New & Noteworthy

Sopping up Uranium with Yeast

March 23, 2012

Yeast may be good for more than making bread and beer or understanding how eukaryotes like humans work.  They may also be useful for cleaning up high volume, low concentration waste uranium (think uranium waste water).

The idea would be to add yeast to the contaminated area, have the yeast take the uranium up, put the yeast into radioactive waste and repeat with new yeast.  This would be a relatively cheap, simple way to detoxify this form of radioactive waste.

An obvious way to improve on this idea is to identify yeast strains that can accumulate more uranium than the wild type strain.  In a new study out in Geomicrobiology Journal, Sakamoto and coworkers have started down this path by identifying genes that allow yeast to grow in the presence of uranium and those involved in uranium accumulation.

They did this with two different screens using a set of 4,098 non-essential gene deletion strains.  In the first they identified 13 strains that grew more poorly than wild type at 0.5 mM uranium.  And in the second, they identified 17 strains that accumulated less uranium than wild type.

There was very little overlap between the two sets of strains suggesting different pathways (or sets of pathways) may be involved in accumulation and growth.  However, there were two deletion strains that showed up in both screens.  Both of the identified genes, PHO86 and PHO2, are involved in phosphate metabolism.

These genes definitely make sense.  A number of previous studies had hinted strongly that uranium accumulates on the surface of yeast in the form of insoluble uranium-phosphate complexes. 

The idea behind the importance of these genes is that yeast deals with higher uranium levels by scavenging more phosphate.  When genes involved in this process are knocked out, the yeast can’t get the extra phosphate it needs to form the insoluble uranium phosphate complexes.  Now it grows poorly and has less uranium on its surface. 

It will be interesting to see how the other genes are involved in uranium survival or accumulation.  Perhaps one day researchers will be able to turn yeast into a grade A uranium sponge.  Here’s hoping they can!

For those really interested, here is a list of the genes identified in each screen:

Uranium sensitive: PHO2, PHO84, PHO86, PHO87, VPS74, ENT5, CPR1, GLO2, OPI1, ATG15, PTC6, SLC1, and uncharacterized ORF, YPR116W.

Uranium accumulation: OPI1, PHO86, APL4, PEX10, VPS74, PHO2, SPT20, GAL11, SWP82, IVY1, FLO1, DIT2, RPL2A, and uncharacterized ORFs, YGL214W, YJR098C, YNL035C, and YPR116W.       

A nice lecture on bioremediation (using biology to clean up toxic waste)

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Not any Cyclin Will Do

March 16, 2012

Getting through a cell cycle is a complicated process. All sorts of proteins need to work and stop working at the right times in the right places to get the DNA copied, get the cells growing larger, have the cells divide and so on.

Key regulators in this process are the cyclins and their dependent kinases. Different cyclins are expressed at different points in the cell cycle, at which time they direct their cyclin-dependent kinase (CDK) to the appropriate subset of proteins to be phosphorylated. A big part of cell cycle regulation, then, comes from when a cyclin is expressed. But this is not the whole story.

In a study out in this month’s issue of GENETICS, DeCesare and Stuart showed that at least for the B-type cyclin Clb5 in the yeast S. cerevisiae, timing isn’t everything. And even more unexpectedly, they found that a key part of this cyclin’s specificity comes from its N terminus.

In yeast, Clb5 is involved in premeiotic DNA synthesis. Many researchers had previously argued that any B-type cyclin expressed at the right time would be sufficient to promote this function. DeCesare and Stuart were able to show that this was not the case by putting two different cyclins, Clb1 and Clb3, under the control of the CLB5 promoter. These cyclins were now expressed at the right time but neither could substitute for Clb5.

The authors next set out to discover what part of Clb5 conferred this specificity by creating chimeric versions of Clb3 and Clb5. They identified two regions in Clb5 important for premeiotic DNA synthesis — a hydrophobic patch and the N terminus.

The hydrophobic patch was expected; this region is highly conserved in all cyclins and has previously been shown in to be involved in interacting with protein substrates. But the N terminus was a surprise. It was thought to be involved primarily in cyclin stability and/or subcellular localization and not protein-protein interactions.

The authors were not able to identify which specific part of the N terminus of Clb5 was involved in conferring specificity. In their experiments, there was a gradual decline in the ability of the Clb3-Clb5 chimera to promote premeiotic DNA synthesis as more and more of Clb5 was replaced with Clb3. It is as if the whole region is involved in determining specificity.

And the decreasing ability of the Clb3-Clb5 chimera to induce premeiotic DNA synthesis was not due to the loss of kinase activity. When paired with Cdc28 (also known as Cdk1), all of the chimeras in the experiment were equal or even more active than the wild type Clb5/Cdc28 pair.

