, and that this elaborate mechanism regulates the utilization of food supplies and possibly other important functions, thus representing a novel general means for regulating gene expression in microbes.

Phylogenetic relationships of fungal cytochromes c.

The CYC1 gene encoding cytochrome c in the yeast Candida albicans was cloned by complementation of a cytochrome c-deficient mutant of Saccharomyces cerevisiae, and its DNA sequence was determined. The analysis of the amino acid sequences of cytochrome c from 14 fungal species and two isoforms from S. cerevisiae revealed sequences unique to fungi, and revealed a phylogenetic relationship with a pronounced divergence between Schizosaccharomyces pombe and other ascomycetous budding yeast.

Cloning and sequencing of the beta-glucosidase-encoding gene from Candida molischiana strain 35M5N.

We have isolated a gene (bgln) encoding beta-glucosidase (beta Glu) from a cosmid library of the yeast, Candida molischiana 35M5N. The nucleotide sequence of bgln and its flanking regions was determined. This gene was found to be composed of 2289 bp and 763 amino acid (aa) residues encoding an 83.3-kDa protein. The aa sequence shared eleven putative N-glycosylation sites. Homology comparisons showed that this enzyme can be considered as a new member of the family-3 glycosyl hydrolases. Multiple alignment experiments revealed four conserved regions on aa sequences from beta Glu of this family.

Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation.

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.

A very stable beta-glucosidase from a Candida molischiana mutant strain: enzymatic properties, sequencing, and homology with other yeast beta-glucosidases.

We purified a beta-glucosidase from the mutant strain Candida molischiana 35M5N. Analysis of the kinetic properties of this enzyme did not show any differences between the previously purified wild-type enzyme and that of the mutant. Nevertheless, a study of the stability of the enzyme at different pH levels and temperatures showed the increase resistance of this protein. This enzyme was found to be stable at pH 5 for 145 h and retained 78% of its initial activity after the same time at pH 3.5 (optimal pH) and 30 degrees C. This difference between the wild-type and the mutant enzyme could be explained by differences in the quantity or quality of glycosylation. This glycoprotein showed different forms after deglycosylation. Some peptides from this protein were also sequenced. An homology analysis found similarities between this beta-glucosidase and beta-glucosidases of Candida pelliculosa and Schizophyllum commune.

Karyotype studies on different strains of Candida molischiana by pulsed-field gel electrophoresis.

We used pulsed-field gel electrophoresis to compare the electrophoretic karyotype of a Candida molischiana mutant, de-repressed for beta-glucosidase production, with a wild-type strain and a reference strain. The chromosomal organization in this mutant yeast was found to be quite different. Hybridization patterns and the relative fluorescence of all bands indicated eight chromosomes in the mutant strain and seven in the other two. All three strains seemed to be haploid, with an estimated genome size of 12 Mb; the beta-glucosidase gene was on the same chromosome in all of them and the SfiI restriction patterns of this chromosome indicated that it is not affected by the mutation.

Selection and study of a Candida molischiana mutant derepressed for beta-glucosidase production.

A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of beta-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.