June 05, 2017
Back in 1915, writer Elbert Hubbard coined the phrase, “When life gives you lemons make lemonade.” (His actual quote was “He picked up the lemons that Fate had sent him and started a lemonade-stand.”)
The idea of course is to take something bad and make it into something good. Like, if your research gives you terribly weak glue, invent Post-It notes.
Or as Hope and coworkers show in a new study in GENETICS, when your yeast experiment gets gummed up because the yeast evolves a sticky trait, do additional experiments to learn about the evolution of that complex trait. And “invent” a way to make the yeast less likely to flocculate, or stick together.
This new “invention” will be very useful for anyone trying to evolve yeast to create new products or to study how evolution works. It also showed that for this trait, under these conditions with this strain, there was one major way to get to flocculence—up-regulating the FLO1 gene. This meant they could greatly reduce the risk of this trait popping up by simply deleting FLO1.
Studying evolution in yeast often involves using a chemostat, an automated way to keep the yeast growing through repeated dilutions with fresh media. Scientists use this method to study how yeast evolves under varying conditions over hundreds of generations.
An unfortunate side effect of this method is that it also tends to select for yeast that stick together. These yeast are diluted away less, often meaning they become more common over the generations.
In this study, Hope and coworkers ran 96 chemostats under three different conditions for 300 generations and found that in 34.7% of the cultures, the yeast ended up aggregated. This was even though they used a strain of S288c in which the FLO8 gene was mutated. This strain flocculates less often than wild type!
These authors picked the 23 most aggregated cultures to study in more detail. They found that 2 out of 23 strains aggregated because of a mother/daughter separation defect. The rest were more run-of-the-mill flocculent strains.
They next used whole genome sequencing to try to identify which genes when mutated caused flocculence in the strains. They saw no FLO8 revertants.
The two strains with a mother/daughter separation defect both had mutations in the ACE2 gene, which encodes an important transcription factor for septation as well as other processes.
FLO1-related mutations dominated the other 21. They found a couple of different Ty insertions in the promoter region of FLO1 in 12 of the strains and mutations in TUP1 in 5 more. Tup1p is a general repressor known to repress FLO1, so it looks like up-regulating FLO1 leads to flocculent cultures. Other candidate mutations were found in FLO9 and ROX3.
They wanted to try to identify the responsible gene(s) in strains where there was no obvious candidate gene and also to confirm that the genes they identified really caused the trait, so they next did backcrosses between each mutant strain and a wild type strain.
The backcrosses identified two other ways to get flocculence— by mutating either CSE2 or MIT1. The researchers also confirmed that the mutations they found, including these two new ones, were probably the main cause of the flocculence in each individual strain. This trait co-segregated with the appropriate mutation in a 2:2 pattern for 20/21 strains as is predicted for a single causal mutation.
The results are even more FLO1-heavy than they appear. Further studies showed that ROX3, CSE2, and MIT1 all require a functional FLO1 to see their effects.
So FLO1 appears to be the main route to flocculence in this strain. And the next set of experiments confirmed this.
Hope and coworkers ran 32 wild type and 32 FLO1 knockout strains in separate chemostats for 250 generations and found that 8 wild-type strains flocculated while only 1 FLO1 knockout strain did. Knocking out FLO1 seems to make for a more well-behaved yeast (at least in terms of evolving a flocculent trait in a chemostat).
And the strain can probably be improved upon even more. For example, researchers may want to limit Ty mobility as this was a major way that FLO1 was up-regulated, increase the copy number of key repressors or link those repressors to essential genes. Another possibility is to also mutate FLO9 as its up-regulation was the cause of that one aggregated strain in the FLO1 knockout experiment.
Researchers no longer need to “settle” (subtle, huh?) for big parts of their experiments being hampered by gummy yeast. Hope and coworkers have created a strain that is less likely to flocculate by simply knocking out FLO1. Not as ubiquitously useful as a Post-It note but a potential Godsend for scientists using yeast to understand evolution.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
October 14, 2015
All over the world and through the ages, people have moved from the country into the big city to look for a better life. These folks often find that even though they can adapt to city life and city ways, they still hang on to their core country values. As the old saying goes, “You can take the boy out of the country, but you can’t take the country out of the boy.”
Our friend Saccharomyces cerevisiae didn’t migrate voluntarily into the lab. But it ended up there, and has been as lonely as a new migrant in a big city.
Which is of course how we need it to be. One of the basic tenets of classical microbiology is that you can’t begin to study an organism until you’ve isolated it in a pure culture.
And studying pure S. cerevisiae has yielded a huge body of knowledge about molecular biology, cell biology, and genetics. But by not studying yeast in the context of its old country home, we may have missed a few things.
In a new article in PLOS ONE, Rossouw and colleagues uncover one of them. S. cerevisiae has a family of FLO genes that promote flocculation, the adherence of yeast cells to each other. It has always been a bit puzzling why a whole family of genes that are pretty much redundant with each other would be maintained through evolution.
When the researchers took S. cerevisiae out of its lab isolation by mixing it with other yeast species, they found that the different flocculation genes actually determine which species it can co-flocculate with. Different Flo proteins prefer different partners.
This discovery helps us understand the evolution of this gene family and also opens the door to further study of inter-species interactions in the vineyard. And since flocculation is an important property in winemaking and brewing, there could even be tasty practical applications of this knowledge.
The researchers started by surveying 18 non-Saccharomyces yeast strains that are found in vineyards. They looked at the ability of the yeasts to flocculate both as pure strains and when mixed with either of two S. cerevisiae wine strains.
