Loss and fragmentation of chromosome 5 are major events linked to the adaptation of rad52-DeltaDelta strains of Candida albicans to sorbose.

Candida albicans can adapt and grow on sorbose plates by losing one copy of Chr5. Since rad52 mutants of Saccharomyces cerevisiae lose chromosomes at a high rate, we have investigated the ability of C. albicans rad52 to adapt to sorbose. Carad52-DeltaDelta mutants generate Sou(+) strains earlier than wild-type but the final yield is lower, probably because they die at a higher rate in sorbose. As other strains of C. albicans, CAF2 and rad52-DeltaDelta derivatives generate Sou(+) strains by a loss of one copy of Chr5 about 75% of the time. In addition, rad52 strains were able to produce Sou(+) strains by a fragmentation/deletion event in one copy of Chr5, consisting of loss of a region adjacent to the right telomere. Finally, both CAF2 and rad52-DeltaDelta produced Sou(+) strains with two apparent full copies of Chr5, suggesting that additional genomic changes may also regulate adaptation to sorbose.

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.

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.

Specific chromosome alterations in fluconazole-resistant mutants of Candida albicans.

The exposure of Candida albicans to fluconazole resulted in the nondisjunction of two specific chromosomes in 17 drug-resistant mutants, each obtained by an independent mutational event. The chromosomal changes occurred at high frequencies and were related to the duration of the drug exposure. The loss of one homologue of chromosome 4 occurred after incubation on a fluconazole medium for 7 days. A second change, the gain of one copy of chromosome 3, was observed after exposure for 35 or 40 days. We found that the mRNA levels of ERG11, CDR1, CDR2, and MDR1, the candidate fluconazole resistance genes, remained either the same or were diminished. The lack of overexpression of putative drug pumps or the drug target indicated that some other mechanism(s) may be operating. The fluconazole resistance phenotype, electrophoretic karyotypes, and transcript levels of mutants were stable after growth for 112 generations in the absence of fluconazole. This is the first report to demonstrate that resistance to fluconazole can be dependent on chromosomal nondisjunction. Furthermore, we suggest that a low-level resistance to fluconazole arising during the early stages of clinical treatment may occur by this mechanism. These results support our earlier hypothesis that changes in C. albicans chromosome number is a common means to control a resource of potentially beneficial genes that are related to important cellular functions.

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.

Variation in assimilating functions occurs in spontaneous Candida albicans mutants having chromosomal alterations.

In this study, four clinical isolates and over 100 colony morphology mutants, previously derived spontaneously from strain 3153A during growth on glucose medium, were examined for their utilization of 21 carbon and 3 nitrogen sources at various growth temperatures. The results demonstrated extensive variability in the pattern of assimilation among the mutants and strains, including both the gain and loss of assimilating functions. The persistent alterations in assimilation patterns observed in sequentially produced subclones illustrated an extensive ability of C. albicans populations to constantly produce new combinations of assimilating functions. The variability among spontaneous mutants derived from a single strain explains the well documented variability among natural isolates. From these results we established a relationship between the previously documented broad spectrum of spontaneous chromosomal aberrations in these mutants to the expression of genes controlling the utilization of alternative carbon and nitrogen sources. The existence of cryptic genes, responsible for growth on alternative substrates, was previously deduced from the analysis of other mutants obtained as a response to the restrictive condition on media containing non-assimilating carbon sources. Thus, mutants with altered assimilation functions can arise either on glucose medium or by selection on restricted media. Extensive differences between the patterns of chromosomal aberrations and the distribution of correlated phenotypes in the two groups of mutants indicated that the same phenotypes may be produced by two different mechanisms involving the same or different genes.

Physical constitution of ribosomal genes in common strains of Saccharomyces cerevisiae.

Several recent investigations, employing restriction endonucleases that do not cleave within rDNA units, revealed that a number of laboratory strains of Saccharomyces cerevisiae apparently contains a single tandem array of approximately 50 to 200 rDNA units on each chromosome XII homolog. The number of these rDNA units varies from strain to strain, among subclones of the same strain, and after different conditions of growth. In contrast, the commonly-used strain S288C and its derivatives contain two clusters on each chromosome XII homolog. Although the two clusters are stably maintained, the number of rDNA units within each cluster can vary as in strains with single clusters.

Chromosomal alterations of Candida albicans are associated with the gain and loss of assimilating functions.

We have demonstrated that a normal laboratory strain of Candida albicans spontaneously produces mutants which acquire the ability to assimilate certain carbon sources that are not utilized by the parental strain. The examination of mutants acquiring the ability to utilize either sorbose or D-arabinose revealed a few additional phenotypic changes, including the gain and loss of the capacity to assimilate other carbon sources. The change of assimilation patterns resembled the polymorphic variation of assimilation patterns found among different wild-type strains of C. albicans. Most importantly, these sorbose- and D-arabinose-positive mutants were associated with chromosomal rearrangements, with each class of positive mutants having alterations of specific chromosomes. These findings demonstrated for the first time that chromosomal alterations in C. albicans are involved in genetic variation of fundamental functions of this asexual microorganism.

Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae.

Naturally occurring strains of Candida albicans are opportunistic pathogens that lack a sexual cycle and that are usually diploids with eight pairs of chromosomes. C. albicans spontaneously gives rise to a high frequency of colonial morphology mutants with altered electrophoretic karyotypes, involving one or more of their chromosomes. However, the most frequent changes involve chromosome VIII, which contains the genes coding for ribosomal DNA (rDNA) units. We have used restriction fragment lengths to analyze the number and physical array of the rDNA units on chromosome VIII in four normal clinical strains and seven morphological mutants derived spontaneously from one of the clinical isolates. HindIII does not cleave the rDNA repeats and liberates the tandem rDNA cluster from each homolog of chromosome VIII as a single fragment, whereas the cleavage at a single site by NotI reveals the size of the single rDNA unit. All clinical strains and morphological mutants differed greatly in the number of rDNA units per cluster and per cell. The four clinical isolates differed additionally among themselves by the size of the single rDNA unit. For a total of 25 chromosome VIII homologs in a total of 11 strains considered, the variability of chromosome VIII was exclusively due to the length of rDNA clusters (or the number of rDNA units) in approximately 92% of the cases, whereas the others involved other rearrangements of chromosome VIII. Only slight variations in the number of rDNA units were observed among 10 random C. albicans subclones and 10 random Saccharomyces cerevisiae subclones grown for a prolonged time at 22 degrees C. However, when grown faster at optimal temperatures of 37 and 30 degrees C, respectively, both fungi accumulated higher numbers of rDNA units, suggesting that this condition is selected for in rapidly growing cells. The morphological mutants, in comparison with the C. albicans subclones, contained a markedly wider distribution of the number of rDNA units, suggesting that a distinct process may be involved in altering the number of rDNA units in these mutants.