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PubMed1978

Effect of clindamycin and lincomycin therapy on faecal flora.

Bacterial counts were carried out on the faeces of 160 patients receiving clindamycin or lincomycin treatment for bacterial infections. In all the patients the total bacteroides count was significantly reduced while strains of Enterobacteriaciae, yeasts, and streptococci were correspondingly increased. Severe diarrhoea developed in 25 (16%) patients, but this could not be related to a change in faecal flora. Diarrhoea was most common when clindamycin was given prophylactically, women were more affected than men, and the incidence was highest in those aged over 60 years. No cases of pseudomembranous colitis were seen. Although clindamycin is a valuable antibiotic for treating established severe anaerobic bacterial infections it should be used cautiously in elderly patients.

Bacterial Infections↗

PubMed2003

Preparation of yeast strains displaying IgG binding domain ZZ and enhanced green fluorescent protein for novel antigen detection systems.

To develop novel immunofluorescence-labeling and antigen-detection systems, the ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G, and enhanced green fluorescent protein (EGFP) were displayed on the cell surface of Saccharomyces cerevisiae by cell-surface engineering using the C-terminus 318 amino acids of Flo1 protein. Two systems were constructed, one for co-display of ZZ and EGFP, and one for display of a fusion protein of the two. In both cases, two proteins on the cell surface successfully retain their activities.

Journal Article↗

PubMedDate not supplied

Characterization of absorption and scattering properties for various yeast strains by time-resolved spectroscopy.

An understanding of the optical properties of biological media and cells is essential to the development of noninvasive optical studies of tissues. Unicellular organisms offer a unique opportunity to investigate the factors affecting light propagation, since they can be manipulated in ways impossible for more complex biological samples. In this study, we examined optical absorption and scattering properties of strongly multiple scattering yeast suspensions by means of near-infrared (NIR) time-resolved spectroscopy (TRS) and a sample substitution method. We determined the critical parameters for photon migration by varying the cell organelle content, the cell ploidy, the cell size, and the concentration of suspended cells. The results indicate that the photon absorption is insensitive to cell differentiation and that the cell volume is the primary factor determining light-scattering property.

Cell Count↗

PubMed1988

Hepatitis B vaccine produced in yeast.

A gene encoding the 226 amino acid hepatitis B surface antigen (HBsAg), subtype adw, was cloned into a generalized vector for the expression of heterologous genes in Saccharomyces cerevisiae. The 5' end of the genomic HBsAg gene was replaced with a chemically synthesized DNA segment that conserved the amino acid sequence of the protein but utilized DNA sequences that optimize translation initiation in yeast. High-cell-density fermentations of laboratory strains of Saccharomyces cerevisiae have been developed in which HBsAg production increases linearly with respect to cell mass. The HBsAg is present as a lipoprotein particle in cell lysates and has been purified to homogeneity. The evidence presented indicates that the HBsAg particles may be formed during lysis of the yeast cells. The purified HBsAg particles have a morphology similar to that of the 22 nm particles present in the serum of human chronic carriers of hepatitis B. The reactivity of the yeast-derived HBsAg particles with a series of monoclonal antibodies is essentially identical to that of human plasma HBsAg. By this analysis, therefore, the structure of the HBsAg protein is similar in yeast and in human particles. The purified yeast HBsAg particles were formulated with alum adjuvant and subsequently were shown to confer immunity in chimpanzees to challenge with two heterologous serotypes (adr, ayw) of hepatitis B virus.

Animals↗

PubMed1996

SCD5, a suppressor of clathrin deficiency, encodes a novel protein with a late secretory function in yeast.

