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1.  The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica 
Brazilian Journal of Microbiology  2011;42(3):937-947.
The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.
PMCID: PMC3768797  PMID: 24031709
yeasts; Antarctica; diversity; extracellular enzymes; mycosporines
2.  Cold-adapted yeasts from Antarctica and the Italian Alps—description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov. 
Extremophiles   2009;14(1):47-59.
Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20°C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.
Electronic supplementary material
The online version of this article (doi:10.1007/s00792-009-0286-7) contains supplementary material, which is available to authorized users.
PMCID: PMC2797416  PMID: 19898737
Antarctic microbiology; Cold adaptation; Molecular phylogeny; Psychrophiles; Taxonomy
3.  Diversity of Both the Cultivable Protease-Producing Bacteria and Bacterial Extracellular Proteases in the Coastal Sediments of King George Island, Antarctica 
PLoS ONE  2013;8(11):e79668.
Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 105 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.
PMCID: PMC3817139  PMID: 24223990
4.  Genome Sequence of Exiguobacterium antarcticum B7, Isolated from a Biofilm in Ginger Lake, King George Island, Antarctica 
Journal of Bacteriology  2012;194(23):6689-6690.
Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e.g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.
PMCID: PMC3497522  PMID: 23144424
5.  Taxonomic Richness of Yeasts in Japan within Subtropical and Cool Temperate Areas 
PLoS ONE  2012;7(11):e50784.
An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks.
Methodology/Principal Findings
A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan’s subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared.
Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an “indicator” to recognize species, genera and higher taxonomic ranks.
PMCID: PMC3511277  PMID: 23226383
6.  Extracellular protease from the antarctic yeast Candida humicola. 
The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.
PMCID: PMC195704  PMID: 1622266
7.  Recovering Greater Fungal Diversity from Pristine and Diesel Fuel Contaminated Sub-Antarctic Soil Through Cultivation Using Both a High and a Low Nutrient Media Approach 
Novel cultivation strategies for bacteria are widespread and well described for recovering greater diversity from the “hitherto” unculturable majority. While similar approaches have not yet been demonstrated for fungi it has been suggested that of the 1.5 million estimated species less than 5% have been recovered into pure culture. Fungi are known to be involved in many degradative processes, including the breakdown of petroleum hydrocarbons, and it has been speculated that in Polar Regions they contribute significantly to bioremediation of contaminated soils. Given the biotechnological potential of fungi there is a need to increase efforts for greater species recovery, particularly from extreme environments such as sub-Antarctic Macquarie Island. In this study, like the yet-to-be cultured bacteria, high concentrations of nutrients selected for predominantly different fungal species to that recovered using a low nutrient media. By combining both media approaches to the cultivation of fungi from contaminated and non-contaminated soils, 91 fungal species were recovered, including 63 unidentified species. A preliminary biodegradation activity assay on a selection of isolates found that a high proportion of novel and described fungal species from a range of soil samples were capable of hydrocarbon degradation and should be characterized further.
PMCID: PMC3219075  PMID: 22131985
novel cultivation; fungi; sub-Antarctic; soil; diversity; hydrocarbon degradation
8.  Up, Down, and All Around: Scale-Dependent Spatial Variation in Rocky-Shore Communities of Fildes Peninsula, King George Island, Antarctica 
PLoS ONE  2014;9(6):e100714.
