The phenotypic heterogeneity of saccharomyces cerevisiae strains from natural environments

Painel apresentado por Ricardo Franco DuarteSaccharomyces cerevisiae is the model organism par excellence and stands today at the forefront of molecular biology, genetics and genomics. However, as for many other laboratory model organisms, understanding of the ecological, evolutionary and population genetic features that shaped the biology of S. cerevisiae is underscored by a wealth of knowledge on molecular and cellular biology, mainly obtained from a very limited number of reference laboratory strains. In the last few years, yeast researchers developed a keen interest to identify genomic variability between wild-type yeast strains from different ecological niches or strains that are used for different technological applications. The aim of the present work was to constitute a S. cerevisiae wine yeast strain collection for the conservation of biodiversity, sustainable development of genetic resources and for the identification of strains for biotechnological applications. The S. cerevisiae strains that constitute our strain collection were obtained from grape varieties collected in the Vinho Verde appellation of origin in Portugal. Grape samples were collected in consecutive years since 2001, from multiple sampling sites of several vineyards at harvest time. A total of 2520 yeast isolates were obtained during 2001 – 2006, and were assigned to 350 different strains, based on genetic analysis by a set of highly polymorphic microsatellites. Using neural networks, a subset of 103 genetically most diverse strains was chosen for phenotypic analysis, that included growth in synthetic must media at various temperatures, utilization of carbon sources (glucose, ribose, arabinose, xylose, saccharose, galactose, rafinose, maltose, glycerol, potassium acetate and pyruvic acid), growth in ethanol containing media, evaluation of osmotic and oxidative stress resistance, H2S production and utilization of different nitrogen sources. Significant heterogeneity was apparent among strains and we identified numerous strains with the capability to grow in media containing ribose, arabinose and xylose, not previously described for S. cerevisiae.Financially supported by the program POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004)

Fungal infections diagnosis - Past, present and future

Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the stan-dard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient's treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as "gold standard", to the molecular methods currently used, and finally mentioning some of the more futuristic approaches.(c) 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.This work was supported by the UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019 strategic programs, which is funded by national funds through the FCT-Fundacao para a Ciência e Tecnologia, and by the ERDF-European Regional Devel-opment Fund through the COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI) and Sistema de Apoio à Investigacao Cientifica e Tecnol?ogica (SAICT)

Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus

Introduction Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. Methods A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. Results Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/mu L), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. Discussion This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions

Contributions of adaptive laboratory evolution towards the enhancement of the biotechnological potential of non-conventional yeast species

Changes in biological properties over several generations, induced by controlling shortterm evolutionary processes in the laboratory through selective pressure, and whole-genome re-sequencing, help determine the genetic basis of microorganism’s adaptive laboratory evolution (ALE). Due to the versatility of this technique and the imminent urgency for alternatives to petroleum-based strategies, ALE has been actively conducted for several yeasts, primarily using the conventional species Saccharomyces cerevisiae, but also non-conventional yeasts. As a hot topic at the moment since genetically modified organisms are a debatable subject and a global consensus on their employment has not yet been attained, a panoply of new studies employing ALE approaches have emerged and many different applications have been exploited in this context. In the present review, we gathered, for the first time, relevant studies showing the ALE of non-conventional yeast species towards their biotechnological improvement, cataloging them according to the aim of the study, and comparing them considering the species used, the outcome of the experiment, and the employed methodology. This review sheds light on the applicability of ALE as a powerful tool to enhance species features and improve their performance in biotechnology, with emphasis on the non-conventional yeast species, as an alternative or in combination with genome editing approaches.This work was supported by the UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019 strategic programs and the project PTDC/BIA-MIC/32059/2017, which is funded by national funds through the FCT-Fundação para a Ciência e Tecnologia and by the ERDF-European Regional Development Fund through the COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and Sistema de Apoio à Investigação Científica e Tecnológica (SAICT). T.F. was supported by Fellowship 2021.04595.BD from Fundação para a Ciência e Tecnologia, Portugal

ITS rDNA barcodes clarify molecular diversity of aquatic hyphomycetes

Aquatic hyphomycetes are key microbial decomposers of allochthonous organic matter in freshwater ecosystems. Although their importance in carbon flow and food webs in streams is widely recognized, there are still gaps in our understanding of their molecular diversity and distribution patterns. Our study utilized the growing database of ITS rDNA barcodes of aquatic hyphomycetes (1252 sequences) and aimed to (i) produce new barcodes for some lesser-known taxa; (ii) clarify the taxonomic placement of some taxa at the class or order level, based on molecular data; and (iii) provide insights into the biogeographical origins of some taxa. This study increased the number of aquatic hyphomycete species with available ITS barcodes from 119 (out of ~300 species described) to 136. Phylogenetically, the 136 species were distributed between 2 phyla, 6 classes, and 10 orders of fungi. Future studies should strive to increase the database of ITS sequences, especially focusing on species with unclear phylogenetic relationships (incertae sedis) and with few sequences available. The geographical distribution of species with available ITS sequences included 50 countries from five continents, but 6 countries had more than 20 species associated, showing a bias toward the northern hemisphere, likely due to sampling bias.This work is supported by the project STREAMECO—Biodiversity and ecosystem functioning under climate change: from the gene to the stream: PTDC/CTA-AMB/31245/2017 funded by the Portuguese Foundation for Science and Technology (FCT) and by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. Additional support from the National Science Foundation (NSF DEB-1655797) to V.G. is gratefully acknowledged

Uncovering the promiscuous activity of IL-6 proteins: a multi-dimensional analysis of phylogeny, classification and residue conservation

The IL-6 family of cytokines, known for their pleiotropic behavior, share binding to the gp130 receptor for signal transduction with the necessity to bind other receptors. Leukemia inhibitory factor receptor is triggered by the IL-6 family proteins: leukemia inhibitory factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine factor 1 (CLCF1). Besides the conserved binding sites to the receptor, not much is known in terms of the diversity and characteristics of these proteins in different organisms. Herein, we describe the sequence analysis of LIF, OSM, and CT-1 from several organisms, and m17, a LIF ortholog found in fishes, regarding its phylogenetics, intrinsic properties, and the impact of conserved residues on structural features. Sequences were identified in seven classes of vertebrates, showing high conservation values in binding site III, but protein-dependent results on binding site II. GRAVY, isoelectric point, and molecular weight parameters were relevant to differentiate classes in each protein and to enable, for the first time and with high fidelity, the prediction of both organism class and protein type just using machine learning approaches. OSM sequences from primates showed an increased BC loop when compared to the remaining mammals, which could influence binding to OSM receptor and tune signaling pathways. Overall, this study highlights the potential of sequence diversity analysis to understand IL-6 cytokine family evolution, showing the conservation of function-related motifs and evolution of class and protein-dependent characteristics. Our results could impact future medical treatment of disorders associated with imbalances in these cytokines.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. and project FUN2CYT: Harnessing the potential for biomedical applications of pleiotropic cytokines LIF and oncostatin M (POCI-01-0145-FEDER-030568) supported by Programa Operacional Competitividade e Internacionalizaçao (FEDER) and FCT, I.P. Raul ˜ Machado acknowledges FCT I.P. for funding within the Scientific Employment Stimulus instrument (CEECIND/00526/2018)

Aquatic hyphomycete taxonomic relatedness translates into lower genetic divergence of the nitrate reductase gene

Aquatic hyphomycetes are key microbial decomposers in freshwater that are capable of producing extracellular enzymes targeting complex molecules of leaf litter, thus, being crucial to nutrient cycling in these ecosystems. These fungi are also able to assimilate nutrients (e.g., nitrogen) from stream water, immobilizing these nutrients in the decomposing leaf litter and increasing its nutritional value for higher trophic levels. Evaluating the aquatic hyphomycete functional genetic diversity is, thus, pivotal to understanding the potential impacts of biodiversity loss on nutrient cycling in freshwater. In this work, the inter- and intraspecific taxonomic (ITS1-5.8S-ITS2 region) and functional (nitrate reductase gene) diversity of 40 aquatic hyphomycete strains, belonging to 23 species, was evaluated. A positive correlation was found between the taxonomic and nitrate reductase gene divergences. Interestingly, some cases challenged this trend: Dactylella cylindrospora (Orbiliomycetes) and Thelonectria rubi (Sordariomycetes), which were phylogenetically identical but highly divergent regarding the nitrate reductase gene; and Collembolispora barbata (incertae sedis) and Tetracladium apiense (Leotiomycetes), which exhibited moderate taxonomic divergence but no divergence in the nitrate reductase gene. Additionally, Tricladium chaetocladium (Leotiomycetes) strains were phylogenetically identical but displayed a degree of nitrate reductase gene divergence above the average for the interspecific level. Overall, both inter- and intraspecific functional diversity were observed among aquatic hyphomycetes.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the Portuguese Foundation for Science and Technology (FCT) I.P and by the project STREAMECO-Biodiversity and eco-system functioning under climate change: from the gene to the stream: PTDC/CTA-AMB/31245/2017 funded by FCT

O gene da nitrato redutase é diverso entre as espécies de hifomicetos aquáticos?

