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JCVI Employs Yeast as Go-Between in Bacterial Genome Transplant

NEW YORK (GenomeWeb News) – Researchers from the J. Craig Venter Institute demonstrated online in Science today that they were again able to transplant the genome of one bacterial species into another — this time with a brief stopover in yeast.

The team cloned the Mycoplasma mycoides genome into a yeast centromeric vector and transformed it into yeast, where they tweaked the sequence before transplanting it into M. capricolum.

"The maintenance of the M. mycoides genome in yeast allowed us to access the powerful repertoire of yeast genetic methods and produce an M. mycoides strain that had not previously existed," senior author John Glass, a senior scientist with JCVI's synthetic biology group, and his co-authors wrote. "Thus, we report engineering of a bacterial cell by altering its genome outside of its native cellular environment."

Sanjay Vashee, the corresponding author on the paper, told GenomeWeb Daily News via e-mail that by moving the bacterial chromosome to yeast, "we can use the powerful suite of yeast genetic tools to manipulate it and then boot it up to obtain our mutant strains."

This approach, he added, "will enable us to more easily create a minimal cell that only has genes that is essential for life. Also, there are many microbes, some that are important industrially or medically in nature that are not genetically tractable. If we can extend this technology to these organisms then it may be possible to bring genetic tools to them so we can understand them better."

JCVI researchers have been working towards developing synthetic organisms — which they say could be useful for everything from bioenergy production to carbon sequestration — for several years.

In 2007, they showed that they could transfer the genome from one bacteria, M. mycoides, to another, M. capricolum. The following year the team described an approach for creating a synthetic M. genitalium genome — a method they streamlined in late 2008 by finding a way to co-transform 25 overlapping pieces of DNA into yeast in one go.

In the current study, JCVI researchers report that by using yeast as in intermediate step in bacterial genome transplants they can not only take advantage of genetic tools in this system but also improve the transplantation process. The paper also describes tricks for overcoming bacterial restriction modification systems — restriction enzymes that bacteria use as a protective mechanism against invading foreign DNA.

The team cloned the 1.1 million base M. mycoides genome into a yeast centromeric plasmid and transformed it into Saccharomyces cerevisiae. Their subsequent multiplex PCR and gel electrophoresis experiments indicated that growing the sequence in yeast didn't lead to deletions or other detectable changes to the genome.

Once the genome was in yeast, the team knocked out a M. mycoides gene coding for a non-essential restriction endonuclease. Meanwhile, to prevent the M. capricolum cells from degrading the M. mycoides genome, they inactivated M. capricolum's restriction enzyme by popping an antibiotic resistance marker into the gene's coding region.

Because bacteria methylate their own DNA in such a way that protective enzymes cannot attack their own genome, the team also tried methylating M. mycoides DNA using extracts from M. mycoides or M. capricolum cells or with purified M. mycoides methyltransferase enzymes.

The researchers were able to transplant the M. mycoides genome into M. capricolum cells missing the restriction enzyme regardless of whether the transplanted genome was methylated or not. But when the M. capricolum restriction enzyme was present, only the methylated M. mycoides genomes could be transferred into the recipient cells, they reported.

When they sequenced the genome of a transplanted cell, the researchers found that the sequence represented M. mycoides and yeast vector DNA but not yeast or M. capricolum, suggesting the transplanted genome did not recombine with the yeast genome or recipient cell genomes.

Describing the advantages of the current method over direct M. mycoides genome transfer to M. capricolum cells, the authors explained, "We previously reported transplantation of naked genomic DNA purified from M. mycoides cells. The transplant events were rare, and there remained the possibility that they resulted from damaged cells that could be somehow repaired in the presence of recipient cells, or from genomes that were complexed with some M. mycoides component other than genomic DNA."

"Transplantation from yeast of the non-methylated M. mycoides genome into M. capricolum [restriction enzyme negative] cells eliminates the possibility that components of the M. mycoides cells are required for transplantation," they added.

To accomplish their goal of creating a synthetic organism, the team will have to go a step further: transferring a synthetic rather than a natural genome from yeast to a recipient bacterial cell. But those involved say the method used in the current paper should make it possible to engineer M. mycoides strains with a host of genetic changes.

"It is now possible to readily generate M. mycoides strains with multiple targeted gene deletions, insertions, and rearrangements," they wrote. "It would also be possible to engineer bacterial genomes in yeast using random methods, to yield populations of altered bacteria upon transplantation. After screening for a desired trait, these methods could be re-applied in a cyclical manner to introduce new traits."

Vashee said that the researchers plan to use knowledge gleaned from the study to create a synthetic cell.

"Using the methods developed here, we can more easily begin to make a minimal cell, one that only has genes that are necessary for life and allow us to understand better what constitutes life," he said.

In addition, "there are countless other species of bacteria for which we have no tools that might make them useful to the biotechnology industry," Vashee said. "Once we learn how to apply the methods we developed using mycoplasmas to other kinds of bacteria, a whole world of new possibilities open up for using microbes to solve human needs in bioenergy, medicine, and industry."

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