Clathrin and its associated proteins constitute a major class of coat proteins involved in vesicle budding during membrane transport. An interesting characteristic of the yeast clathrin heavy chain gene (CHC1) is that in some strains a CHC1 deletion is lethal, while in others it is not. Recently, our laboratory developed a screen that identified five multicopy suppressors that can rescue lethal strains of clathrin heavy chain-deficient yeast (Chc - scd1-i) to viability. One of these suppressors, SCD5, encodes a novel protein of 872 amino acids containing two regions of repeated motifs of unknown function. Deletion of SCD5 has shown that it is essential for cell growth at 30 degrees C. scd5-delta strains carrying low copy plasmids encoding C-terminal truncations of Scd5p are temperature sensitive for growth at 37 degrees C. At the nonpermissive temperature, cells expressing a 338-amino acid deletion (Scd5P-delta 338) accumulate an internal pool of fully glycosylated invertase and mature alpha-factor, while processing and sorting of the vacuolar hydrolase carboxypeptidase Y is normal. The truncation mutant also accumulates 80- to 100-nm vesicles similar to many late sec mutants. Moreover, at 34 degrees C, overexpression of Scd5p suppresses the temperature sensitivity of a sec2 mutant, which is blocked at a post-Golgi step of the secretory pathway. Biochemical analyses indicate that approximately 50% of Scd5p sediments with a 100,000 x g membrane fraction and is associated as a peripheral membrane protein. Overall, these results indicate that Scd5p is involved in vesicular transport at a late stage of the secretory pathway. Furthermore, this suggests that the lethality of clathrin-deficient yeast can be rescued by modulation of vesicular transport at this late secretory step.

Amino Acid Sequence↗

PubMed1982

Sexual development in a homothallic fission yeast: synthesis of readiness proteins resolved by gel electrophoresis.

Sexual development of a homothallic strain of Schizosaccharomyces pombe was monitored by radiolabelling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Of more than 60 bands detected by Coomassie brilliant blue and by autoradiography, about 30 bands synthesized during development were discrete enough for experimental analysis. About a dozen bands are preferentially vegetative, another dozen preferentially developmental. However, vegetative bands as a group are also synthesized during development. Their synthesis is relatively unaffected by low concentrations of cycloheximide or by chloramphenicol and is not temperature sensitive at 37 degrees C nor catabolite repressible. Only band 40 (ca. 40 000 daltons) seems to be exclusively vegetative. The synthesis of developmental bands 13, 18, 24, 30, and alpha, all of which first appear during late-log phase, is catabolite repressible. Developmental band 51 is also synthesized throughout the vegetative phase. The synthesis of bands 24, 30, 51, and alpha is temperature sensitive at 37 degrees C during the development, but that of band 18 is not. The synthesis of band 13 during development is not temperature sensitive, but its earlier synthesis during late-log phase is. The synthesis of all these six developmental bands is immediately inhibited by cycloheximide, but not by chloramphenicol. Their appearance as a group of radioactive bands is greatly diminished in cultures grown in cycloheximide, in chloramphenicol, or in ethidium bromide. Developmental bands 13, 18, 24, and 30 may be called readiness proteins. They first appear prior to the earliest morphological signs of sexual activity. Their developmental synthesis is inhibited by conditions that inhibit sexual development. Such inhibitory conditions include anaerobiosis, restrictive temperature, aging in stationary phase, the presence of inhibitors of cytoplasmic protein synthesis and of mitochondrial function, and catabolite repression. Readiness proteins may be regulating the switch from vegetative metabolism.

Ascomycota↗

PubMed2005

CARE-2 fingerprinting of Candida albicans isolates.

The emergence of resistance to antifungal drugs in medically important fungi has become a significant problem in recent years. Probably the best-studied example is the development of resistance to the widely used antifungal agent fluconazole in the yeast Candida albicans. The availability of matched series of clinical isolates representing the same strain in which drug resistance developed over time has provided opportunities to detect cellular alterations that are correlated with drug resistance. We describe a method for DNA fingerprinting of C. albicans isolates based on Southern hybridization of genomic DNA with the C. albicans-specific repetitive DNA element CARE-2. Molecular typing with CARE-2 permits highly reliable discrimination of unrelated strains to ascertain that serial isolates recovered from individual patients indeed represent the same C. albicans strain.

Antifungal Agents↗

PubMed1988

A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae.

A simple assay called the sectoring shuffle was developed to monitor the mutational state of essential genes cloned into yeast centromeric plasmids. The essence of this assay is the creation of a conditional phenotype, colony color sectoring, for an essential gene in the absence of conditional thermosensitive or cold-sensitive alleles of that gene. This allows the quick determination of the mutational state of a cloned essential gene by observing its effect on the sectoring phenotype of the tester strain. During the course of this work we developed a family of 20 Escherichia coli-yeast shuttle vectors, pUN plasmids, containing ARS1 CEN4 and a variety of selectable markers as well as the SUP11 gene which can act as a color marker in the proper background. These vectors are compact and have been very useful for the sectoring-shuffle assay and for gene analysis in general. This paper describes these vectors, the sectoring shuffle and several applications of sectoring phenotypes.

Centromere↗

PubMedDate not supplied

Susceptibility of yeast isolates from defined German patient groups to 5-fluorocytosine.

Susceptibility and development of resistance to 5-fluorocytosine (5-FC) in Candida strains isolated from defined German groups of probands was investigated. 5-FC susceptibility was determined in a microdilution assay in yeast nitrogen base after 24 h of incubation at 37 degrees C. The range of 5-FC concentration investigated was between 0.015 and 16 micrograms ml-1. Isolates with a minimum inhibitory concentration (MIC) of > 16 micrograms ml-1 were regarded as 5-FC resistant. In total 335 Candida isolates were investigated, and 20 of them (6.0%) were found to be resistant. The Candida isolates were rather different with respect to their origin: out of 57 vaginal isolates from non-risk patients from Southern Germany 3.5% were 5-FC-resistant strains. One hundred and fifty-nine isolates from the urine of long-term intensive care patients from the whole of Germany showed 5-FC resistance (6.3%). Out of 74 isolates of different localization from intensive care patients of the University Clinics of Freiburg, 10.8% showed 5-FC resistance. Among 45 isolates from the oral cavity of HIV-positive patients from the Frankfurt region, no 5-FC-resistant strain was found. The epidemiology of 5-FC resistance is based mainly on the percentage of non-albicans isolates of the proband groups (C. tropicalis, C. krusei and others), and is less based on the frequency of Candida albicans serotype B isolates. In sequential observations with individual intensive care patients, no increase of 5-FC resistance in their Candida isolates could be observed with longer periods of hospitalization.

Candida↗

PubMed2006

Biodegradation of oil tank bottom sludge using microbial consortia.

We present a rationale for the selection of a microbial consortia specifically adapted to degrade toxic components of oil refinery tank bottom sludge (OTBS). Sources such as polluted soils, petrochemical waste, sludge from refinery-wastewater plants, and others were used to obtain a collection of eight microorganisms, which were individually tested and characterized to analyze their degradative capabilities on different hydrocarbon families. After initial experiments using mixtures of these strains, we developed a consortium consisting of four microorganisms (three bacteria and one yeast) selected in the basis of their cometabolic effects, emulsification properties, colonization of oil components, and degradative capabilities. Although the specific contribution each of the former parameters makes is not clearly understood, the activity of the four-member consortium had a strong impact not only on linear alkane degradation (100%), but also on the degradation of cycloalkanes (85%), branched alkanes (44%), and aromatic and sulphur-aromatic compounds (31-55%). The effectiveness of this consortium was significantly superior to that obtained by individual strains, commercial inocula or an undefined mixture of culturable and non-culturable microorganisms obtained from OTBS-polluted soil. However, results were similar when another consortium of four microorganisms, previously isolated in the same OTBS-polluted soil, was assayed.

Bacteria↗

PubMed1980

Strain variation and morphogenesis of yeast- and mycelial-phase Candida albicans in low-sulfate, synthetic medium.

A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.

Amino Acids↗

PubMed1983

Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding.

Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-sister crosses. Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced. To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.

Ascomycota↗

PubMed2005

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal.

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.

Agriculture↗

PubMed2000

Transformation of Endomyces fibuliger based on its gene for orotidine-5'-phosphate decarboxylase.

