Molecular and Epidemiological Characterization of Vaginal Saccharomyces cerevisiae Isolates

Although vaginitis caused by Saccharomyces cerevisiae is extremely rare, in recent years we have experienced an increasing frequency of S. cerevisiae isolation from the vaginas of fertile-age women. In order to investigate the epidemiology of these vaginal infections, a total of 40 isolates of S. cerevisiae derived from symptomatic and asymptomatic women were characterized by two DNA typing approaches, named ribosomal DNA (rDNA) hybridization and Ty917 hybridization, based on the Southern blotting technique. After transfer, the polymorphic DNA restriction fragments were hybridized with the entire repeat of S. cerevisiae rDNA for one method and with the entire sequence of the Ty917 retrotransposon for the other. After elaboration with computer-assisted analysis, the results of each method showed that Ty917 hybridization is endowed with a discriminatory power higher than that of rDNA hybridization. With the Ty917 hybridization method, all of the S. cerevisiae isolates tested appeared very heterogeneous, with the exception of those collected from individual patients with recurrent vaginitis. This allowed us to exclude a possible common source of infection while the high relatedness among S. cerevisiae sequential isolates from recurrent-vaginitis patients could suggest a pattern of relapse rather than frequent reinfection.     

Investigation of Batten disease with the yeast Saccharomyces cerevisiae

The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

Phenotypic and Genotypic Analysis of Clostridium difficile Isolates: a Single-Center Study

Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution''s C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level.     

Formation of circular amplifications in Saccharomyces cerevisiae by a breakage-fusion-bridge mechanism

Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge.     

A new killer toxin produced by Saccharomyces cerevisiae

A wine-making Saccharomyces cerevisiae yeast strain isolated in our laboratory produces two different killer toxins, each one encoded by one dsRNA plasmid. One toxin has the same specificity as the one produced by strain M437 described by Naumov, but the dsRNA plasmid which encodes it migrates slightly faster in poly acrylamide gel electrophoresis. The other toxin has not been previously described, and is encoded by a dsRNA fraction which migrates at a lower rate than the × fraction of M437. These two dsRNA plasmids can be maintained separately in different yeast strains.     

Uptake of DNA by fragile mutants of Saccharomyces cerevisiae

Summary When Saccharomyces cerevisiae SY15 rho° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60–70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 g/ml DNA and 10 mM CaCl₂. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells.     

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP

Summary Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5 monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup ^-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.     

Depression by miconazole of heat-induced respiratory-deficiency in Saccharomyces cerevisiae

Miconazole, at 0.2 μM , decreased, by two orders of magnitude, the specific mutation rate of Saccharomyces cerevisiae to respiratory-deficient mutants (petites), which had been induced at either 37 °C or 39 °C. Identical concentrations of ketoconazole did not change the mutation rate. The results fit in the mechanisms of action which have been proposed for imidazole antimycotics, and constitute further support for the hypothesis that the targets of thermal death, in petite-positive associately-profiled yeasts, lie in the mitochondria.     

Some aspects of the phenylacetylcarbinol biosynthesis by Saccharomyces cerevisiae

The biosynthesis of phenylacetylcarbinol (PAC) by Saccharomyces cerevisiae in media containing benzaldehyde and fermentable sugars was studied under different experimental conditions. In shaking cultures with small volumes of medium a sigmoidal production curve for PAC was obtained. During the first and second part of the fermentation period (5 to 10 hours), the number of living yeast cells increased. In the third period (from the 10th to the 20th hour), the number of living yeast cells decreased rapidly. In agitated batch cultures the PAC produced during the first 5 hours of fermentation decreased only slowly on further incubation. In agitated but non aerated batch cultures, the PAC production increased rapidly, but the yields were much lower than in aerated batches. Acetone dried cells of baker's yeast produced PAC from benzaldehyde but no benzylalcohol. Besides PAC and acetylbenzoyl, a new biosynthetic product, trans-cinnam-aldehyde was determined.     

