A Systematic Review on Quiescent State Research Approaches in S. cerevisiae.

Quiescence, the temporary and reversible arrest of cell growth, is a fundamental biological process. However, the lack of standardization in terms of reporting the experimental details of quiescent cells and populations can cause confusion and hinder knowledge transfer. We employ the systematic review methodology to comprehensively analyze the diversity of approaches used to study the quiescent state, focusing on all published research addressing the budding yeast Saccharomyces cerevisiae. We group research articles into those that consider all cells comprising the stationary-phase (SP) population as quiescent and those that recognize heterogeneity within the SP by distinguishing phenotypically distinct subpopulations. Furthermore, we investigate the chronological age of the quiescent populations under study and the methods used to induce the quiescent state, such as gradual starvation or abrupt environmental change. We also assess whether the strains used in research are prototrophic or auxotrophic. By combining the above features, we identify 48 possible experimental setups that can be used to study quiescence, which can be misleading when drawing general conclusions. We therefore summarize our review by proposing guidelines and recommendations pertaining to the information included in research articles. We believe that more rigorous reporting on the features of quiescent populations will facilitate knowledge transfer within and between disciplines, thereby stimulating valuable scientific discussion.

A pluralistic view of holobionts in the context of process ontology.

Developing precise definitions and fine categories is an important part of the scientific endeavour, enabling fidelity of transfers of knowledge and the progress of science. Currently, as a result of research on symbiotic microorganisms, science has been flooded with discoveries which appear to undermine many commonly accepted concepts and to introduce new ones that often require updated conceptualisations. One question currently being debated concerns whether or not a holobiont can be considered an organism. Based on which concept, physiology or evolutionary, of the organism is chosen, the verdict differs. We attempt here to show how a change in perspective, from that of substance ontology into that of process ontology, is capable of reconciling opposing positions within the existing discussion and enabling the implementation of conceptual pluralism.

On the Ecological Significance of Phenotypic Heterogeneity in Microbial Populations Undergoing Starvation.

To persist in variable environments, populations of microorganisms have to survive periods of starvation and be able to restart cell division in nutrient-rich conditions. Typically, starvation signals initiate a transition to a quiescent state in a fraction of individual cells, while the rest of the cells remain nonquiescent. It is widely believed that, while quiescent (Q) cells help the population to survive long starvation, the nonquiescent (NQ) cells are a side effect of imperfect transition. We analyzed the regrowth of starved monocultures of Q and NQ cells compared to that of mixed, heterogeneous cultures from simple and complex starvation environments. Our experiments, as well as mathematical modeling, demonstrate that Q monocultures benefit from better survival during long starvation and from a shorter lag phase after resupply of rich medium. However, when the starvation period is very short, the NQ monocultures outperform Q and mixed cultures due to their short lag phase. In addition, only NQ monocultures benefit from complex starvation environments, where nutrient recycling is possible. Our study suggests that phenotypic heterogeneity in starved populations could be a form of bet hedging that is adaptive when environmental determinants, such as the length of the starvation period, the length of the regrowth phase, and the complexity of the starvation environment, vary over time. IMPORTANCE Nongenetic cell heterogeneity is present in glucose-starved yeast populations in the form of quiescent (Q) and nonquiescent (NQ) phenotypes. There is evidence that Q cells help the population survive long starvation. However, the role of the NQ cell type is not known, and it has been speculated that the NQ phenotype is just a side effect of the imperfect transition to the Q phenotype. Here, we show that, in contrast, there are ecological scenarios in which NQ cells perform better than monocultures of Q cells or naturally occurring mixed populations containing both Q and NQ cells. NQ cells benefit when the starvation period is very short and environmental conditions allow nutrient recycling during starvation. Our experimental and mathematical modeling results suggest a novel hypothesis: the presence of both Q and NQ phenotypes within starved yeast populations may reflect a form of bet hedging where different phenotypes provide fitness advantages depending on the environmental conditions.

Forest Saccharomyces paradoxus are robust to seasonal biotic and abiotic changes.