Os hifomicetos aquáticos (AQH) são os principais decompositores microbianos de folhada em ribeiros, sendo capazes de produzir enzimas extracelulares que degradam as moléculas complexas da folhada, desempenhando portanto um papel crucial no ciclo de nutrientes nestes ecossistemas. Estes fungos também assimilam nutrientes (azoto e fósforo) da água, imobilizando-os na folhada em decomposição e aumentando seu valor nutricional para níveis tróficos superiores. A nitrato redutase é a enzima responsável por iniciar o processo metabólico de assimilação de nitrato por meio da conversão de nitrato em nitrito. O objetivo deste estudo foi avaliar a diversidade do gene que codifica a nitrato redutase em espécies de hifomicetos aquáticos. Os resultados revelaram diferenças intra- e interespecíficas entre os isolados fúngicos, tanto no fragmento do gene como na proteína para a qual ele se traduz. Isto levanta questões sobre se estas diferenças genéticas afetam a funcionalidade da enzima, e quais os possíveis impactos para o ciclo do azoto em ecossistemas de água doce.Este trabalho foi suportado pela FCT (projeto STREAMECO - PTDC/CTA-AMB/31245/2017)

Bioinformatic approaches for the genetic and phenotypic characterization of a Saccharomyces cerevisiae wine yeast collection

Apresentação efectuada nas "Jornadas de Biologia de Leveduras Professor Nicolau van Uden, 15, Porto, Portugal, 2007."In the years following the publication of the Saccharomyces cerevisiae genome, enough evidence was provided showing substantial genetic differences among wine yeast strains. At present, about 200 S. cerevisiae strains are commercially available, whereas each strain harbours a combination of specific traits that are of interest for winemakers. Exploring the biodiversity and genetic constitution of indigenous fermentative strains is an important contribution towards the selection of strains with specific phenotypes. From 54 spontaneous fermentations of grapes collected in three vineyards of the Vinho Verde Region during three consecutive harvests, a total of 1620 yeast isolates were obtained and categorized in 300 strains based on mitochondrial DNA RFLP patterns. For each pattern a unique allelic combination for six polymorphic microsatellite loci (ScAAT1 – ScAAT6) was found. No correlation was apparent between geographic distance and genetic similarity among individual isolates. However, genetic differences among S. cerevisiae populations were apparent from gradations in allele frequencies rather than from distinctive “diagnostic” genotypes, and the accumulation of small allele-frequency differences across six loci allowed the identification of population structures. Additional bioinformatics approaches (Bionumerics software) were performed with a subset of 47 strains in order to search for associations between microsatellite data and phenotypic traits, using semiquantitative results from an enzymatic activities test panel (api®ZYM, bioMérieux® SA). Most strains showed similar phenotypic profiles, whereas five strains were clearly distinctive based on the activity for acid phosphatase, naphthol-phosphohydrolase and β-galactosidase. Three of them showed rare alleles (allelic frequency 0.05 - 0.2) for the more polymorphic microsatellites ScAAT1 and ScAAT4.Fundação para a Ciência e a Tecnologia (FCT) - POCI 2010 program (FEDER/FCT, POCI/AGR/56102 /2004)

Single cell oil production by oleaginous yeasts grown in synthetic and waste-derived volatile fatty acids

Four yeast isolates from the species—Apiotrichum brassicae, Candida tropicalis, Metschnikowia pulcherrima, and Pichia kudriavzevii—previously selected by their oleaginous character and growth flexibility in different carbon sources, were tested for their capacity to convert volatile fatty acids into lipids, in the form of single cell oils. Growth, lipid yields, volatile fatty acids consumption, and long-chain fatty acid profiles were evaluated in media supplemented with seven different volatile fatty acids (acetic, butyric, propionic, isobutyric, valeric, isovaleric, and caproic), and also in a dark fermentation effluent filtrate. Yeasts A. brassicae and P. kudriavzevii attained lipid productivities of more than 40% (w/w), mainly composed of oleic (>40%), palmitic (20%), and stearic (20%) acids, both in synthetic media and in the waste-derived effluent filtrate. These isolates may be potential candidates for single cell oil production in larger scale applications by using alternative carbon sources, combining economic and environmental benefits.This work was supported by the European project "VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks" (Call H2020-NMBP-BIO-2016 Grant agreement No. 720777) and by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P., by the ERDF through the COMPETE2020