Understanding the variation of biodiversity along environmental gradients and multiple spatial scales is relevant for theoretical and management purposes. Hereby, we analysed the spatial variability in diversity and structure of intertidal and subtidal macrobenthic Antarctic communities along vertical environmental stress gradients and across multiple horizontal spatial scales. Since biotic interactions and local topographic features are likely major factors for coastal assemblages, we tested the hypothesis that fine-scale processes influence the effects of the vertical environmental stress gradients on the macrobenthic diversity and structure. We used nested sampling designs in the intertidal and subtidal habitats, including horizontal spatial scales ranging from few centimetres to 1000s of metres along the rocky shore of Fildes Peninsula, King George Island. In both intertidal and subtidal habitats, univariate and multivariate analyses showed a marked vertical zonation in taxon richness and community structure. These patterns depended on the horizontal spatial scale of observation, as all analyses showed a significant interaction between height (or depth) and the finer spatial scale analysed. Variance and pseudo-variance components supported our prediction for taxon richness, community structure, and the abundance of dominant species such as the filamentous green alga Urospora penicilliformis (intertidal), the herbivore Nacella concinna (intertidal), the large kelp-like Himantothallus grandifolius (subtidal), and the red crustose red alga Lithothamnion spp. (subtidal). We suggest that in coastal ecosystems strongly governed by physical factors, fine-scale processes (e.g. biotic interactions and refugia availability) are still relevant for the structuring and maintenance of the local communities. The spatial patterns found in this study serve as a necessary benchmark to understand the dynamics and adaptation of natural assemblages in response to observed and predicted environmental changes in Antarctica.
PMCID: PMC4067381  PMID: 24956114
9.  Characterization of the fungal microflora in raw milk and specialty cheeses of the province of Quebec 
Dairy Science & Technology  2011;92(5):455-468.
The cheese microbial ecosystem is complex, and the presence of non-starter adventitious microorganisms in milk may have an influence on the organoleptic characteristics of cheese. The aim of this study was to analyze the composition and diversity of the fungal flora of raw milk destined for cheesemaking from 19 dairy farms in Quebec and to monitor their evolution throughout ripening. Six hundred ten yeast and mold isolates were collected from raw milk and raw milk cheeses over a 9-month period. Based on the sequences of the rDNA ITS1-5.8S-ITS2 region, 67% of the raw milk isolates were yeasts, which were assigned to 37 species across 11 genera, while 33% were molds, which were assigned to 33 species across 25 genera. A semi-quantitative analysis of the yeasts and molds in the raw milk from four farms was performed over a 5-month period. The composition and diversity of the fungal microflora were totally different for each farm, each of which had a unique species profile. To determine whether adventitious yeast strains from the milk could develop in raw milk cheese, a multilocus-sequence-typing (MLST) analysis was performed on 13 Issatchenkia orientalis (syn. Pichia kudriavzevii, anamorph: Candida krusei) isolates. The same MLST genotypes were identified for strains independently isolated from raw milk and raw milk cheese from a farm processing its own milk. This study contributes to the understanding of the natural fungal microflora of raw milk and suggests that non-starter yeasts and molds can transfer from raw milk to raw milk cheese and may influence cheese ripening.
Electronic supplementary material
The online version of this article (doi:10.1007/s13594-011-0051-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3478505  PMID: 23125908
Artisanal cheese; Fungi; Yeast; Molds; Candida krusei; Issatchenkia orientalis; Pichia kudriavzevii; Multilocus sequence typing; MLST; Non-starter yeasts and molds; 手工干酪; 真菌; 酵母; 霉菌; 多位点序列分型; 非发酵剂酵母和霉菌; 真菌群落
10.  Enzymatic Hydrolysis of Yeast Cell Walls I. Isolation of Wall-Decomposing Organisms and Separation and Purification of Lytic Enzymes 
Journal of Bacteriology  1965;89(6):1570-1580.
Tanaka, Hirosato (University of California, Davis), and Herman J. Phaff. Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J. Bacteriol. 89:1570–1580. 1965.—A number of microorganisms, able to decompose and grow on yeast cell walls, were isolated from soil. These isolates demonstrated various types of attack on yeast walls. A bacterium, identified as Bacillus circulans, and a species of Streptomyces produced clear, lysed zones when grown on an agar medium containing baker's yeast cell walls. The streptomycete formed glucanase, mannanase, and protease, but B. circulans produced only glucanases. Purified mannan could be prepared from the culture fluid of B. circulans grown on baker's yeast cell walls. In a liquid, mineral medium, extracellular lytic enzyme production by B. circulans was optimal after 3 days of aerobic growth at 30 C with 0.5% baker's yeast cell walls as the carbon source. Twelve other carbon sources were ineffective as inducers. Among a number of polysaccharides tested, the crude enzymes of B. circulans hydrolyzed only β-1→3 glucan (laminarin) and β-1→6 glucan (pustulan), both by a random mechanism, to a mixture of dimer and glucose. The β-1→3 and β-1→6 glucanases were separated from each other by diethylaminoethyl cellulose column chromatography. Water-soluble oat glucan, which contains in the linear chain both β-1→3 and β-1→4 bonds, was also hydrolyzed by the bacterial β-1→3 glucanase. The products of this reaction indicated that this enzyme hydrolyzes β-1→3 or β-1→4 glucosidic linkages, provided the β-glucopyranosyl units composing these bonds are substituted in the 3 position by another glucose unit.
PMCID: PMC277693  PMID: 14291597
11.  Indole-3-Acetic Acid-Producing Yeasts in the Phyllosphere of the Carnivorous Plant Drosera indica L 
PLoS ONE  2014;9(12):e114196.
Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.
PMCID: PMC4252105  PMID: 25464336
12.  A Deviation from the Bipolar-Tetrapolar Mating Paradigm in an Early Diverged Basidiomycete 
PLoS Genetics  2010;6(8):e1001052.
In fungi, sexual identity is determined by specialized genomic regions called MAT loci which are the equivalent to sex chromosomes in some animals and plants. Usually, only two sexes or mating types exist, which are determined by two alternate sets of genes (or alleles) at the MAT locus (bipolar system). However, in the phylum Basidiomycota, a unique tetrapolar system emerged in which four different mating types are generated per meiosis. This occurs because two functionally distinct molecular recognition systems, each encoded by one MAT region, constrain the selection of sexual partners. Heterozygosity at both MAT regions is a pre-requisite for mating in both bipolar and tetrapolar basidiomycetes. Tetrapolar mating behaviour results from the absence of genetic linkage between the two regions bringing forth up to thousands of mating types. The subphylum Pucciniomycotina, an early diverged lineage of basidiomycetes encompassing important plant pathogens such as the rusts and saprobes like Rhodosporidium and Sporidiobolus, has been so far poorly explored concerning the content and organization of MAT loci. Here we show that the red yeast Sporidiobolus salmonicolor has a mating system unlike any previously described because occasional disruptions of the genetic cohesion of the bipolar MAT locus originate new mating types. We confirmed that mating is normally bipolar and that heterozygosity at both MAT regions is required for mating. However, a laboratory cross showed that meiotic recombination may occur within the bipolar MAT locus, explaining tetrapolar features like increased allele number and evolution rates of some MAT genes. This pseudo-bipolar system deviates from the classical bipolar–tetrapolar paradigm and, to our knowledge, has never been observed before. We propose a model for MAT evolution in the Basidiomycota in which the pseudo-bipolar system may represent a hitherto unforeseen gradual form of transition from an ancestral tetrapolar system to bipolarity.
Author Summary
Sexual reproduction in fungi is regulated by genomic regions called MAT loci that determine sexual identity in a manner comparable to that of sex chromosomes in animal and plants. In most fungi, sexual reproduction is bipolar, i.e., two alternate and distinct sets of genes at the MAT locus determine two mating types (the equivalent to sexes). In the Basidiomycota, which is the fungal lineage that includes the mushrooms, a unique (tetrapolar) sexual reproduction system evolved in which the mating type is determined by two functionally independent and genetically unlinked regions. In addition, the tetrapolar system functions with multiple alleles for at least one the two classes of MAT genes. This potentially generates thousands of mating types, as observed for many mushroom species. Here we report on the molecular characterization of the mating system in the basidiomycetous red yeast Sporidiobolus salmonicolor, which belongs to the Pucciniomycotina, the earliest derived lineage of the Basidiomycota that has remained virtually unexplored with respect to gene content and structure of MAT loci. Our results revealed for the first time a mating system that is neither tetrapolar nor strictly bipolar, and we propose a model for the evolution of basidiomycete MAT loci that accommodates this novel finding.
PMCID: PMC2916851  PMID: 20700437
13.  Genomic and phenotypic insights into the ecology of Arthrobacter from Antarctic soils 
BMC Genomics  2015;16(1):36.
Members of the bacterial genus Arthrobacter are both readily cultured and commonly identified in Antarctic soil communities. Currently, relatively little is known about the physiological traits that allow these bacteria to survive in the harsh Antarctic soil environment. The aim of this study is to investigate if Antarctic strains of Arthrobacter owe their resilience to substantial genomic changes compared to Arthrobacter spp. isolated from temperate soil environments.
Quantitative PCR-based analysis revealed that up to 4% of the soil bacterial communities were comprised of Arthrobacter spp. at four locations in the Ross Sea Region. Genome analysis of the seven Antarctic Arthrobacter isolates revealed several features that are commonly observed in psychrophilic/psychrotolerant bacteria. These include genes primarily associated with sigma factors, signal transduction pathways, the carotenoid biosynthesis pathway and genes induced by cold-shock, oxidative and osmotic stresses. However, these genes were also identified in genomes of seven temperate Arthrobacter spp., suggesting that these mechanisms are beneficial for growth and survival in a range of soil environments. Phenotypic characterisation revealed that Antarctic Arthrobacter isolates demonstrate significantly lower metabolic versatility and a narrower salinity tolerance range compared to temperate Arthrobacter species. Comparative analyses also revealed fewer protein-coding sequences and a significant decrease in genes associated with transcription and carbohydrate transport and metabolism in four of the seven Antarctic Arthrobacter isolates. Notwithstanding genome incompleteness, these differences together with the decreased metabolic versatility are indicative of genome content scaling.
The genomes of the seven Antarctic Arthrobacter isolates contained several features that may be beneficial for growth and survival in the Antarctic soil environment, although these features were not unique to the Antarctic isolates. These genome sequences allow further investigations into the expression of physiological traits that enable survival under extreme conditions and, more importantly, into the ability of these bacteria to respond to future perturbations including climate change and human impacts.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1220-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4326396  PMID: 25649291
Antarctica; Comparative genomics; Arthrobacter
14.  Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study 
PLoS ONE  2014;9(5):e96595.
Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.
PMCID: PMC4015989  PMID: 24816850
15.  Identification of Mating Type Genes in the Bipolar Basidiomycetous Yeast Rhodosporidium toruloides: First Insight into the MAT Locus Structure of the Sporidiobolales▿ †  
Eukaryotic Cell  2008;7(6):1053-1061.
Rhodosporidium toruloides is a heterothallic, bipolar, red yeast that belongs to the Sporidiobolales, an order within a major lineage of basidiomycetes, the Pucciniomycotina. In contrast to other basidiomycetes, considerably less is known about the nature of the mating type (MAT) loci that control sexual reproduction in this lineage. Three genes (RHA1, RHA2, and RHA3) encoding precursors of the MAT A1 pheromone (rhodotorucine A) were previously identified and formed the basis for a genome walking approach that led to the identification of additional MAT genes in complementary mating strains of R. toruloides. Two mating type-specific alleles encoding a p21-activated kinase (PAK; Ste20 homolog) were found between the RHA2 and RHA3 genes, and identification in MAT A2 strains of a gene encoding a presumptive pheromone precursor enabled prediction of the structure of rhodotorucine a. In addition, a putative pheromone receptor gene (STE3 homolog) was identified upstream of RHA1. Analyses of genomic data from two closely related species, Sporobolomyces roseus and Sporidiobolus salmonicolor, identified syntenic regions that contain homologs of all the above-mentioned genes. Notably, six novel pheromone precursor genes were uncovered, which encoded, similarly to the RHA genes, multiple tandem copies of the peptide moiety. This suggests that this structure, which is unique among fungal lipopeptide pheromones, seems to be prevalent in red yeasts. Species comparisons provided evidence for a large, multigenic MAT locus structure in the Sporidiobolales, but no putative homeodomain transcription factor genes (which are present in all basidiomycetous MAT loci characterized thus far) could be found in any of the three species in the vicinity of the MAT genes identified.
PMCID: PMC2446649  PMID: 18408057
16.  Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole 
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
Chemical screens with a library enriched for known drugs identified a diverse set of 148 compounds that potentiated the action of the antifungal drug fluconazole against the fungal pathogens Cryptococcus neoformans, Cryptococcus gattii and Candida albicans, and the model yeast Saccharomyces cerevisiae, often in a species-specific manner.Chemogenomic profiles of six confirmed hits in S. cerevisiae revealed different modes of action and enabled the prediction of additional synergistic combinations; three-way synergistic interactions exhibited even stronger synergies at low doses of fluconazole.The synergistic combination of fluconazole and the antidepressant sertraline was active against fluconazole-resistant clinical fungal isolates and in an in vivo model of Cryptococcal infection.
Rising fungal infection rates, especially among immune-suppressed individuals, represent a serious clinical challenge (Gullo, 2009). Cancer, organ transplant and HIV patients, for example, often succumb to opportunistic fungal pathogens. The limited repertoire of approved antifungal agents and emerging drug resistance in the clinic further complicate the effective treatment of systemic fungal infections. At the molecular level, the paucity of fungal-specific essential targets arises from the conserved nature of cellular functions from yeast to humans, as well as from the fact that many essential yeast genes can confer viability at a fraction of wild-type dosage (Yan et al, 2009). Although only ∼1100 of the ∼6000 genes in yeast are essential, almost all genes become essential in specific genetic backgrounds in which another non-essential gene has been deleted or otherwise attenuated, an effect termed synthetic lethality (Tong et al, 2001). Genome-scale surveys suggest that over 200 000 binary synthetic lethal gene combinations dominate the yeast genetic landscape (Costanzo et al, 2010). The genetic buffering phenomenon is also manifest as a plethora of differential chemical–genetic interactions in the presence of sublethal doses of bioactive compounds (Hillenmeyer et al, 2008). These observations frame the difficulty of interdicting network functions in eukaryotic pathogens with single agent therapeutics. At the same time, however, this genetic network organization suggests that judicious combinations of small molecule inhibitors of both essential and non-essential targets may elicit additive or synergistic effects on cell growth (Sharom et al, 2004; Lehar et al, 2008). Unbiased screens for drugs that synergistically enhance a specific bioactive effect, but which are not themselves individually active—termed a syncretic combination—are one means to substantially elaborate chemical space (Keith et al, 2005). Indeed, compounds that enhance the activity of known agents in model yeast and cancer cell line systems have been identified both by focused small molecule library screens and by computational methods (Borisy et al, 2003; Lehar et al, 2007; Nelander et al, 2008; Jansen et al, 2009; Zinner et al, 2009).
To extend the stratagem of chemical synthetic lethality to clinically relevant fungal pathogens, we screened a bioactive library of known drugs for synergistic enhancers of the widely used fungistatic drug fluconazole against the clinically relevant pathogens C. albicans, C. neoformans and C. gattii, as well as the genetically tractable budding yeast S. cerevisiae. Fluconazole is an azole drug that inhibits lanosterol 14α-demethylase, the gene product of ERG11, an essential cytochrome P450 enzyme in the ergosterol biosynthetic pathway (Groll et al, 1998). We identified 148 drugs that potentiate the antifungal action of fluconazole against the four species. These syncretic compounds had not been previously recognized in the clinic as antifungal agents, and many acted in a species-specific manner, often in a potent fungicidal manner.
To understand the mechanisms of synergism, we interrogated six syncretic drugs—trifluoperazine, tamoxifen, clomiphene, sertraline, suloctidil and L-cycloserine—in genome-wide chemogenomic profiles of the S. cerevisiae deletion strain collection (Giaever et al, 1999). These profiles revealed that membrane, vesicle trafficking and lipid biosynthesis pathways are targeted by five of the synergizers, whereas the sphingolipid biosynthesis pathway is targeted by L-cycloserine. Cell biological assays confirmed the predicted membrane disruption effects of the former group of compounds, which may perturb ergosterol metabolism, impair fluconazole export by drug efflux pumps and/or affect active import of fluconazole (Kuo et al, 2010; Mansfield et al, 2010). Based on the integration of chemical–genetic and genetic interaction space, a signature set of deletion strains that are sensitive to the membrane active synergizers correctly predicted additional drug synergies with fluconazole. Similarly, the L-cycloserine chemogenomic profile correctly predicted a synergistic interaction between fluconazole and myriocin, another inhibitor of sphingolipid biosynthesis. The structure of genetic networks suggests that it should be possible to devise higher order drug combinations with even greater selectivity and potency (Sharom et al, 2004). In an initial test of this concept, we found that the combination of a non-synergistic pair drawn from the membrane active and sphingolipid target classes exhibited potent three-way synergism with a low dose of fluconazole. Finally, the combination of sertraline and fluconazole was active in a G. mellonella model of Cryptococcal infection, and was also efficacious against fluconazole-resistant clinical isolates of C. albicans and C. glabrata.
Collectively, these results demonstrate that the combinatorial redeployment of known drugs defines a powerful antifungal strategy and establish a number of potential lead combinations for future clinical assessment.
Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
PMCID: PMC3159983  PMID: 21694716
antifungal; combination; pathogen; resistance; synergism
17.  Ecology of pathogenic yeasts in Amazonian soil. 
In an investigation of Amazonian soil as a natural reservoir for pathogenic fungi, 1,949 soil samples collected from diverse geographical and ecological settings of the Brazilian Amazon Basin were analyzed for the presence of non-keratinophilic fungi by the indirect mouse inoculation procedure and for the presence of keratinophilic fungi by the hair bait technique. All soil samples were acidic with low pH values. From 12% of the soil samples, 241 yeast and yeastlike isolates pertaining to six genera and 82 species were recovered, of which 63% were Torulopsis and 26% were Candida species. Nine fungi with known pathogenic potentials were encountered among 43% (104) of the isolates: T. glabrata, C. guilliermondii, C. albicans, C. pseudotropicalis, C. stellatoidea, C. tropicalis, Rhodotorula rubra, and Wangiella dermatitidis. The yeast flora was marked by species diversity, low frequency of each species, random geographical distribution, and an apparent lack of species clustering. The composition and distribution of the yeast flora in soil differed from those of the yeast flora harbored by bats, suggesting that the Amazonian external environment and internal bat organs act as independent natural habitats for yeasts.
PMCID: PMC239680  PMID: 6538774
18.  An Application of Wastewater Treatment in a Cold Environment and Stable Lipase Production of Antarctic Basidiomycetous Yeast Mrakia blollopis 
PLoS ONE  2013;8(3):e59376.
Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to Mrakia blollopis CBS8921T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.
PMCID: PMC3597603  PMID: 23516630
19.  Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes 
Journal of Clinical Microbiology  2000;38(6):2302-2310.
Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by ≤2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of ≥99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.
PMCID: PMC86787  PMID: 10834993
20.  Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp. 
Applied and Environmental Microbiology  1995;61(12):4251-4257.
The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.
PMCID: PMC167736  PMID: 8534092
21.  Mitochondrial Genome Evolution in a Single Protoploid Yeast Species 
G3: Genes|Genomes|Genetics  2012;2(9):1103-1111.
Mitochondria are organelles, which play a key role in some essential functions, including respiration, metabolite biosynthesis, ion homeostasis, and apoptosis. The vast numbers of mitochondrial DNA (mtDNA) sequences of various yeast species, which have recently been published, have also helped to elucidate the structural diversity of these genomes. Although a large corpus of data are now available on the diversity of yeast species, little is known so far about the mtDNA diversity in single yeast species. To study the genetic variations occurring in the mtDNA of wild yeast isolates, we performed a genome-wide polymorphism survey on the mtDNA of 18 Lachancea kluyveri (formerly Saccharomyces kluyveri) strains. We determined the complete mt genome sequences of strains isolated from various geographical locations (in North America, Asia, and Europe) and ecological niches (Drosophila, tree exudates, soil). The mt genome of the NCYC 543 reference strain is 51,525 bp long. It contains the same core of genes as Lachancea thermotolerans, the nearest relative to L. kluyveri. To explore the mt genome variations in a single yeast species, we compared the mtDNAs of the 18 isolates. The phylogeny and population structure of L. kluyveri provide clear-cut evidence for the existence of well-defined geographically isolated lineages. Although these genomes are completely syntenic, their size and the intron content were found to vary among the isolates studied. These genomes are highly polymorphic, showing an average diversity of 28.5 SNPs/kb and 6.6 indels/kb. Analysis of the SNP and indel patterns showed the existence of a particularly high overall level of polymorphism in the intergenic regions. The dN/dS ratios obtained are consistent with purifying selection in all these genes, with the noteworthy exception of the VAR1 gene, which gave a very high ratio. These data suggest that the intergenic regions have evolved very fast in yeast mitochondrial genomes.
PMCID: PMC3429925  PMID: 22973548
mitochondrial DNA; intraspecific diversity; population structure; purifying selection; Lanchancea kluyveri
22.  Plant and Bird Presence Strongly Influences the Microbial Communities in Soils of Admiralty Bay, Maritime Antarctica 
PLoS ONE  2013;8(6):e66109.
Understanding the environmental factors that shape microbial communities is crucial, especially in extreme environments, like Antarctica. Two main forces were reported to influence Antarctic soil microbes: birds and plants. Both birds and plants are currently undergoing relatively large changes in their distribution and abundance due to global warming. However, we need to clearly understand the relationship between plants, birds and soil microorganisms. We therefore collected rhizosphere and bulk soils from six different sampling sites subjected to different levels of bird influence and colonized by Colobanthus quitensis and Deschampsia antarctica in Admiralty Bay, King George Island, Maritime Antarctic. Microarray and qPCR assays targeting 16S rRNA genes of specific taxa were used to assess microbial community structure, composition and abundance and analyzed with a range of soil physico-chemical parameters. The results indicated significant rhizosphere effects in four out of the six sites, including areas with different levels of bird influence. Acidobacteria were significantly more abundant in soils with little bird influence (low nitrogen) and in bulk soil. In contrast, Actinobacteria were significantly more abundant in the rhizosphere of both plant species. At two of the sampling sites under strong bird influence (penguin colonies), Firmicutes were significantly more abundant in D. antarctica rhizosphere but not in C. quitensis rhizosphere. The Firmicutes were also positively and significantly correlated to the nitrogen concentrations in the soil. We conclude that the microbial communities in Antarctic soils are driven both by bird and plants, and that the effect is taxa-specific.
PMCID: PMC3688718  PMID: 23840411
23.  Evolutionary principles of modular gene regulation in yeasts 
eLife  2013;2:e00603.
Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles.
eLife digest
The incredible diversity of living creatures belies the fact that their genes are quite similar. In the 1970s Mary-Claire King and Allan Wilson proposed that a process called gene regulation—which determines when, where and how genes are expressed as proteins—is responsible for this diversity. Four decades later, the central role of gene regulation in evolution has been confirmed in a wide range of species including bacteria, fungi, flies and mammals, although the details remain poorly understood. In recent years it has been suggested that the duplication of genes—and sometimes the duplication of whole genomes—has had a crucial influence on the part played by gene regulation in the evolution of many different species.
Ascomycota fungi are uniquely suited to the study of genetics and evolution because of their diversity—they include C. albicans, a fungus that is found in the human mouth and gut, and various species of yeast—and because many of their genomes have already been sequenced. Moreover, their genomes are relatively small, which simplifies the task of working out how it has changed over the course of evolution. It is also known that species in this branch of the tree of life diverged before and after an event in which a whole genome was duplicated.
Ascomycota fungi use glucose as a source of carbon in different ways during aerobic growth. Most, including C. albicans, are respiratory and rely on oxidative phosphorylation processes to produce energy. However, a small number—including S. cerevisiae and S. pombe, two types of yeast that are widely used as model organisms—prefer to ferment glucose, even when oxygen is available. Species that favor the latter respiro-fermentative lifestyle have evolved independently at least twice: once after the whole genome duplication event that lead to S. cerevisiae, and once when S. pombe and the other fission yeasts evolved.
Thompson et al. have measured mRNA profiles in 15 different species of yeast and reconstructed how the regulation of groups of genes (modules) have evolved over a period of more than 300 million years. They found that modules have diverged proportionally to evolutionary time, with prominent changes in gene regulation being associated with changes in lifestyle (especially changes in carbon metabolism) and a whole genome duplication event.
Gene duplication events result in gene paralogs—identical genes at different places in the genome—and these have made significant contributions to the evolution of different forms of gene regulation, especially just after the duplication event. Moreover, the paralogs produced in whole genome duplication events have resulted in bigger changes over longer periods of time. Similar patterns were observed in the regulation of the genes involved in the response to heat shock in eight of the species, which suggests that these are general evolutionary principles.
The changes in gene expression associated with the respiro-fermentative lifestyle may also have implications for our understanding of cancer: healthy cells rely on oxidative phosphorylation to produce energy whereas, similar to yeast cells, most cancerous cells rely on respiro-fermentation. Furthermore, yeast cells and cancer cells both support their rapid growth and proliferation by using glucose for biosynthesis to support cell division, although this process is not fully understood. Normal cells, on the other hand, use glucose primarily for energy and tend not to divide rapidly.
Thompson et al. found that the genes encoding enzymes in two biosynthetic pathways—one that produces the nucleotides necessary for DNA replication, and one that synthesizes glycine—are induced in respiro-fermentative yeasts but repressed in respiratory yeast cells. The fact that similar changes are observed in the same two pathways when normal cells become cancer cells suggests that these pathways have an important role in the development of cancer. The framework developed by Thompson et al. could also be used to explore the evolution of gene regulation in other species and biological processes.
PMCID: PMC3687341  PMID: 23795289
regulatory evolution; duplication; divergence; carbon lifestyle; module; gene expression; S. cerevisiae; S. pombe
24.  Isolation and enzyme bioprospection of endophytic bacteria associated with plants of Brazilian mangrove ecosystem 
SpringerPlus  2014;3:382.
The mangrove ecosystem is a coastal tropical biome located in the transition zone between land and sea that is characterized by periodic flooding, which confers unique and specific environmental conditions on this biome. In these ecosystems, the vegetation is dominated by a particular group of plant species that provide a unique environment harboring diverse groups of microorganisms, including the endophytic microorganisms that are the focus of this study. Because of their intimate association with plants, endophytic microorganisms could be explored for biotechnologically significant products, such as enzymes, proteins, antibiotics and others. Here, we isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. After identifying the isolates, the bacterial communities were evaluated for enzyme production. Protease activity was observed in 75% of the isolates, while endoglucanase activity occurred in 62% of the isolates. Bacillus showed the highest activity rates for amylase and esterase and endoglucanase. To our knowledge, this is the first reported diversity analysis performed on endophytic bacteria obtained from the branches of mangrove trees and the first overview of the specific enzymes produced by different bacterial genera. This work contributes to our knowledge of the microorganisms and enzymes present in mangrove ecosystems.
PMCID: PMC4125609  PMID: 25110630
Mangrove; Endophytes; Bacteria; Biotechnological potential; Enzymes
25.  Production of extracellular ribonuclease by yeasts and yeastlike fungi, and its repression by orthophosphate in species of Cryptococcus and Tremella. 
Journal of Bacteriology  1976;125(3):955-960.
A strain of Cryptococcus laurentii and a haploid isolate of Tremella foliacea were shown to produce orthophosphate-repressible ribonuclease in liquid culture. Addition of as little as 1 mM K2HPO4, pH 7.0, completely repressed enzyme production by both fungi. The orthophosphate-repressible enzyme was not produced by other species of the two genera tested. These results, together with other findings, suggest a close phylogenetic relationship between Cryptococcus laurentii and Tremella foliacea. The ability of other yeasts and yeastlike fungi to hydrolyze ribonucleic acid in a solid test medium was assessed. Based on the limited number of organisms available for study, extracellular ribonuclease activity was found in species having close affinity to the Basidiomycetes and in yeasts classified in the ascomycetous genera, Endomycopsis, Hansenula, and Kluyveromyces. Other ascomycetous yeasts did not exhibit extracellular ribonuclease.
PMCID: PMC236171  PMID: 1254561

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