Microorganisms are famous for adapting quickly to new environments. However, most evidence for rapid microbial adaptation comes from laboratory experiments or domesticated environments, and it is unclear how rates of adaptation scale from human-influenced environments to the great diversity of wild microorganisms. We examined potential monthly-scale selective pressures in the model forest yeast Saccharomyces paradoxus. Contrary to expectations of seasonal adaptation, the S. paradoxus population was stable over four seasons in the face of abiotic and biotic environmental changes. While the S. paradoxus population was diverse, including 41 unique genotypes among 192 sampled isolates, there was no correlation between S. paradoxus genotypes and seasonal environments. Consistent with observations from other S. paradoxus populations, the forest population was highly clonal and inbred. This lack of recombination, paired with population stability, implies that selection is not acting on the forest S. paradoxus population on a seasonal timescale. Saccharomyces paradoxus may instead have evolved generalism or phenotypic plasticity with regard to seasonal environmental changes long ago. Similarly, while the forest population included diversity among phenotypes related to intraspecific interference competition, there was no evidence for active coevolution among these phenotypes. At least ten percent of the forest S. paradoxus individuals produced "killer toxins," which kill sensitive Saccharomyces cells, but the presence of a toxin-producing isolate did not predict resistance to the toxin among nearby isolates. How forest yeasts acclimate to changing environments remains an open question, and future studies should investigate the physiological responses that allow microbial cells to cope with environmental fluctuations in their native habitats.

Hypersensitive SSY1 mutations negatively influence transition to quiescence in yeast Saccharomyces cerevisiae.

Most cells spend the majority of their life in the non-proliferating, quiescent state. Transition to this state is crucial for microorganisms to survive long starvation periods and restart divisions afterwards. Experimental evolution allowed us to identify several mutation in genes that are presumably important for such transition in yeast cells. Most of these candidate genes belong to the SPS amino acid sensing pathway or to the SIR complex. We assembled these mutations on the ancestral strain background. Analysis of the quiescent/non-quiescent cell ratio of the starved yeast populations confirmed the crucial role of SSY1, the primary receptor component of the SPS sensor, in transition to the Q state. The evolved SSY1 mutations increased yeast sensitivity to amino acid presence in the environment. This resulted in decreased quiescent cell fraction and a 5.14% increase of the total amino acid content in the starved populations. We discuss external amino acid sensing via the SPS pathway as one of the mechanisms influencing transition to quiescence.

Aspects of Multicellularity in Saccharomyces cerevisiae Yeast: A Review of Evolutionary and Physiological Mechanisms.

The evolutionary transition from single-celled to multicellular growth is a classic and intriguing problem in biology. Saccharomyces cerevisiae is a useful model to study questions regarding cell aggregation, heterogeneity and cooperation. In this review, we discuss scenarios of group formation and how this promotes facultative multicellularity in S. cerevisiae. We first describe proximate mechanisms leading to aggregation. These mechanisms include staying together and coming together, and can lead to group heterogeneity. Heterogeneity is promoted by nutrient limitation, structured environments and aging. We then characterize the evolutionary benefits and costs of facultative multicellularity in yeast. We summarize current knowledge and focus on the newest state-of-the-art discoveries that will fuel future research programmes aiming to understand facultative microbial multicellularity.

Correction to: Some theoretical insights into the hologenome theory of evolution and the role of microbes in speciation.

The original version of this article unfortunately contained a mistake.

Some theoretical insights into the hologenome theory of evolution and the role of microbes in speciation.

Research on symbiotic communities (microbiomes) of multicellular organisms seems to be changing our understanding of how species of plants and animals have evolved over millions of years. The quintessence of these discoveries is the emergence of the hologenome theory of evolution, founded on the concept that a holobiont (a host along with all of its associated symbiotic microorganisms) acts a single unit of selection in the process of evolution. Although the hologenome theory has become very popular among certain scientific circles, its principles are still being debated. In this paper, we argue, firstly, that only a very small number of symbiotic microorganisms are sufficiently integrated into multicellular organisms to act in concert with them as units of selection, thus rendering claims that holobionts are units of selection invalid. Secondly, even though holobionts are not units of selection, they can still constitute genuine units from an evolutionary perspective, provided we accept certain constraints: mainly, they should be considered units of co-operation. Thirdly, we propose a reconciliation of the role of symbiotic microorganisms with the theory of speciation through the use of a developed framework. Mainly, we will argue that, in order to understand the role of microorganisms in the speciation of multicellular organisms, it is not necessary to consider holobionts units of selection; it is sufficient to consider them units of co-operation.

Division of labour in the yeast: Saccharomyces cerevisiae.

Division of labour between different specialized cell types is a central part of how we describe complexity in multicellular organisms. However, it is increasingly being recognized that division of labour also plays an important role in the lives of predominantly unicellular organisms. Saccharomyces cerevisiae displays several phenotypes that could be considered a division of labour, including quiescence, apoptosis and biofilm formation, but they have not been explicitly treated as such. We discuss each of these examples, using a definition of division of labour that involves phenotypic variation between cells within a population, cooperation between cells performing different tasks and maximization of the inclusive fitness of all cells involved. We then propose future research directions and possible experimental tests using S. cerevisiae as a model organism for understanding the genetic mechanisms and selective pressures that can lead to the evolution of the very first stages of a division of labour. Copyright © 2017 John Wiley & Sons, Ltd.

Adaptive Roles of SSY1 and SIR3 During Cycles of Growth and Starvation in Saccharomyces cerevisiae Populations Enriched for Quiescent or Nonquiescent Cells.

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth-starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population's fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients (ECM21, RSP5, MSN1, SIR4, and IRA2) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1 Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures.

Rapid multiple-level coevolution in experimental populations of yeast killer and nonkiller strains.

Coevolution between different biological entities is considered an important evolutionary mechanism at all levels of biological organization. Here, we provide evidence for coevolution of a yeast killer strain (K) carrying cytoplasmic dsRNA viruses coding for anti-competitor toxins and an isogenic toxin-sensitive strain (S) during 500 generations of laboratory propagation. Signatures of coevolution developed at two levels. One of them was coadaptation of K and S. Killing ability of K first increased quickly and was followed by the rapid invasion of toxin-resistant mutants derived from S, after which killing ability declined. High killing ability was shown to be advantageous when sensitive cells were present but costly when they were absent. Toxin resistance evolved via a two-step process, presumably involving the fitness-enhancing loss of one chromosome followed by selection of a recessive resistant mutation on the haploid chromosome. The other level of coevolution occurred between cell and killer virus. By swapping the killer viruses between ancestral and evolved strains, we could demonstrate that changes observed in both host and virus were beneficial only when combined, suggesting that they involved reciprocal changes. Together, our results show that the yeast killer system shows a remarkable potential for rapid multiple-level coevolution.

Sociobiology of the budding yeast.

Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitnessbased Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis.

Generation of stable, non-aggregating Saccharomyces cerevisiae wild isolates.

Cellular aggregates observed during growth of Saccharomyces cerevisiae strains derived from various natural environments makes most laboratory techniques optimized for non-aggregating laboratory strains inappropriate. We describe a method to reduce the size and percentage of the aggregates. This is achieved by replacing the native allele of the AMN1 gene with an allele found in the W303 laboratory strain. The reduction in aggregates is consistent across various environments and generations, with no change in maximum population density or strain viability, and only minor changes in maximum growth rate and colony morphology.

Effect of dispersal and nutrient availability on the competitive ability of toxin-producing yeast.

The ecological role of interference competition through toxin production is not well understood. In particular, it is unclear under what conditions the benefits of toxic killing outweigh the metabolic costs involved. A killer advantage has been suggested to rely on local competitive interactions where the benefits of killing accrue to the toxin producer preferentially, but this notion has little empirical support. In addition, contrasting predictions exist about the effect of resource abundance on the benefits of toxin production; this benefit should either be highest when resources are abundant and metabolic costs are relatively low or when resources are scarce and toxic killing is a 'last resort strategy' to obtain nutrients. Here, we test these predictions for one aspect of competitive ability, that is, the ability of toxin producers to invade a population of sensitive non-producers from a low initial frequency. We use competition experiments between isogenic K1 toxin-producing and non-producing strains of Saccharomyces cerevisiae, where we manipulate dispersal under two extreme nutrient conditions: one environment with and the other without replenishment of nutrients. We find that toxin production is beneficial when dispersal is limited under both nutrient conditions, but only when resources are abundant these outweigh its cost and allow invasion of the producer.