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One hundred and twenty Candida albicans clinical isolates from the late 1980s and early 1990s were examined for homozygosity at the MTL locus. Of these, 108 were heterozygous ( MTLa/ MTLα), whereas seven were MTLa and five were MTLα. Five of the homozygous isolates were able to switch to the opaque cell morphology, while opaque cells were not detectable among the remaining seven. Nevertheless, all but one of the isolates homozygous at the MTL locus were shown to mate and to yield cells containing markers from both parents; the non-mater was found to have a frameshift in the MTLα 1 gene. In contrast to Saccharomyces cerevisiae, C. albicans homozygotes with no active MTL allele failed to mate rather than mating as a cells. There was no correlation between homozygosity and fluconazole resistance, mating and fluconazole resistance or switching and fluconazole resistance, in part because most of the strains were isolated before the widespread use of this antifungal agent, and only three were in fact drug resistant. Ten of the 12 homozygotes had rearranged karyotypes involving one or more homologue of chromosomes 4, 5, 6 and 7. We suggest that karyotypic rearrangement, drug resistance and homozygosity come about as the result of induction of hyper-recombination during the infection process; hence, they tend to occur together, but each is the independent result of the same event. Furthermore, as clinical strains can mate and form tetraploids, mating and marker exchange are likely to be a significant part of the life cycle of C. albicans in vivo. [ABSTRACT FROM AUTHOR]

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis. [ABSTRACT FROM AUTHOR]

Summary Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT ) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae . We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTL a and MTL α strains, whereas disruptions in STE20 had no effect. A disruption in KEX2 , a gene involved in processing the S. cerevisiae pheromone Mfα, prevented mating in MTL α but not MTL a cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTL a but not in MTL α cells. Disruption of two cell wall genes, ALS1 and INT1 , had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1 . The results reveal that these two diverged yeasts show a surprising similarity in their mating processes. [ABSTRACT FROM AUTHOR]

Regulation of activity of acetohydroxyacid synthetase has been studied. L-Valine, one of the end-products of the combined pathway, is the only amino acid to inhibit strongly the enzyme (Ki = 5 × 10-5 M). Sensitivity to the inhibitor is rapidly lost by heating at 35°. The desensitized enzyme is stable at this temperature. In crude extracts, the enzyme is always partly desensitized, but 100% inhibition by valine is reached when the enzyme is assayed in situ, in benzene permeabilized cells. In addition, valine inhibition is more strictly dependent upon pH than activity itself. Kinetic studies of affinity for the substrate or inhibitor show no cooperative effects and valine inhibition seems truly competitive with pyruvate. Specific feedback inhibition by valine constitutes an additional proof that the acetohydroxyacid synthetase activity characterized hi studies reported in the preceding paper, is operative for valine biosynthesis. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthetase activity has been characterized, independently of the acetoin-forming system in Saccharomyces cerevisiae by a differential colorimetric assay. Maximum pH and Km values have been established for both enzymes with pyruvate as the substrate. Acetolactate formation by acetohydroxyacid synthetase has been shown directly by the separation of dinitrophenylosazones by thin-layer chromatography. Evidence for the biosynthetic role of the activity measured resides in its presence in wild-type strains, in its absence in the is2 class of mutants (isoleucine-valine auxotrophs) and in its reappearance in prototrophic revertants of these mutants. [ABSTRACT FROM AUTHOR]

A study of threonine deaminase from Saccharomyces cerevisiae has been undertaken. The enzyme has been purified 50-fold, with a low resolution of pyridoxal phosphate coenzyme. The purified enzyme is very unstable under all conditions tried so far. Properties have been studied with crude extracts. Optimum pH has been found to be 8.2. Isoleucine has complex effects on the enzyme activity. At a concentration of 0.1 mM, it is able to stimulate the enzyme. At higher concentrations, isoleucine inhibits. The Ki values increase with increasing pH, but 100% inhibition can be reached at all ranges of pH studied. Affinity of the enzyme for its substrate is even more strongly modified by pH. a) Substrate cooperativity, nil at pH 7, intermediate at pH 8, seems to be maximum at pH 9. At any pH, the substrate cooperativity can be suppressed by addition of L-valine. In addition, rather low concentrations of isoleucine also act partly as valine does. b) Apparent Km values, measured in the presence of valine, vary with pH in the opposite direction to Ki values. Hill's coefficients for substrate and inhibitor reflect these findings. For threonine alone, they vary from 1.2 at pH 7 to 3 at pH 9, In the presence of valine, they remain close to 1, at all pH. Isoleucine, 0.1 mM, produces an incomplete effect compared to valine. For isoleucine inhibition, the cooperativity coefficients remain close to 3 at all pH. The molecular weight of the enzyme has been determined to be roughly 1.2 × 105, at, pH 6.8 and at pH 8.7. Thermal inactivation is unimodal and fails to show desensitization of the enzyme in any of the conditions tried. The implications of these results on the structure of the enzyme have been discussed. [ABSTRACT FROM AUTHOR]

The virulence of Candida albicans, a major human fungal pathogen, has been considered dependent on the ability to transition between different morphologies. A new study reports a screen of C. albicans mutants that demonstrates that pathogenesis can be dissociated from morphological switching and in vitro growth rate. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented discussing the use of needle cricothyroidotomy to allow oxygenation and ventilation to the patient.