Effects of Hydrocarbons on the Morphology of Candida tropicalis pK 233

Candida tropicalis pK 233 exhibited marked morphological changes depending upon carbon sources for growth. Although the yeast showed a typical yeast-like development when grown on glucose, the cells grown on hydrocarbon or ethanol were composed of a mixture of filamentous-form (F-cells) and yeast-form cells (Y-cells). The carbon chain lengths of n-alkanes tested as growth substrates had a significant influence on the ratio of F-cells to Y-cells. Electronmicroscopic observation revealed that a hypha was divided by septa into several cells.

Separation of Y-cells and F-cells was achieved by using a suitable filter cloth. F-cells gave a high Qo₂ value compared with Y-cells when hydrocarbon was used as oxidation substrate, even though there was little difference between the respiratory activities of these two cells measured with glucose.     

Morphological change in Candida tropicalis pK 233 caused by ethanol and its prevention by myo-inositol

The cells of Candida tropicalis pK 233 grew in filamentous form when cultivated in a synthetic medium supplemented with ethanol. The ethanol-grown cells excreted significant amounts of polysaccharides into culture medium. Myo-inositol added simultaneously with ethanol prevented both the morphological change and the extracellular production of polysaccharides.     

The primary structure of a peroxisomal fatty acyl-CoA oxidase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a gene encoding peroxisomal fatty acyl-CoA oxidase (AOx) from the yeast Candida tropicalis pK233. The AOx gene contains no intervening sequences and has a single open reading frame of 2127 nt encoding a protein of 708 amino acids (aa), not including the initiator methionine. The Mr of the protein is 79,155. Codon utilization in the gene is not random, with 87.4% of the aa specified by 25 principal codons. The principal codons used in the expression of AOx in C. tropicalis are similar to those used in highly expressed genes of Saccharomyces cerevisiae. The AOx protein shows a 94.2% homology with POX4 protein of C. tropicalis. One stretch of 36 aa shows no homology between the two proteins.     

cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.     

The nucleotide sequence of complementary DNA and the deduced amino acid sequence of peroxisomal catalase of the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of cDNA encoding peroxisomal catalase (Cat) from the yeast Candida tropicalis pK233. The catalase cDNA (Cat) has a single open reading frame (ORF) of 1455 nt, encoding a protein of 484 amino acids (aa), not including the initiator methionine. The Mr of the protein is 54767. Codon use in the gene is not random, with 90.9% of the aa specified by 25 principal codons. The principal codons used in the expression of Cat in C. tropicalis are similar to those used in the expression of the fatty acyl-CoA oxidase gene of C. tropicalis and of highly expressed genes in Saccharomyces cerevisiae. Cat shows 48.0%, 49.7%, and 48.3% aa identity with human, bovine, and rat catalases, respectively, and 44.3% aa identity with catalase T of S. cerevisiae. The 3 aa of bovine liver catalase previously postulated to participate in catalysis and 79.5% of those aa in the immediate environment of hemin, the prosthetic group of catalase, are conserved in Cat of C. tropicalis.     

Transglycosyl-Amylase of Candida tropicalis

Transglucosyl-amylase was purified 96-fold and partially characterized. The K_m value with dextrin as substrate was 9.1 mg/ml. Glycerol, an acceptor of d-glucose, appeared to inhibit dextrin hydrolysis noncompetitively. The energy of activation of the enzyme was 7,920 cal/mole. Indirect determinations showed that synthesis of d-glucosyl glycerol was significantly affected by the nature of the amylaceous substrate. Glucosyl-glycerol synthesis did not increase as incubation temperature was raised from 50 to 60 C. Direct determinations by gas-liquid chromatography indicated that the synthesis of glucosyl glycerol, as a function of the concentration of either enzyme, substrate, or glycerol, traced a curvilinear path approaching 15 mg/ml as the maximum. When enzyme, substrate, and glycerol at high concentrations were varied in all possible combinations, however, conditions for producing as much as 47.5 mg/ml of glucosyl glycerol were established.     

Trnasglucosyl-amylase of Candida tropicalis

Transglucosyl-amylase was purified 96-fold and partially characterized. The K(m) value with dextrin as substrate was 9.1 mg/ml. Glycerol, an acceptor of d-glucose, appeared to inhibit dextrin hydrolysis noncompetitively. The energy of activation of the enzyme was 7,920 cal/mole. Indirect determinations showed that synthesis of d-glucosyl glycerol was significantly affected by the nature of the amylaceous substrate. Glucosyl-glycerol synthesis did not increase as incubation temperature was raised from 50 to 60 C. Direct determinations by gas-liquid chromatography indicated that the synthesis of glucosyl glycerol, as a function of the concentration of either enzyme, substrate, or glycerol, traced a curvilinear path approaching 15 mg/ml as the maximum. When enzyme, substrate, and glycerol at high concentrations were varied in all possible combinations, however, conditions for producing as much as 47.5 mg/ml of glucosyl glycerol were established.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

The morphology of colony variants of three species of Candida.

The colonies of 12 isolates of 3 Candida spp. with variant colony forms were studied by scanning and transmission electron microscopy. Small colonies were formed by 4 isolates each of C. albicans and C. parapsilosis and by 1 of C. tropicalis. These had an abnormally high proportion of degenerate yeast cells with an associated increase in granular cytoplasmic material intercellularly. The increased matrix in these small colonies formed a thick superficial coat over the organisms. Rough colonies were formed by 1 isolate each of C. albicans, C. tropicalis and C. parapsilosis. The convoluted regions of these colonies contained many pseudohyphal cells but few degenerate cells and little granular or fibrillar material in their intercellular matrices. The shape of colonies of Candida spp. may be altered by variations in the viability or the morphology of the organisms.     

Phagocyte-mediated killing of Candida tropicalis

Human peripheral monocytes (MO), neutrophils (PMN), and lymphocytes (PBL) were tested for their ability to kill Candida tropicalis. With incubation times between 30 min and 2 h, unstimulated MO and PMN, but not PBL, were efficient killers of C. tropicalis. Both leukocyte subsets were able to kill at minimum 2.5 1 effector to target ratios. Pre-incubation of MO for 24 h with interferon-gamma or tumor necrosis factor (TNF) increased their ability to kill yeast targets. TNF alone had no effect on C. tropicalis targets at concentrations up to 1000 U/ml. PBL activated for 4 d with interleukin-2 did not kill yeast targets. PMN exhibited more cytocidal efficiency per cell than MO in these assays. Direct contact of effectors and targets was required; no significant killing by PMN or MO supernatants was measured. PMN-mediated killing, but not MO killing, was inhibited by a mixture of catalase and Superoxide dismutase suggesting that oxygen-dependent killing mechanisms were partially responsible for candidacidal activity.     

Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.     

Isolation of Candida tropicalis auxotrophic mutants

An enrichment scheme using nystatin was designed for the isolation of auxotrophic mutants from the diploid-alkane-utilizing yeast Candida tropicalis. A collection of 194 auxotrophs representing 7 phenotypes was isolated. One class of mutants was identified as having a defect in histidinol dehydrogenase activity and a second class of mutants was identified as having a defect in orotidine monophosphate decarboxylase activity. These strains are good candidates to be carrying mutations corresponding to the HIS4 and URA3 genes of Saccharomyces cerevisiae.