ABSTRACT
Methods for isolating genomic DNA from yeasts are optimized for strains of Saccharomyces cerevisiae. The DNeasy tissue kit proved to be effective with 65 additional yeast species, providing 0.1 to 4.7 microg DNA/ml culture with sufficient purity to give reproducible amplified fragment length polymorphism (AFLP) profiles, but was unsuccessful with 13 other species. Two alternative yeast DNA purification kits, MasterPure and Y-DER, were effective with 6 of these and 2 additional species, leaving only 9 species that remained recalcitrant to yielding sufficient amounts of DNA with the required purity.
Subject(s)
DNA, Fungal/isolation & purification , Genome, Fungal/genetics , Molecular Biology/methods , Polymorphism, Restriction Fragment Length , Saccharomyces cerevisiae/genetics , Fluorescence , Reagent Kits, Diagnostic , Reproducibility of Results , Species SpecificityABSTRACT
Ten medically important Saccharomyces strains, comprising six clinical isolates of Saccharomyces cerevisiae and four probiotic strains of Saccharomyces boulardii, were characterized at the genetic and metabolic level and compared with non-medical, commercial yeast strains used in baking and wine-making. Strains were compared by genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis, by ribosomal DNA ITS1 sequencing and by metabolic footprinting using both direct injection mass spectrometry (DIMS) and gas chromatography-time of flight-mass spectrometry (GC-ToF-MS). Overall, the clinical isolates fell into different groupings when compared with the non-medical strains, with good but not perfect correlation amongst strains at both the genetic and metabolic levels. Probiotic strains of S. boulardii that are used therapeutically to treat human gastro-intestinal tract disorders showed tight clustering both genetically and metabolically. Metabolomics was found to be of value both as a taxonomic tool and as a means to investigate anomalous links between genotype and phenotype. Key discriminatory metabolites were identified when comparing the three main groups of clinical, probiotic and non-medical strains and included molecules such as trehalose, myo-inositol, lactic acid, fumaric acid and glycerol 3-phosphate. This study confirmed the link between a subset of clinical isolates and baking or probiotic strains but also highlighted that in general the clinical strains were more diverse at both the genomic and metabolic levels.
Subject(s)
DNA, Fungal/analysis , Metabolic Networks and Pathways , Probiotics , DNA, Ribosomal Spacer/analysis , Gas Chromatography-Mass Spectrometry/methods , Mycological Typing Techniques , Phylogeny , Random Amplified Polymorphic DNA Technique , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/geneticsABSTRACT
The characterization of industrial yeast strains by examining their metabolic footprints (exometabolomes) was investigated and compared to genome-based discriminatory methods. A group of nine industrial brewing yeasts was studied by comparing their metabolic footprints, genetic fingerprints and comparative genomic hybridization profiles. Metabolic footprinting was carried out by both direct injection mass spectrometry (DIMS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), with data analysed by principal components analysis (PCA) and canonical variates analysis (CVA). The genomic profiles of the nine yeasts were compared by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis and microarray comparative genome hybridizations (CGH). Metabolomic and genomic analysis comparison of the nine brewing yeasts identified metabolomics as a powerful tool in separating genotypically and phenotypically similar strains. For some strains discrimination not achieved genomically was observed metabolomically.
Subject(s)
Metabolic Networks and Pathways , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/metabolism , Beer/microbiology , Genome, Fungal , Mass Spectrometry , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Temperature , Terminology as TopicABSTRACT
Twenty-four isolates of Aspergillus sojae, A. parasiticus, A. oryzae and A. flavus, including a number that have the capacity to produce aflatoxin, have been compared using amplified fragment length polymorphisms (AFLPs). Based on analysis of 12 different primer combinations, 500 potentially polymorphic fragments have been identified. Analysis of the AFLP data consistently and clearly separates the A. sojae/A. parasiticus isolates from the A. oryzae/A. flavus isolates. Furthermore. there are markers that can be used to distinguish the A. sojae isolates from those of A. parasiticus, which form the basis for species-specific markers. However, whilst there were many polymorphisms between isolates within the A. oryzae/A. flavus subgroup, no markers could be identified that distinguish between the two species. Sequencing of the ribosomal DNA ITS (internal transcribed spacers) from selected isolates also separated the A. sojae/A. parasiticus subgroup from the A. oryzae/A. flavus subgroup, but was unable to distinguish between the A. sojae and A. parasiticus isolates. Some ITS variation was found between isolates within the A. oryzae/A. flavus subgroup, but did not correlate with the species classification, indicating that it is difficult to use molecular data to separate the two species. In addition, sequencing of ribosomal ITS regions and AFLP analysis suggested that some species annotations in public culture collections may be inaccurate.
