Browsing by Author "Cubillos, Francisco A."

Now showing 1 - 13 of 13
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    An integrative taxonomy approach reveals Saccharomyces chiloensis sp. nov. as a newly discovered species from Coastal Patagonia
    (2024) Pena, Tomas A.; Villarreal, Pablo; Agier, Nicolas; De Chiara, Matteo; Barria, Tomas; Urbina, Kamila; Villarroel, Carlos A.; Santos, Ana R. O.; Rosa, Carlos A.; Nespolo, Roberto F.; Liti, Gianni; Fischer, Gilles; Cubillos, Francisco A.
    Species delineation in microorganisms is challenging due to the limited markers available for accurate species assignment. Here, we applied an integrative taxonomy approach, combining extensive sampling, whole-genome sequence-based classification, phenotypic profiling, and assessment of interspecific reproductive isolation. Our work reveals the presence of a distinct Saccharomyces lineage in Nothofagus forests of coastal Patagonia. This lineage, designated Saccharomyces chiloensis sp. nov., exhibits 7% genetic divergence from its sister species S. uvarum, as revealed by whole-genome sequencing and population analyses. The South America-C (SA-C) coastal Patagonia population forms a unique clade closely related to a previously described divergent S. uvarum population from Oceania (AUS, found in Australia and New Zealand). Our species reclassification is supported by a low Ortho Average Nucleotide Identity (OANI) of 93% in SA-C and AUS relative to S. uvarum, which falls below the suggested species delineation threshold of 95%, indicating an independent evolutionary lineage. Hybrid spore viability assessment provided compelling evidence that SA-C and AUS are reproductively isolated from S. uvarum. In addition, we found unique structural variants between S. chiloensis sp. nov. lineages, including large-scale chromosomal translocations and inversions, together with a distinct phenotypic profile, emphasizing their intraspecies genetic distinctiveness. We suggest that S. chiloensis sp. nov diverged from S. uvarum in allopatry due to glaciation, followed by post-glacial dispersal, resulting in distinct lineages on opposite sides of the Pacific Ocean. The discovery of S. chiloensis sp. nov. illustrates the uniqueness of Patagonia's coastal biodiversity and underscores the importance of adopting an integrative taxonomic approach in species delineation to unveil cryptic microbial species. The holotype of S. chiloensis sp. nov. is CBS 18620(T).
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (Public Library Science, 2024) Cerda Rojas, Ariel Patricio; Rivera, Maira; Armijo, Grace; Ibarra-Henríquez, Catalina; Reyes, Javiera; Blázquez Sánchez, Paula; Avilés, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vásquez, Yesseny; Cortez-San Martín, Marcelo; Cubillos, Francisco A.; García, Patricia; Ferrés, Marcela; Ramírez Sarmiento, César Antonio; Federici, Fernan; Gutiérrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (2024) Cerda, Ariel; Rivera, Maira; Armijo, Grace; Ibarra-Henriquez, Catalina; Reyes, Javiera; Blazquez-Sanchez, Paula; Aviles, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vasquez, Yesseny; Cortez-San Martin, Marcelo; Cubillos, Francisco A.; Garcia, Patricia; Ferres, Marcela; Ramirez-Sarmiento, Cesar A.; Federici, Fernan; Gutierrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    Climate change and population persistence in a hibernating marsupial
    (2024) Nespolo, Roberto F.; Quintero-Galvis, Julian F.; Fonturbel, Francisco E.; Cubillos, Francisco A.; Vianna, Juliana; Moreno-Meynard, Paulo; Rezende, Enrico L.; Bozinovic, Francisco
    Climate change has physiological consequences on organisms, ecosystems and human societies, surpassing the pace of organismal adaptation. Hibernating mammals are particularly vulnerable as winter survival is determined by short-term physiological changes triggered by temperature. In these animals, winter temperatures cannot surpass a certain threshold, above which hibernators arouse from torpor, increasing several fold their energy needs when food is unavailable. Here, we parameterized a numerical model predicting energy consumption in heterothermic species and modelled winter survival at different climate change scenarios. As a model species, we used the arboreal marsupial monito del monte (genus Dromiciops), which is recognized as one of the few South American hibernators. We modelled four climate change scenarios (from optimistic to pessimistic) based on IPCC projections, predicting that northern and coastal populations (Dromiciops bozinovici) will decline because the minimum number of cold days needed to survive the winter will not be attained. These populations are also the most affected by habitat fragmentation and changes in land use. Conversely, Andean and other highland populations, in cooler environments, are predicted to persist and thrive. Given the widespread presence of hibernating mammals around the world, models based on simple physiological parameters, such as this one, are becoming essential for predicting species responses to warming in the short term.
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    Domestication signatures in the non-conventional yeast Lachancea cidri
    (2024) Villarreal, Pablo; O'Donnell, Samuel; Agier, Nicolas; Munoz-Guzman, Felipe; Benavides-Parra, Jose; Urbina, Kami; Pena, Tomas A.; Solomon, Mark; Nespolo, Roberto F.; Fischer, Gilles; Varela, Cristian; Cubillos, Francisco A.
    Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (similar to 16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.
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    Fungal Diversity Analysis of Grape Musts from Central Valley-Chile and Characterization of Potential New Starter Cultures
    (2020) Mandakovic, Dinka; Pulgar, Rodrigo; Maldonado, Jonathan; Mardones, Wladimir; Gonzalez, Mauricio; Cubillos, Francisco A.; Cambiazo, Veronica
    Autochthonous microorganisms are an important source of the distinctive metabolites that influence the chemical profile of wine. However, little is known about the diversity of fungal communities associated with grape musts, even though they are the source of local yeast strains with potential capacities to become starters during fermentation. By using internal transcribed spacer (ITS) amplicon sequencing, we identified the taxonomic structure of the yeast community in unfermented and fermented musts of a typicalVitis viniferaL. var. Sauvignon blanc from the Central Valley of Chile throughout two consecutive seasons of production. Unsurprisingly,Saccharomycesrepresented the most abundant fungal genus in unfermented and fermented musts, mainly due to the contribution ofS. uvarum(42.7%) andS. cerevisiae(80%). Unfermented musts were highly variable between seasons and showed higher values of fungal diversity than fermented musts. Since microbial physiological characterization is primarily achieved in culture, we isolated nine species belonging to six genera of fungi from the unfermented must samples. All isolates were characterized for their potential capacities to be used as new starters in wine. Remarkably, onlyMetschnikowia pulcherrimacould co-exist with a commercialSaccharomyces cerevisiaestrain under fermentative conditions, representing a feasible candidate strain for wine production.
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    Nakazawaea atacamensis f.a., sp. nov. a novel nonconventional fermentative ascomycetous yeast species from the Atacama Desert
    (2024) Araya, Macarena; Villarreal, Pablo; Moyano, Tomas; Santos, Ana R. O.; Diaz, Francisca P.; Bustos-Jarufe, Andrea; Urbina, Kamila; del Pino, Javier E.; Groenewald, Marizeth; Gutierrez, Rodrigo A.; Rosa, Carlos A.; Cubillos, Francisco A.
    In this study, we describe Nakazawaea atacamensis f. a., sp. nov., a novel species obtained from Neltuma chilensis plant samples in Chile's hyperarid Atacama Desert. In total, three strains of N. atacamensis were obtained from independent N. chilensis samples (synonym Prosopis chilensis, Algarrobo). Two strains were obtained from bark samples, while the third strain was obtained from bark-exuded gum from another tree. The novel species was defined using molecular characteristics and subsequently characterized with respect to morphological, physiological, and biochemical properties. A neighbor-joining analysis using the sequences of the D1/D2 domains of the large subunit ribosomal RNA gene revealed that N. atacamensis clustered with Nakazawaea pomicola. The sequence of N. atacamensis differed from closely related species by 1.3%-5.2% in the D1/D2 domains. A phylogenomic analysis based on single-nucleotide polymorphism's data confirms that the novel species belongs to the genus Nakazawaea, where N. atacamensis clustered with N. peltata. Phenotypic comparisons demonstrated that N. atacamensis exhibited distinct carbon assimilation patterns compared to its related species. Genome sequencing of the strain ATA-11A-BT revealed a genome size of approximately 12.4 Mbp, similar to other Nakazawaea species, with 5116 protein-coding genes annotated using InterProScan. In addition, N. atacamensis exhibited the capacity to ferment synthetic wine must, representing a potential new yeast for mono or co-culture wine fermentations. This comprehensive study expands our understanding of the genus Nakazawaea and highlights the ecological and industrial potential of N. atacamensis in fermentation processes. The holotype of N. atacamensis sp. nov. is CBS 18375T. The Mycobank number is MB 849680.
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    Identification of new ethanol-tolerant yeast strains with fermentation potential from central Patagonia
    (2022) Villarreal, Pablo; Quintrel, Pablo A.; Olivares-Munoz, Sebastian; Ruiz, Jose J.; Nespolo, Roberto F.; Cubillos, Francisco A.
    The quest for new wild yeasts has increasingly gained attention because of their potential ability to provide unique organoleptic characters to fermented beverages. In this sense, Patagonia offers a wide diversity of ethanol-tolerant yeasts and stands out as a bioprospecting alternative. This study characterized the genetic and phenotypic diversity of yeast isolates obtained from Central Chilean Patagonia and analyzed their fermentation potential under different fermentative conditions. We recovered 125 colonies from Nothofagus spp. bark samples belonging to five yeast species: Saccharomyces eubayanus, Saccharomyces uvarum, Lachancea cidri, Kregervanrija delftensis, and Hanseniaspora valbyensis. High-throughput microcultivation assays demonstrated the extensive phenotypic diversity among Patagonian isolates, where Saccharomyces spp and L. cidri isolates exhibited the most outstanding fitness scores across the conditions tested. Fermentation performance assays under wine, mead, and beer conditions demonstrated the specific potential of the different species for each particular beverage. Saccharomyces spp. were the only isolates able to ferment beer wort. Interestingly, we found that L. cidri is a novel candidate species to ferment wine and mead, exceeding the fermentation capacity of a commercial strain. Unlike commercial strains, we found that L. cidri does not require nutritional supplements for efficient mead fermentation. In addition, L. cidri produces succinic and acetic acids, providing a distinct profile to the final fermented product. This work demonstrates the importance of bioprospecting efforts in Patagonia to isolate novel wild yeast strains with extraordinary biotechnological potential for the fermentation industry.
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    Modeling heterothermic fitness landscapes in a marsupial hibernator using changes in body composition
    (2023) Abarzua, Tamara; Camus, Isidora; Ortiz, Felipe; Nunque, Abel; Cubillos, Francisco A.; Sabat, Pablo; Nespolo, Roberto F.
    Hibernation is an adaptive strategy that allows animals to enter a hypometabolic state, conserving energy and enhancing their fitness by surviving harsh environmental conditions. However, addressing the adaptive value of hibernation, at the individual level and in natural populations, has been challenging. Here, we applied a non-invasive technique, body composition analysis by quantitative magnetic resonance (qMR), to calculate energy savings by hibernation in a population of hibernating marsupials (Dromiciops gliroides). Using outdoor enclosures installed in a temperate rainforest, and measuring qMR periodically, we determined the amount of fat and lean mass consumed during a whole hibernation cycle. With this information, we estimated the daily energy expenditure of hibernation (DEEH) at the individual level and related to previous fat accumulation. Using model selection approaches and phenotypic selection analysis, we calculated linear (directional, beta), quadratic (stabilizing or disruptive, gamma) and correlational (p) coefficients for DEEH and fat accumulation. We found significant, negative directional selection for DEEH (beta(DEEH) = - 0.58 +/- 0.09), a positive value for fat accumulation (beta(FAT) = 0.34 +/- 0.07), and positive correlational selection between both traits (p(DEEH x FAT) = 0.24 +/- 0.07). Then, individuals maximizing previous fat accumulation and minimizing DEEH were promoted by selection, which is visualized by a bi-variate selection surface estimated by generalized additive models. At the comparative level, results fall within the isometric allometry known for hibernation metabolic rate in mammals. Thus, by a combination of a non-invasive technique for body composition analysis and semi-natural enclosures, we were characterized the heterothermic fitness landscape in a semi-natural population of hibernators.
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    Molecular profiling of beer wort fermentation diversity across natural Saccharomyces eubayanus isolates
    (2020) Mardones, Wladimir; Villarroel, Carlos A.; Krogerus, Kristoffer; Tapia, Sebastian M.; Urbina, Kamila; Oporto, Christian I.; O'Donnell, Samuel; Minebois, Romain; Nespolo, Roberto; Fischer, Gilles; Querol, Amparo; Gibson, Brian; Cubillos, Francisco A.
    The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating 'end-to-end' genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2-methylpropanol and 3-methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.
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    Natural Variation in Non-coding Regions Underlying Phenotypic Diversity in Budding Yeast
    (2016) Salinas, Francisco; De Boer, Carl G.; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo Castro, Luis Fernando; Martínez, Claudio; Cubillos, Francisco A.
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    Rapid selection response to ethanol in Saccharomyces eubayanus emulates the domestication process under brewing conditions
    (2022) Mardones, Wladimir; Villarroel, Carlos A.; Abarca, Valentina; Urbina, Kamila; Pena, Tomas A.; Molinet, Jennifer; Nespolo, Roberto F.; Cubillos, Francisco A.
    Although the typical genomic and phenotypic changes that characterize the evolution of organisms under the human domestication syndrome represent textbook examples of rapid evolution, the molecular processes that underpin such changes are still poorly understood. Domesticated yeasts for brewing, where short generation times and large phenotypic and genomic plasticity were attained in a few generations under selection, are prime examples. To experimentally emulate the lager yeast domestication process, we created a genetically complex (panmictic) artificial population of multiple Saccharomyces eubayanus genotypes, one of the parents of lager yeast. Then, we imposed a constant selection regime under a high ethanol concentration in 10 replicated populations during 260 generations (6 months) and compared them with propagated controls exposed solely to glucose. Propagated populations exhibited a selection differential of 60% in growth rate in ethanol, mostly explained by the proliferation of a single lineage (CL248.1) that competitively displaced all other clones. Interestingly, the outcome does not require the entire time-course of adaptation, as four lineages monopolized the culture at generation 120. Sequencing demonstrated that de novo genetic variants were produced in all propagated lines, including SNPs, aneuploidies, INDELs and translocations. In addition, the different propagated populations showed correlated responses resembling the domestication syndrome: genomic rearrangements, faster fermentation rates, lower production of phenolic off-flavours and lower volatile compound complexity. Expression profiling in beer wort revealed altered expression levels of genes related to methionine metabolism, flocculation, stress tolerance and diauxic shift, likely contributing to higher ethanol and fermentation stress tolerance in the evolved populations. Our study shows that experimental evolution can rebuild the brewing domestication process in 'fast motion' in wild yeast, and also provides a powerful tool for studying the genetics of the adaptation process in complex populations.
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    RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast
    (2016) Kessi Pérez, Eduardo I.; Araos, Sebastián; García, Verónica; Salinas, Francisco; Abarca, Valentina; Larrondo Castro, Luis Fernando; Martínez, Claudio; Cubillos, Francisco A.