Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Biochemical studies carried out on different groups ofCandida parapsilosis andCandida rhagii strains by comparing some cellular and mitochondrial activities

A comparative biochemical study was performed on some strains ofCandida rhagii and on strains belonging to different subgroups ofCandida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within theC. parapsilosis species.     

Ultrastructure of chitin in hyphae ofCandida albicans and other dimorphic and mycelial fungi

Summary Chitin microfibrils exposed by chemical extraction of hyphal walls ofCandida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus andMucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the mouldsC. cinereus andM. mucedo but were similar to those reported for the yeastSaccharomyces cerevisiae. InC. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthasein vitro were very much longer than any observed from hyphal preparations.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Immunochemical studies on mannans of the genusSaccharomyces

Antigenic mannans isolated from the cells ofSaccharomyces fermentati, Saccharomyces rosei,Saccharomyces delbrueckii, Torulopsis colliculosa, Candida albicans andSaccharomyces cerevisiae were examined for their reactivity withSaccharomyces fermentati andCandida albicans antisera. Mannans ofTorulaspora as well asCandida albicans showed high cross-reactivity with the investigated antisera, which could be due to the presence of long side chains established by the partial acetolysis method. The low specific rotations ofSaccharomyces fermentati, Saccharomyces rosei andTorulopsis colliculosa mannans indicate a predominance of β-glycosidio linkages, whereasSaccharomyces delbrueckii andCandida albicans mannans possess predominantly α-linkages.Saccharomyces cerevisiae mannan showed different structural and immunological properties.     

Rapid and extensive vacuolation of the budding yeastsSaccharomyces cerevisiae andCandida albicans by amphotericin B

The effect of amphotericin B (AMPH) on vacuolation in the budding yeastsSaccharomyces cerevisiae andCandida albicans was studied. The minimum inhibitory concentration of AMPH for growth ofS. cerevisiae andC. albicans was 1 µg/ml. In untreated control cultures, mature cells had large central vacuoles in the exponential phase, which hampered the detection of vacuolation effect. Small buds in untreated exponential phase cells, however, only rarely showed vacuoles under the light microscope. Treatment with 0.2 µg/ml of AMPH for 20–30 min induced extensive vacuolation not only in mothers but also buds ofS. cerevisiae. Extensive vacuolation lasted 4 h or more, and growth rate of the cells was much reduced for 8 h or more. Vacuolation itself was not fatal: on removal of the drug most cells gradually recovered from vacuolation and eventually multiplied. A similar effect of AMPH was also observed inC. albicans but at a higher concentration (0.5 µg/ml).     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

A possible application of ale brewery strains ofSaccharomyces cerevisiae in lager beer production

The physiological characteristics of two strains of brewery ale yeasts,Saccharomyces cerevisiae, with sedimentation abilities, were investigated to see if the strains were suitable for lager beer production. Compared with typical industrial ale strains ofS. cerevisiae and lager strains ofS. uvarum (nowS. cerevisiae), the investigated strains differ in fermentation dynamics, as well as in biological properties. The differences, however, particularly between the two strains and the lager brewing yeasts, were not significant.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis,Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.     

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

Analytical differentiation of wine fermentations using pure and mixed yeast cultures

Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Cloning of theHIS3 gene ofYarrowia lipolytica

TheHIS3 gene of the yeastYarrowia lipolytica has been cloned from a genomic library by complementation of thehis3 mutation ofSaccharomyces cerevisiae. The gene was subsequently subcloned inEscherichia coli and characterized by restriction enzyme mapping.     

Electrophoretic karyotyping and antifungal susceptibility patterns of Candida parapsilosis clinical isolates causing deep and superficial fungal infections

Forty-six isolates of Candida parapsilosis, each from a single patient, were collected from July 1993 through March 1999 at the University of Ancona Hospitals and Clinics. Twenty-eight strains were isolated from superficial lesioned sites, including skin, nails and other sources while 18 strains were isolated from blood. The isolates were typed by electrophoretickaryotyping (EK) and tested for their susceptibility to fluconazole (FLC),itraconazole (ITC), flucytosine (5-FC), and amphotericin B (AMB). Ourdata confirmed that EK is a useful technique for DNA typing of isolates ofCandida parapsilosis and showed that the source of isolation is notassociated with a given DNA type. Although strains belonging to this speciesof Candida are susceptible to the most common antifungals, including the triazoles, the degree of ITC susceptibility was dose dependent (MIC rangingfrom 0.25–0.5 μg/ml) for 98% of the isolates. This revised version was published online in June 2006 with corrections to the Cover Date.     

An effective automated glucose sensor for fermentation monitoring and control

An industrial glucose analyser was partnered to an automated injection system to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures ofSchizosaccharomyces pombe and continuous and fed-batch cultures ofSaccharomyces cerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

Use of starter cultures containingStreptococcus diacetilactis,Lactobacillus brevis andSaccharomyces cerevisiae for fermenting milk for production of Nigeriannono

Starter cultures consisting ofStreptococcus diacetilactis, Strept. cremoris, Lactobacillus brevis andSaccharomyces cerevisiae were tested singly and in mixtures for ability to ferment milk to producenono with organoleptically acceptable qualities. Only mixed cultures containing eitherStrept. diacetilactis orStrept. cremoris andL. brevis were suitable. Presence of yeast adversely affected either acid formation or diacetyl production.Nono containingStrept. diacetilactis was acceptable, even in the presence ofSacch. cerevisiae, because of the high diacetyl production. A mixed starter containing two of these organisms,Strept. diacetilactis orStrept. cremoris andL. brevis, is recommended fornono production.Sacch. cerevisiae is not essential.R. N. Okagbue and M. O. Bankole were formerly with the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.