Seven years ago, researchers showed that they could strip cells down to their barest fundamentals, creating a life form with the smallest genome that still allowed it to grow and divide in the lab. But in shedding half its genetic load, that “minimal” cell also lost some of the hardiness and adaptability that natural life evolved over billions of years. That left biologists wondering whether the reduction might have been a one-way trip: In pruning the cells down to their bare essentials, had they left the cells incapable of evolving because they could not survive a change in even one more gene?
Now we have proof that even one of the weakest, simplest self-replicating organisms on the planet can adapt. During just 300 days of evolution in the lab, the generational equivalent of 40,000 human years, measly minimal cells regained all the fitness they had sacrificed, a team at Indiana University recently reported in the journal Nature. The researchers found that the cells responded to selection pressures about as well as the tiny bacteria from which they were derived. A second research group at the University of California, San Diego came to a similar conclusion independently in work that has been accepted for publication.
“It turns out life, even such simple wimpy life as a minimal cell, is much more robust than we thought,” said Kate Adamala, a biochemist and assistant professor at the University of Minnesota who was not involved in either study. “You can throw rocks at it, and it’s still going to survive.” Even in a genome where every single gene serves a purpose, and a change would seemingly be detrimental, evolution molds organisms adaptively.
“It’s a stunning achievement,” said Roseanna Zia, a physicist at the University of Missouri whose research aims to build a physics-based model of a minimal cell and who was not involved in the study. The new work showed that even without any genome resources to spare, she said, the minimal cells could increase their fitness with random changes in essential genes.
The new evolution experiments are starting to provide insights into how the smallest, simplest organisms might evolve — and how principles of evolution unite all forms of life, even genetic novelties developed in labs. “Increasingly, we are seeing evidence that this [minimal cell] is an organism that is not something bizarro and unlike the rest of life on Earth,” said John Glass, an author on the Nature study and the leader of the synthetic biology group at the J. Craig Venter Institute (JCVI) in California that first engineered the minimal cell.
What If We ‘Let It Loose’?
Just as 19th- and 20th-century physicists used hydrogen, the simplest of all the atoms, to make seminal discoveries about matter, synthetic biologists have been developing minimal cells to study the basic principles of life. That goal was realized in 2016 when Glass and his colleagues produced a minimal cell, JCVI-syn3.0. They modeled it after Mycoplasma mycoides, a goat-dwelling parasitic bacterium that already gets by with a very small genome. In 2010, the team had engineered JCVI-syn1.0, a synthetic version of the natural bacterial cell. Using it as a guide, they drew up a list of genes known to be essential, assembled them in a yeast cell and then transferred that new genome into a closely related bacterial cell that was emptied of its original DNA.
Two years later at a conference in New England, Jay Lennon, an evolutionary biologist at Indiana University Bloomington, listened to a talk from Clyde Hutchison, a professor emeritus at JCVI who had led the team engineering the minimal cell. Afterward, Lennon asked him, “What happens when you let this organism loose?” That is, what would happen to the minimal cells if they were subjected to natural selection pressures like bacteria in the wild?
For Lennon as an evolutionary biologist, the question was an obvious one. But after he and Hutchison both pondered it for a few minutes, it became apparent that the answer wasn’t.
The minimal cell “is a type of life — it’s an artificial type of life, but it’s still life,” Lennon said, because it fulfills the most basic definition of life as something able to reproduce and grow. It should therefore respond to evolutionary pressures just as gorillas, frogs, fungi and all other organisms do. But the overarching hypothesis was that the streamlined genome might “cripple the ability of this organism to adaptively evolve,” Lennon said.
No one had a clue what would really happen, however, because researchers have generally taken great care to keep minimal cells from evolving. When samples of the cells are distributed by JCVI to any of the roughly 70 labs that now work with them, they’re delivered pristine and frozen at minus 80 degrees Celsius. When you take them out, it’s like their first day on Earth, Lennon said: “These are brand new cells that had never seen a day of evolution.”
Shortly after their encounter, Hutchison put Lennon in touch with Glass, who shared samples of his team’s minimal cells with Lennon’s lab in Indiana. Then Lennon and Roy Moger-Reischer, his graduate student at the time, got to work.
Testing the Streamlined Cells
They began with an experiment aimed at measuring mutation rates in the minimal cells. They repeatedly transferred a sliver of the growing minimal cell population into petri dishes, which freed the cells to grow without constraining influences like competition. They found that the minimal cell mutated at a rate comparable to that of the engineered M. mycoides — which is the highest of any recorded bacterial mutation rate.
The mutations in the two organisms were fairly similar, but the researchers noticed that a natural mutational bias was exaggerated in the minimal cell. In the M. mycoides cells, a mutation was 30 times more likely to switch an A or a T in the genetic code for a G or a C than the other way around. In the minimal cell, it was 100 times more likely. The probable explanation is that some genes removed during the minimization process normally prevent that mutation.
In a second series of experiments, rather than bringing over a small group of cells, the researchers transferred dense populations of cells for 300 days and 2,000 generations. That allowed more competition and natural selection to occur, favoring beneficial mutations and the emergence of genetic variants that eventually ended up in all the cells.
To measure the fitness of the cells, they calculated their maximum growth rate every 65 to 130 generations. The faster the cells grew, the more daughter cells they produced for the next generation. To compare the fitness of evolved and unevolved minimal cells, the researchers made them compete against the ancestral bacteria. They measured how abundant the cells were at the start of the experiment and after 24 hours.
They calculated that the original minimal cell had lost 53% of its relative fitness along with its nonessential genes. The minimization had “made the cell sick,” Lennon said. Yet by the end of the experiments, the minimal cells had evolved all that fitness back. They could go toe-to-toe against the ancestral bacteria.
“That blew my mind,” said Anthony Vecchiarelli, a microbiologist at the University of Michigan who was not involved in the study. “You would think that if you have only essential genes, now you’ve really limited the amount of evolution that … can go in the positive direction.”
Yet the power of natural selection was clear: It rapidly optimized fitness in even the simplest autonomous organism, which had little to no flexibility for mutation. When Lennon and Moger-Reischer adjusted for the relative fitness of the organisms, they found that the minimal cells evolved 39% faster than the synthetic M. mycoides bacteria from which they were derived.
The Fear-Greed Trade-Off
The study was an “incredibly thought-provoking” first step, Vecchiarelli said. It’s uncertain what would happen if the cells were to keep evolving: Would they gain back some of the genes or complexity that they lost in the minimization process? After all, the minimal cell itself is still a bit of a mystery. About 80 of the genes essential to its survival have no known function.
The findings also raise questions about which genes need to stay in the minimal cell for natural selection and evolution to proceed.
Since 2016, the JCVI team has added back some nonessential genes to help the minimal cell lines grow and divide more like natural cells. Before they did that, JCVI-syn3.0 was growing and dividing into weird shapes, a phenomenon that Glass and his team are investigating to see if their minimal cells divide the way primordial cells did.
The researchers found that most of the beneficial mutations favored by natural selection in their experiments were in essential genes. But one critical mutation was in a nonessential gene called ftsZ, which codes for a protein that regulates cell division. When it mutated in M. mycoides, the bacterium grew 80% larger. Curiously, the same mutation in the minimal cell didn’t increase its size. That shows how mutations can have different functions depending on the cellular context, Lennon said.
In a complementary study, which has been accepted by iScience but not yet published, a group led by Bernhard Palsson at the University of California, San Diego reported similar results from experiments on a variant of the same minimal cell. They didn’t find an ftsZ mutation in their evolved minimal cells, but they did find similar mutations in other genes that govern cell division, emphasizing the point that there are multiple ways to achieve a biological outcome, Palsson said.
They didn’t look at cell size, but they checked which genes were expressed before, during and after the episode of evolution. They observed a “fear-greed trade-off,” a tendency also seen in natural bacteria to evolve mutations in genes that will help it grow rather than mutations that would produce more DNA repair proteins to correct the errors.
Here you can see that “mutations tend to reflect the cellular processes that are needed to improve a function,” Palsson said.
Demonstrating that the minimal cell can evolve like cells with a more natural genome was important because it validated “how well it represents life in general,” Zia said. For many researchers, the entire point of a minimal cell is to serve as a critically useful guide to understanding more complex natural cells and the rules they follow.
Other studies are also beginning to probe how minimal cells respond to natural pressures. A group reported in iScience in 2021 that minimal cells can quickly evolve resistance to different antibiotics, just like bacteria.
Knowing which genes are more likely to mutate and lead to useful adaptations could someday help researchers design drugs that get better at what they do in the body over time. To build robust synthetic life forms that have very different abilities, evolutionary biologists and synthetic biologists must work together, “because no matter how much you engineer it, it’s still biology, and biology evolves,” Adamala said.