Endomyces fibuliger is a yeast used in the production of Chinese rice wine. It secretes enzymes such as glucoamylase, alpha-amylase and acid protease. Very little is known of the genetics of E. fibuliger. In order to develop a transformation system for this yeast, orotidine-5'-phosphate decarboxylase mutant strains were obtained and characterized. Transformation of the E. fibuliger ura3 mutant F1 with an integrative plasmid that carried the wild-type URA3 gene of E. fibuliger gave complementation of this mutation. The E. fibuliger gene encodes the orotidine-5'-phosphate decarboxylase enzyme consisting of 266 amino acid residues with a 69.4% sequence identity with orotidine-5'-phosphate decarboxylase of Saccharomyces cerevisiae. Our finding that E. fibuliger URA3 complements the ura3 mutation in S. cerevisiae confirms that the URA3 gene of E. fibuliger encodes a protein that exerts a similar function.

Amino Acid Sequence↗

PubMed2000

Access to Wieland-Miescher ketone in an enantiomerically pure form by a kinetic resolution with yeast-mediated reduction.

Both enantiomers of Wieland-Miescher ketone [3,4,8, 8a-tetrahydro-8a-methyl-1,6(2H,7H)-naphthalenedione], in a highly enantiomerically enriched form, became readily available by a newly developed kinetic resolution with yeast-mediated reduction. From a screening of yeast strains, Torulaspora delbrueckii IFO 10921 was selected. The collected cells of this strain, obtained by an incubation in a glucose medium, smoothly reduced only the isolated carbonyl group of the (S)-enantiomer, while the (R)-enantiomer remained intact. Starting from both enantiomers ( approximately 70% ee) prepared by an established proline-mediated asymmetric Robinson annulation, the reduction with T. delbrueckii gave the (R)-enantiomer (98% ee) and the corresponding alcohol (4aS,5S)-4,4a, 5,6,7,8-hexahydro-5-hydroxy-4a-methyl-2(3H)-naphthalenone (94% ee, 94% de) in preparative scale in nearly quantitative yields. An approach for the asymmetric synthesis of the Wieland-Miescher ketone was also successful. 2-Methyl-2-(3-oxobutyl)-1,3-cyclohexanedione, the prochiral precursor, was reduced with this strain to give a cyclic acetal form of (2S, 3S)-3-hydroxy-2-methyl-2-(3-oxobutyl)cyclohexanone, in a stereomerically pure form.

Ketones↗

PubMed1995

High yield expression of functionally active human liver CYP2D6 in yeast cells.

In order to develop a model system for studying drug metabolism, we constructed recombinant yeast strains expressing human liver cytochromes P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimization of the initiation ATG codon context. The PCR-based site-mutagenesis method was used to introduce an AAA sequence immediately before the initiation codon resulting in increased translation of the GAL10-CYC1-derived mRNA. The use of a peptidase-deficient yeast strain also helped to increase the CYP2D6 content. A P450 content of 250 +/- 30 pmol per mg of microsomal protein was achieved. HPLC analysis confirmed that heterologously expressed CYP2D6 catalysed the oxidation of debrisoquine and dextromethorphan, two prototype substrates for CYP2D6. The Km for debrisoquine 4-hydroxylase was found to be 50 microM and Vmax 7.5 pmol mg-1 min-1. Dextromethorphan O-demethylase activity in CYP2D6-containing microsomes was characterized by Km 8.5 microM and Vmax 700 pmol mg-1 min-1. Biotransformation of debrisoquine and dextromethorphan was not detected in control microsomes. Yeast synthesizing CYP2D6 represents a useful in vitro system for studying xenobiotic metabolism.

Base Sequence↗

PubMed1996

Genetic characterization of transactivation of the human T-cell leukemia virus type 1 promoter: Binding of Tax to Tax-responsive element 1 is mediated by the cyclic AMP-responsive members of the CREB/ATF family of transcription factors.

To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element-binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax.

Activating Transcription Factors↗

PubMed1993

Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae. Application to the selection of strains containing novel forms of cytochrome-c oxidase.

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain. Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Cytochromes↗