Oxidation of naphthalene by Saccharomyces cerevisiae and Candida utilis

The yeasts Saccharomyces cerevisiae 118 and Candida utilis 128 were examined for their ability to metabolize the polycyclic aromatic hydrocarbons naphthalene and anthracene. The two yeasts tested oxidized these aromatics. Metabolites were extracted and analyzed by thin layer chromatography and gas chromatography. The predominant oxidation product of naphthalene found in the culture medium was 1-naphthol.     

Non-selective transformation of Saccharomyces cerevisiae

Summary Wild or industrial yeast strains cannot be transformed by most selective vectors due to a lack of auxotrophic mutations. To enable identification of transformants of such yeast species, we have developed a 2-µm DNA vector with an indicator gene that can be used without any additional marker. The Escherichia coli gene for -lactamase (bla) was placed under the control of the yeast promoter for the structural gene encoding ADHI. This increased the amount of -lactamase produced in Saccharomyces cerevisiae 100-fold giving an enzyme activity in transformant colonies which is high enough to be detected directly on indicator plates. Non-selectively, the transformation frequency is even higher than under selective conditions indicating that selection does not assist the establishment of new plasmids. Transformants isolated non-selectively were found to retain the endogenous 2-µm DNA. Under control of appropriate promoters, the bacterial bla gene may also provide a convenient marker for other eukaryotic transformation systems.     

Instability of Saccharomyces cerevisiae heterokaryons

Summary We have constructed heterokaryons of Saccharomyces cerevisiae by crossing kar1 — mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from kar1 — crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable.This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability.     

Flow microcalorimetry of a respiration-deficient mutant of Saccharomyces cerevisiae

In aerobic batch cultures in mineral medium with glucose of a respiration-deficient mutant of Saccharomyces cerevisiae, growth parameters were estimated and the heat evolved was measured by a flow microcalorimeter. A growth enthalpy of – 163.6 joule per mole of glucose consumed was measured. Under anaerobic conditions, the value was – 134.6 joule, closer to the expected for alcoholic fermentation alone. The difference was found to be due to cyanide-resistant respiration under aerobic conditions.     

Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases

Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition. Received: 25 January / 15 March 1999     

Phenotypic and Genotypic Characterization of Mycobacterium africanum Isolates from West Africa

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. Most clinical isolates are M. tuberculosis or M. bovis. These species can be distinguished by phenotypes and genotypes. However, there is no simple definition of M. africanum, and some authors question the validity of this species. We analyzed 17 human isolates from Sierra Leone, identified as M. africanum by biochemical and growth characteristics. We sequenced polymorphic genes and intergenic regions. We amplified DNA from six loci with variable numbers of tandem repeats (VNTRs) and determined the exact number of repeats at each locus in each strain. All M. africanum isolates had the ancestral CTG Leu at katG codon 463. Drug-resistant M. africanum isolates had katG and rpoB mutations similar to those found in drug-resistant M. bovis and M. tuberculosis. Fourteen Sierra Leone M. africanum isolates (designated group A) had katG codon 203 ACC Thr, also found in M. africanumT (the T indicates type strain) from Senegal. Group A isolates clustered with M. africanumT by VNTR analysis. Three M. africanum isolates (group B) had katG codon 203 ACT Thr, found in M. tuberculosisT, and clustered with M. tuberculosisT by VNTR analysis. Phenotypic identification of M. africanum yielded a heterogeneous collection of strains. Genotypic analyses identified a cluster (M. africanum group A) which included M. africanumT and was distinct from the rest of the M. tuberculosis complex. Future studies of M. africanum should include both phenotypic and genotypic analyses.     

Genetic transfer mediated by isolated nuclei in Saccharomyces cerevisiae

Summary Viable hybrids of Saccharomyces cerevisiae were obtained by transfer of isolated diploid nuclei into haploid protoplasts using a polyethylene glycol (PEG) fusion procedure.     

A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation

Summary We present a simplified and rapid method for the transformation of yeast cells by electroporation. Stationary cells, scraped off the agar of Petri dish cultures stored in the refrigerator for up to 6 weeks, are suspended in sorbitol buffer, spun down by gentle centrifugation, transferred into the electroporation cuvette, and immediately subjected to transformation via electroporation. Transformation efficiency of this 10-min method, which does not require the preparation of cell cultures, is about 10% of the hitherto best performing transformation procedure using cells of defined growth phase.