What it looks like is happening is that Clb5 uses both its hydrophobic patch and its N terminus to bring appropriate proteins to Cdk1 for phosphorylation. Different parts of each region are used to interact with different subsets of proteins involved in premeiotic DNA synthesis. At least for Clb5 and premeiotic DNA synthesis, it looks like not any cyclin will do.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Stellar Strain Yields Increased SAM Production

March 10, 2012

Yeast geneticists often go the extra mile to get their mutant. But Huang and coworkers went hundreds of extra miles to get theirs. Hundreds of miles straight up, that is.

In a recent study published online in the Journal of Applied Microbiology, Huang and coworkers identified and improved upon a strain of Saccharmoyces cerevisiae that makes increased amounts of S-adenosyl-L-methionine or SAM. This is an important chemical in many pharmacological and medical uses and is primarily made via microbiological synthesis. Increased production would be an obvious boon to researchers and the pharmaceutical industry.

What makes this study interesting is that the researchers obtained their initial strain from outer space. They shot cultures up into space on a satellite where the poor yeast had to endure the harsh environment there for 18 days. Researchers then collected the samples when the satellite returned to Earth.

Out of six hundred random clones from the flight, researchers found 43 that made at least 10% more SAM than their wild type counterpart. A second round of selection yielded strain H5M147 which made 84% more SAM than the wild type strain.

Unfortunately the researchers were not able to (or did not report in this study) why the strain made extra SAM. They used a technique called AFLP that allowed them to see that there were differences in the new and host strain’s genome, but it did not allow them to pinpoint what those differences were nor which ones were significant. That will have to wait for a future study.

They did manage to ramp up SAM production even further in this strain though. First they added an extra copy of a key player in SAM production, the MAT2 gene. Researchers have tried to coax other strains of yeast to make more SAM by adding MAT2 but to no avail. This space strain apparently has genetic mutations that allow extra MAT2 to increase SAM production. The new strain with the integrated version of MAT2 was called H5MR38.

Finally the researchers tinkered with culture conditions to optimize SAM production in H5MR38 further. They found that using sucrose as the carbon source and adding peptone to yeast extract and urea yielded the most SAM.

In the end, Huang and coworkers managed to get 7.76 grams of SAM per liter of culture after 84 hours. This compares to the previous high in Sacchromyces cerevisiae of 5.7 gram per liter after 120 hours and 3.6 grams per liter from a strain of Pichia pastoris in 100 hours. Clearly their space-derived yeast strain is an improvement over anything else identified so far.

Huang and coworkers aren’t the only ones putting yeast into space

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Engineering Magnetic Yeast

March 2, 2012

With a bit of genetic manipulation and a hearty diet of iron, Nishida and Silver report in the latest issue of PLOS Biology that they have caused yeast cells to become magnetic. And this isn’t just a parlor trick. Their research could one day help other scientists create new therapies for the sick and new applications for research and industry.

The first step in the process was to load the yeast up with magnetic iron. The authors took a couple of different approaches.

The simplest was to grow the yeast in lots of iron. Surprisingly, without any other manipulation, this was enough to make the yeast a bit magnetic. But the authors wanted more magnetism.

To accomplish this goal, they needed to keep yeast from transporting excess iron to their vacuole where it is nonmagnetic. They did this by knocking out the gene encoding the vacuolar iron transporter, CCC1.

When grown in lots of ferric citrate, the ccc1Δ strain was about 1.8 times more magnetic than wild type. Nice, but to get even more magnetic yeast, Nishida and Silver added back the three human genes necessary to reconstitute human ferritin. This new strain was now about 2.8 times more magnetic than wild type.

None of this was really earth-shattering yet. Scientists knew that iron was needed to make a cell magnetic and that ferritin-iron complexes were a bit magnetic. What made these initial studies important was that they gave Nishida and Silver the tools to study the underlying mechanisms of magnetism.

The authors took a directed approach to study this problem and knocked out genes known to be involved in iron homeostasis or oxidative stress. Of the 60 knockout strains tested, tco89Δ was the only one to consistently be less magnetic than the wild type strain. On average it was about two fold less magnetic.

Tco89p is a nonessential part of TORC1, a complex involved in the regulation of cell growth in response to nutrients, stress, and redox states. As might be predicted from TORC1 function, the authors determined that nutrients and the redox state of the medium affected the yeast’s magnetism. They then expanded their screen to look for genes involved in carbon metabolism and mitochondrial redox that might affect magnetism and discovered several (POS5, YFH1, SNF1, and ZWF1).

The current model is that the redox state within the cell and in particular, within the mitochondria, impacts the amount of iron precipitation and hence magnetism in yeast. This is consistent with the iron deposits the authors saw in electron micrographs of the mitochondrial membrane of the magnetic yeast.

These findings should help point researchers in productive directions for engineering magnetic cells in other systems but it is only a first step. Science has a long way to go before therapies based on cell magnetism are helping patients.

More details on these magnetic yeast

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics