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

M.D. Pieczynska

Research output: Thesisinternal PhD, WU

Abstract

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.

 

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Zwaan, Bas, Promotor
  • de Visser, Arjan, Co-promotor
Award date9 Jun 2015
Place of PublicationWageningen
Publisher
Print ISBNs9789462573093
Publication statusPublished - 2015

Keywords

  • yeasts
  • rna viruses
  • toxins
  • symbiosis
  • toxicity
  • coevolution
  • phenotypes
  • fermentation

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