Intriguingly, certain species showed a synergistic effect when mixed with S. cerevisiae, flocculating more than either species on its own. Rossouw and colleagues used microscopy to confirm that the “flocs” did indeed contain both yeast species—a simple observation, since the cells of different species have slightly different shapes.
To test the effects of different FLO genes on co-flocculation, the authors assayed the co-flocculation ability of flo1, flo10, and flo11 deletion mutants as well as Flo1, Flo5, and Flo11 overproducers in individual combinations with six of the non-Saccharomyces yeasts.
The results showed that Flo1 has general effects on flocculation. Overproduction increased co-flocculation across the board with all the species tested, while deletion of FLO1 consistently decreased it. In contrast, deletion of FLO10 didn’t have much effect on co-flocculation.
It was a different story for Flo5 and Flo11, though. Overproduction of each of these not only affected co-flocculation, but had species-specific or even strain-specific effects. Flo5 overproduction caused a relative increase in co-flocculation with Metchnikowia fructicola and a substantial decrease in co-flocculation with two different strains of Hanseniaspora opuntiae. Flo11 overproduction reduced co-flocculation with one of the Hanseniaspora opuntiae strains but not with the other.
All of these experiments were done on mixtures of two species at a time. To get S. cerevisiae even further out of the lab, Rossouw and colleagues created a “consortium” of wine yeasts, a mixture of six species that are found in wine must (freshly pressed grapes) at the start of fermentation. They then added the FLO overproducer strains individually to the consortium, to see their effects in a more natural situation.
They let the yeast consortium flocculate, extracted total DNA from the flocculated or supernatant parts of the culture, and then used automated ribosomal intergenic spacer analysis (ARISA) to see which strains had co-flocculated. This technique can determine the relative abundance of different yeast species in a sample by sequencing a particular region of ribosomal DNA.
In this experiment, overexpression of each of the three FLO genes had significant effects on at least one of the species in the consortium. The species composition of the flocculated yeasts was uniquely different, depending on which gene was overexpressed.
The discovery that the flocculation genes have individual effects on association with other species goes a long way towards explaining why S. cerevisiae has maintained this gene family with so many members that apparently have the same function—at least, when you study a pure culture. Differential regulation of the FLO genes could affect the spectrum of other species that our favorite yeast interacts with.
So, our friend S. cerevisiae didn’t actually get out of the lab in these experiments, but at least it got to rub shoulders with some of its old friends (buds?) from the vineyard. These experiments are a good reminder for researchers to think outside the lab.
And when S. cerevisiae and its friends get together outside the lab, beautiful things can happen. We’ll drink a toast to that!
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
If yeast could sing about its forced migration to the lab, it might sound like this.
Categories: Research Spotlight
July 16, 2012
Most strains of Saccharomyces cerevisiae don’t stick together very well. And hardly any of them form biofilms. But it would be very useful to have a better understanding of why some strains like to stick together and others do not.
Stickiness helps in any process where you want the yeast to do something and then get rid of it. An obvious example is ethanol production either for energy or to make our beer and wine. After sticky yeast are done with their job of making the alcohol, they simply fall to the bottom of the fermentor or float on the surface in a biofilm (the “flor”). This makes the step of separating the yeast from the finished product that much easier.
Understanding more details of yeast stickiness would also be useful for studying harmful yeast. Adhesion to other cells and to substrates is an important factor in pathogenesis. It would be nice to investigate this phenomenon in the more tractable brewer’s yeast.
The Ibeas lab has decided to figure out why most strains of S. cerevisiae can’t flocculate by comparing one of the few that can (the “flor” strain used to make sherry) to a reference strain that can’t. They previously showed that a key gene in the process, FLO11 (also known as MUC1), is expressed at much higher levels in flor. They were also able to show that a large part of this increased expression comes from a 111 base pair deletion in the FLO11 promoter in this particular strain.
In a recent paper in GENETICS, Barrales and coworkers set out to investigate why the loss of these 111 base pairs leads to increased gene expression. They were able to conclude that the deletion does not significantly affect histone occupancy at the promoter. What they could see was that histone placement was affected and that PHO23 may play a significant role in this.
The researchers had previously shown that the histone deacetylase complex (HDAC) Rpd3L was important for maximal FLO11 activity. They next wanted to determine if this complex was the major player in explaining the increased activity of the 111 bp deletion FLO11 promoter (Δ111) over the wild type (WT) one. They did this by comparing the level of mRNA made by each promoter in strains lacking either the Pho23p or the Rpd3p subunits of the Rpd3L complex. They found that the Δ111 construct was much more severely compromised by the loss of PHO23 than was the WT one. (A bit confusingly, neither was much affected by the loss of RPD3.)
Given that PHO23 is part of a complex that affects chromatin, the next thing the researchers did was look at the histones in and around both FLO11 promoters. They found that PHO23 was involved in maintaining an open chromatin structure at the FLO11 promoter but that deleting the 111 base pairs didn’t affect this process significantly.
Where they started to see subtle differences was when they looked at histone placement as opposed to occupancy. Using micrococcal nuclease protection to map chromatin structure, they found a number of differences between the two promoters, centered on the deletion and the TATA box, and deleting PHO23 affected the two promoters in different ways.
It appears that FLO11 is upregulated in the flor strain because the deletion of 111 base pairs leads to an altered chromatin structure. The next steps will be to figure out what this means and then to use that knowledge to create stickier yeast. We’ll end up with a better understanding of transcriptional regulation and adhesion, and beer and wine makers may end up with even better self separating yeast.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight