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Natural history of budding yeast

2009; Elsevier BV; Volume: 19; Issue: 19 Linguagem: Inglês

10.1016/j.cub.2009.07.037

1879-0445

Duncan Greig, Jun‐Yi Leu,

Fungal and yeast genetics research

The microbe humans love best is yeast. For thousands of years it has provided our favourite drug, alcohol, and in return we have fed and housed it. Most of our long love affair has been conducted in the dark, but in 1875 a Danish brewer founded the Carlsberg Laboratory to apply science to the brewing industry, and since then we have pored over the inner workings of Saccharomyces in a frenzy of scientific discovery. But despite our extraordinary intimacy with this organism, mysteries remain. Where does yeast go when it is not with us? What does it feed on and what other species does it interact with? How does it travel? When and where does it have sex? Although we have focussed powerful technology on some parts of yeast biology, we've turned a blind eye to the rest. The application of science to commercial brewing can be traced to Emil Christian Hansen who, working in the Carlsberg Laboratory, discovered how to isolate and propagate pure clones of yeast from single colonies. This technological advance enabled breweries to use a single strain of yeast, in Hansen's case named Saccharomyces carsbergensis — now known to be a hybrid and called S. pastorianus — to allow a consistent brew time after time, in any brewery. Although it can be blamed for the boring uniformity of most commercial lager, it can also be thanked for the science of yeast genetics, which flourished under Hansen's successor, Øjvind Winge. The ability to propagate clones is convenient for geneticists and brewers alike, and consequently most modern yeast laboratories use one of only a handful of well-studied strains. These are not S. pastorianus, which is only used to make lager, but S. cerevisiae, which makes other types of alcoholic drinks like ale, wine, and sake, and also raises bread (by producing, in addition to ethanol, carbon dioxide gas). The ability of many scientists, in different times and places, to focus their attention on what is the genetic equivalent of a single individual, has helped elucidate many of the fundamental cellular processes — such as DNA replication, recombination, RNA splicing, and cell cycle regulation — which are shared by more charismatic, but less convenient eukaryotes, such as ourselves. In 1996, the most popular yeast clone, S288c, became the first eukaryote to have its entire genome sequenced. Thus began the current era of yeast genomic high technologies. Can we now look back and congratulate ourselves that we know the biology of yeast better than that of anything else? Is it just a matter of time and technology before we know everything we need to know about yeast? Not really. The S288c clone was made by crossing a strain found on a rotting fig in 1938 with a number of others, including brewing and baking strains, to produce an offspring with properties useful for the lab. This strain has since been domesticated in a laboratory environment that is, one imagines, about as different from its natural environment as it is possible to get. It is imprisoned, starving and chilled (or even frozen solid) for long periods, and then thrown into a warm sweet soup that is enriched with carcasses of its own species ('yeast extract'). Without competition from other organisms (which microbiologists call contaminants), it grows rapidly to colossal density before a single individual, perhaps a useful mutant or transformant, is plucked out from the crowd and saved. This has had strange consequences for evolution. Deleterious mutations that would normally be removed by natural selection can accumulate in lines going through such population bottlenecks, even if they reduce fitness. Traits that are probably important in the wild but undesirable in the laboratory, such as clumpiness that helps cells stick together to survive environmental stress or preference for some potential mates over others, may be selectively removed. Other traits may deteriorate because they are rarely needed in the lab environment. The extent of this problem is illustrated by the fact that scientists working on sex avoid S288c, which can barely be persuaded to do it, in favour of wilder strains. It is easy to find S. cerevisiae in places associated with alcohol making, like vineyards. But it can also be isolated from a wide variety of other habitats, including oak trees, desert soil, flowers, and even infected hospital patients. Several recent experimental evolution studies have shown that yeast populations can evolve rapidly, adapting to new conditions. And domestic strains often carry adaptations that make them useful for specific purposes, for example baking yeast is good at fermenting the maltose produced in bread dough. Yet in the laboratory, strains from very different sources often look quite similar. Even completely different species are hard to tell apart on the basis of their phenotypes. The closest known relative to S. cerevisiae is S. paradoxus. The two species' genomes are about 15% diverged, which is sufficient to prevent homologous recombination between their chromosomes. Because recombination is required for meiotic chromosome segregation in yeast, F1 hybrids made in the laboratory produce aneuploid and inviable gametes. But although the divergence between the species' genomes is sufficient to cause strong post-zygotic reproductive isolation, the two species appear physiologically nearly identical, although S. cerevisiae seems to grow better than S. paradoxus at high temperatures. Remarkably, no major genetic incompatibilities are apparent between the two species; indeed, F1 hybrids are often fitter than their pure species parents. The apparent phenotypic similarity and genetic compatibility between different Saccharomyces isolates and species might be due to the extremely permissive laboratory environment in which we grow yeast. From gene-deletion projects, we know that only about a fifth of the genes in the yeast genome are essential for growth in normal laboratory conditions. The only yeast speciation gene yet identified is the AEP2 gene of S. bayanus, responsible for incompatibility with the mitochondria of S. cerevisiae. This difference appears to have evolved because S. cerevisiae has specialised in anaerobic fermentation, but S. bayanus is better at aerobic respiration. The incompatibility prevents aerobic respiration, so although hybrids can grow by fermentation on normal yeast medium which is rich in glucose, they cannot grow on non-fermentable carbon sources. Changing other environmental conditions to make them more like those that yeast evolved in might reveal other phenotype differences between strains — for example, S. cerevisiae strains from human patients can grow at the fever-temperature of 42°C, but non-pathogenic strains cannot. For these pathogenic strains, the human body can be approximated by propagating them in laboratory mice, but we have no real idea what the natural environment is for free-living strains such as the laboratory strain S288c. The wide range of habitats in which S. cerevisiae can be found might simply be a consequence of its association with us. Some have argued that S. cerevisiae is a domesticated species, and its natural environment is man-made. But recent phylogenetic analyses of multiple strains have found that grape wine strains and rice wine (like Japanese sake) strains form separate clades which probably originated when alcohol production was discovered by pre-historic humans. Isolates from fruit, fruit-flies, nectar, and oak trees are not closely related to yeast in either of these domesticated clades, suggesting that a truly wild population of S. cerevisiae still exists somewhere. Most S. cerevisiae genomes are mosaics, showing that strains from disparate ecological and geographic sources are often mixed. S. paradoxus is not known to be used by humans, and this makes it attractive to scientists wanting to study wild yeast populations. They have begun systematically to isolate S. paradoxus from oak trees around the world, sometimes finding S. cerevisiae alongside at the same sites. In striking contrast to S. cerevisiae, S. paradoxus populations have strong geographical structure. Strains from Europe, Far East Asia, America, and recently a single isolate from Hawaii, are as much as 5% diverged in genomic nucleotide sequence, sufficient to cause antirecombination and reduced fertility in crosses. Most S. paradoxus polymorphisms are shared only with others within the same continental populations, suggesting that geographic barriers such as the oceans or the Ural Mountains between Europe and the Far East prevent migration. There is about as much genetic variation within each continental population of oak-associated S. paradoxus as there is within S. cerevisiae from all sources (Figure 1). Within continents, mutations are found mixed in different combinations, indicating that each continental population of S. paradoxus is a single recombining sexual population. On a smaller scale, however, identical combinations of alleles can be found again on the same tree or nearby trees, suggesting that clones may predominate locally, either because they are well adapted to the micro-environment or because recombination occurs primarily when they disperse to more distant sites. Interestingly, a single example of an inter-continental dispersal event has recently been identified. An S. paradoxus population was identified in North America that is closely related to a European isolate. Although the immigrants are now sympatric with the native North American population, they appear to be sufficiently diverged to be reproductively isolated from it, suggesting that divergence in allopatry is a potential mechanism for speciation in yeast. These differences between S. cerevisiae and S. paradoxus in dispersal and recombination may be due to human influence on S. cerevisiae. We should be cautious about this explanation, however, because there were also many differences in the way that these species were sampled. Whilst S. cerevisiae has been isolated from many different sources, at many different times, by many different people, using many different methods, most well-studied S. paradoxus samples have been isolated in a similar way, by placing material from oak trees (leaves, bark, exudate, or nearby soil) into a sugary liquid medium and incubating it. This method will sample only oak-associated strains that have higher competitive fitness or higher initial abundance than the other microbes present, potentially missing S. paradoxus strains, or other yeast species, that are less fit under this laboratory protocol. It is interesting to note that, in naturally fermented wine, S. cerevisiae is initially undetectably rare, and only comes to dominate the many other microbial species in the grape juice after producing ethanol to favour its own growth. It might be that, like S. cerevisiae, S. paradoxus is present in a wide range of habitats, perhaps even in some human fermentations, but has gone undetected because of the sampling methods used. Conceivably, there may be undiscovered S. paradoxus populations that enjoy intercontinental dispersal and mixing, or undiscovered wild reservoirs of S. cerevisiae with strong population structures, like the oak-associated S. paradoxus populations. How do yeast travel? It has long been noted that yeast is a favourite food of another well-known human commensal and laboratory model organism, the fruit fly Drosophila melanogaster. Yeast and Drosophila researchers who share a building will testify to the remarkable attractiveness of yeast — AWOL flies head for the yeast lab like thirsty sailors flocking to the nearest bar. Fruit flies seem to be excellent yeast dispersal vectors, and are thought to be essential for inoculating damaged grapes with yeast for making naturally fermented wine. Pioneers in the field, including T. Dobzhansky, E.M. Mrak, and H.J. Phaff, conducted many experiments on this interesting interaction as far back as the 1950s, and it would be well worth revisiting this work armed with new technology and our new understanding of the population structure of oak-associated S. paradoxus. But whilst we can observe fruit flies directly, we cannot see most of the other organisms that might interact with yeast in nature. We know that yeast harbour double-stranded RNA viruses, but these viruses appear to be non-infectious, and are passed on only from cell to cell as the host reproduces. Many of these yeast viruses are actually beneficial — they encode chemical weapons that kill their host's microbial competitors. Yeast can also produce 'public goods', such as extracellular glucose generated by the enzyme invertase. Cells that do not make invertase can use the glucose produced by cells that do, producing complex local interactions that probably explain the high genetic variation in genes encoding these types of 'social' trait. Ecological interactions with predators, pathogens, competitors, and cooperators generate complex and dynamic selective forces driving evolution that cannot be understood simply by studying monocultures of yeast in the laboratory. Laboratory studies have shown that Saccharomyces is capable of a complex life cycle, with several possible responses to different conditions (Figure 2). Because yeast are too small to be observed directly in nature, when (or even whether) all these different life cycle events occur is not known. S. paradoxus is always recovered from oak tree samples as a mitotically growing diploid. But we don't know what form the cells in the sample were in before they were sampled; indeed, we don't even know that they actually grow on trees, only that they are found on them. In the laboratory, diploidy seems to be the preferred state under ideal growth conditions, and a population of diploid cells can reproduce by mitosis apparently indefinitely. Each new diploid cell is capable of producing about 20–30 daughter cells before its reproduction ceases. When nutrients become depleted, mitotic cell division stops and some cells may enter meiosis, producing four haploid spores enclosed in an envelope called the ascus and joined by interspore bridges. The mating type of the spores is determined by the allele at the MAT locus: diploids are heterozygous at MAT so two of the spores in the ascus inherit MATα and the other two inherit MATa. In some ascii, one or more of the meiotic products are lost and fewer than four spores are produced. Ascii with only two spores usually contain one of each mating type. Reducing the amount of carbon available during sporulation can increase the proportion of ascii containing fewer than four spores, a phenomenon known as spore number control. Many cells in a nutrient-depleted population do not do meiosis, but can survive in a quiescent state for months or years. Other cells die in a manner that closely resembles programmed cell death in multicellular organisms, which some scientists interpret as a form of altruism directed towards clonemates who might benefit from the scarce resources that are saved or released by the suicide of their kin. These different responses to nutrient depletion are physiologically diverse and depend strongly on both the genetic background and the environmental conditions. It is not clear what the adaptive significance of these different responses is. What is clear is that yeast can survive with little or no nutrients for a long time. Winge was able to start work on the same strains of yeast that Hansen had stored at the Carlsberg Laboratory forty years before, and another brewery now claims to be using yeast isolated from 45 million year old amber! Our lab-centric view, coming from decades of studying dividing cells, that stationary phase cells are just passively awaiting food now seems overly simplistic. In nature it seems more likely that yeast spend most of their time in low nutrient conditions, and adaptations to this environment might be more important for yeast evolution than the high growth rates that laboratory experiments typically focus on. When nutrients become abundant again, haploid spores can germinate and become metabolically active gametes. In the laboratory, if the spores are dissected from an ascus before being placed on nutrient agar, about 90–99% of them germinate synchronously, with the remaining being apparently inviable. The two mating-types produce different pheromones allowing signalling and cell fusion only between haploid gametes of different mating-types. The metabolic cost of signalling is high and haploids evolved in an environment in which the pheromone is not selected readily lose it, enjoying a significantly higher asexual fitness as a result. Detection of the pheromone is exquisitely sensitive, allowing receivers to accurately choose the strongest signaller. This costly mating system may have evolved to allow a cell to choose the best quality mate on the basis of its ability to afford the strongest signal. But the ascus structure would seem to ensure that, in most cases, the choice of mates is only between haploid products of the same meiosis. In the laboratory, unless measures are taken to disrupt tetrads, most mating does indeed take this form of automixis. Like diploids, haploid gametes can also divide by asexual mitosis, and can also survive nutrient depletion by entering a quiescent state. Additionally, haploids that have produced mitotic offspring and that carry a functional HO gene are homothallic — they can switch mating-type by changing the allele at the MAT locus and then, in the most extreme form of inbreeding possible (haplo-selfing), mate with the daughter cell they just produced, making a diploid that is perfectly homozygous except at the MAT locus. Although these different modes of sexual reproduction cannot be observed directly in nature, they can be detected by their effects on the evolution of the genome. Recombination means that different genomic segments have different genealogical histories, so recombination events can be detected by the disagreements between the phylogenetic trees produced using these different segments of the genome. Using this approach on three completely sequenced strains of S. cerevisiae aligned to a single strain of S. paradoxus, it was possible to estimate the rate of between-tetrad mating (outcrossing) in S. cerevisiae as just one event in every 50,000 mitotic generations. Populations of wild S. paradoxus from the Far East and from Europe have now been sampled enough to make more direct population genetic estimates of several other interesting life cycle parameters. From the mutational diversity and the known per-division mutation rate, the effective population sizes were estimated to be about eight million individuals in both populations. This is much lower than the likely number of cells in each population — perhaps because cells in a colony probably all live and die together, the estimate may be closer to the effective number of colonies. From the diversity generated by mitosis (mutation) and the diversity generated by meiosis (recombination), it was possible to estimate that one meiosis occurs approximately every thousand mitoses in Europe and every three thousand mitoses in the Far East. Because mating can occur only between different mating types, the region linked to MAT is more outbred than regions further away. But mating with a clonemate following HO-induced mating-type switching removes this effect — the whole genome is inbred except MAT itself. By measuring how recombination diversity increases with distance from MAT, the ratio of within-tetrad mating to haplo-selfing could be estimated. And from the frequency of heterozygotes it was then possible to estimate the within-tetrad mating rate as 94%, the haplo-selfing mating rate as 5%, and the between-tetrad mating rate as 1% — the first estimates of these important life cycle parameters from wild populations. These estimates of the frequencies of different modes of mating are population averages. Like the different potential responses to nutrient depletion, their different adaptive functions are not clear but it seems likely that they could be modulated according to environmental or ecological cues. For example, the tetrad ascus may normally promote automixis to preserve locally adapted gene complexes. But ascii eaten by Drosophila are digested, freeing the digestion-resistant spores to mate with those from other tetrads, potentially increasing genetic variation in response to dispersal to new habitats. Whilst oak-associated S. paradoxus strains are homothallic homozygous diploids, S. cerevisiae isolates are often heterothallic — with a non-functional HO allele, unable to switch haploid mating type — heterozygous, and polyploid. These differences may reflect the environments that were sampled rather than any intrinsic differences between the species. The genetic redundancy produced by whole genome duplication might confer benefits such as increased resistance to deleterious mutations and greater adaptive potential, so it may be favoured in harsh or novel environments. In laboratory experiments, polyploid or aneuploid cells were often observed when yeast adapt to stressful conditions, suggesting that yeast cells can benefit from gross changes in their genome contents. The fact that the Saccharomyces sensu stricto yeasts have evolved from an ancient tetraploid underscores the potential importance of major chromosomal changes in yeast. Yeast technology began when mankind learned how to brew a pleasant drink, and it has since developed unprecedented scientific power. This power is now being applied to ecology and evolution, with several impressive initial successes. This is not merely a good application of yeast technology to a new field, it is also a way to increase the power of that technology further. We cannot properly interpret the enormous data being produced from model laboratory yeast without knowledge about the environment that yeast evolved in. Determining the natural history of yeast is a daunting challenge but metagenomics, the analysis of environmental DNA without laboratory culturing, offers the possibility of unbiased sampling of the microbial communities in which yeast live. With sufficient knowledge of the abundance of yeast in space and time, combined with further population genetic and experimental methods, we can perhaps start to see the world from a yeast's point of view.