Role of anti-competitor toxins in the origin and maintenance of diversity in Saccharomyces yeast microbial populations

Abstract Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution. In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains). Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4). Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry. </p

Leaf breakdown rates : a measure of water quality?

The breakdown rates of Alnus glutinosa leaves and the structure of macroinvertebrate communities were used to evaluate the impact of the village of Montalegre (Portugal) on the water quality of the Cávado river. Chemical and microbial analyses of stream water indicated a high organic load in the vicinity of the village. The abundance of macroinvertebrates associated with leaves increased along the pollution gradient, whereas richness of the community decreased. Biotic indices and multivariate analysis applied to aquatic macroinvertebrate communities discriminated polluted from non-polluted sites. Exponential breakdown rates of alder leaves were high (0.014 to 0.060 day-1) and the differences observed among sites suggested that nutrient enrichment stimulated leaf breakdown significantly. Leaf breakdown rates have not reflected improved biotic conditions as assessed by biotic indices at the most downstream site. These results suggest that both data from the structure and function of a stream are important for assessing water quality

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Microbes can engage in social interactions ranging from cooperation to warfare. Biofilms are structured, cooperative microbial communities. Like all cooperative communities, they are susceptible to invasion by selfish individuals who benefit without contributing. However, biofilms are pervasive and ancient, representing the first fossilized life. One hypothesis for the stability of biofilms is spatial structure: Segregated patches of related cooperative cells are able to outcompete unrelated cells. These dynamics have been explored computationally and in bacteria; however, their relevance to eukaryotic microbes remains an open question. The complexity of eukaryotic cell signaling and communication suggests the possibility of different social dynamics. Using the tractable model yeast, Saccharomyces cerevisiae, which can form biofilms, we investigate the interactions of environmental isolates with different social phenotypes. We find that biofilm strains spatially exclude nonbiofilm strains and that biofilm spatial structure confers a consistent and robust fitness advantage in direct competition. Furthermore, biofilms may protect against killer toxin, a warfare phenotype. During biofilm formation, cells are susceptible to toxin from nearby competitors; however, increased spatial use may provide an escape from toxin producers. Our results suggest that yeast biofilms represent a competitive strategy and that principles elucidated for the evolution and stability of bacterial biofilms may apply to more complex eukaryotes

Herbivore regulation of plant abundance in aquatic ecosystems.

Herbivory is a fundamental process that controls primary producer abundance and regulates energy and nutrient flows to higher trophic levels. Despite the recent proliferation of small-scale studies on herbivore effects on aquatic plants, there remains limited understanding of the factors that control consumer regulation of vascular plants in aquatic ecosystems. Our current knowledge of the regulation of primary producers has hindered efforts to understand the structure and functioning of aquatic ecosystems, and to manage such ecosystems effectively. We conducted a global meta-analysis of the outcomes of plant-herbivore interactions using a data set comprised of 326 values from 163 studies, in order to test two mechanistic hypotheses: first, that greater negative changes in plant abundance would be associated with higher herbivore biomass densities; second, that the magnitude of changes in plant abundance would vary with herbivore taxonomic identity. We found evidence that plant abundance declined with increased herbivore density, with plants eliminated at high densities. Significant between-taxa differences in impact were detected, with insects associated with smaller reductions in plant abundance than all other taxa. Similarly, birds caused smaller reductions in plant abundance than echinoderms, fish, or molluscs. Furthermore, larger reductions in plant abundance were detected for fish relative to crustaceans. We found a positive relationship between herbivore species richness and change in plant abundance, with the strongest reductions in plant abundance reported for low herbivore species richness, suggesting that greater herbivore diversity may protect against large reductions in plant abundance. Finally, we found that herbivore-plant nativeness was a key factor affecting the magnitude of herbivore impacts on plant abundance across a wide range of species assemblages. Assemblages comprised of invasive herbivores and native plant assemblages were associated with greater reductions in plant abundance compared with invasive herbivores and invasive plants, native herbivores and invasive plants, native herbivores and mixed-nativeness plants, and native herbivores and native plants. By contrast, assemblages comprised of native herbivores and invasive plants were associated with lower reductions in plant abundance compared with both mixed-nativeness herbivores and native plants, and native herbivores and native plants. However, the effects of herbivore-plant nativeness on changes in plant abundance were reduced at high herbivore densities. Our mean reductions in aquatic plant abundance are greater than those reported in the literature for terrestrial plants, but lower than aquatic algae. Our findings highlight the need for a substantial shift in how biologists incorporate plant-herbivore interactions into theories of aquatic ecosystem structure and functioning. Currently, the failure to incorporate top-down effects continues to hinder our capacity to understand and manage the ecological dynamics of habitats that contain aquatic plants

Phytol: A review of biomedical activities

© 2018 Elsevier Ltd. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (Auguist 2018) in accordance with the publisher’s archiving policyPhytol (PYT) is a diterpene member of the long-chain unsaturated acyclic alcohols. PYT and some of its derivatives, including phytanic acid (PA), exert a wide range of biological effects. PYT is a valuable essential oil (EO) used as a fragrance and a potential candidate for a broad range of applications in the pharmaceutical and biotechnological industry. There is ample evidence that PA may play a crucial role in the development of pathophysiological states. Focusing on PYT and some of its most relevant derivatives, here we present a systematic review of reported biological activities, along with their underlying mechanism of action. Recent investigations with PYT demonstrated anxiolytic, metabolism-modulating, cytotoxic, antioxidant, autophagy- and apoptosis-inducing, antinociceptive, anti-inflammatory, immune-modulating, and antimicrobial effects. PPARs- and NF-κB-mediated activities are also discussed as mechanisms responsible for some of the bioactivities of PYT. The overall goal of this review is to discuss recent findings pertaining to PYT biological activities and its possible applications

Role of anti-competitor toxins in the origin and maintenance of diversity in Saccharomyces yeast microbial populations

Abstract Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution. In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains). Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4). Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry