Legendary Bacterial Evolution Experiment Enters New Era

E. coli cells used in the Long Term Evolution Experiment (LTEE) on an agar plate.Credit: Jeffrey Barrick

On February 24, 1988, evolutionary biologist Richard Lenski filled 12 flasks with sugary growth medium and inoculated each with Escherichia coli bacteria. For the past 34 years, Lenski of Michigan State University in East Lansing and his colleagues have been feeding bacterial cultures, refreshing growth media daily and freezing samples for future study every two months.

The Long-Term Evolution Experiment (LTEE) has become a cornerstone of evolutionary biology that researchers continue to exploit for insights. During their 75,000 generations of growth, bacteria have made huge strides in their physical condition – how fast they grow compared to other bacteria – and have evolved some surprising traits.

Last month, Lenski and his lab took care of LTEE for the last time. The 12 E.coli the lines are now frozen in cryoprotective media and will soon be revived to start new life in the lab of Jeffrey Barrick, an evolutionary biologist at the University of Texas at Austin who first worked on the experiment in the 2000s as a postdoc in Lenski’s laboratory.

The two spoke to Nature about the past, present, and future of one of biology’s longest-running experiments.

Composite image of two portraits of Jeffrey Barrick and Richard Lenski.

Jeffrey Barrick (left) will take over leadership of the E. coli experiment from mentor Richard Lenski, who started the experiment in 1988.Credit: Laura Gerson/UT College of Natural Sciences, Sipa US/Alamy

What prompted you to start the experiment?

Richard Lenski: I like big open questions. I wanted a long, very simple experiment to see how reproducible evolution was. The initial goal was 2000 generations. And I thought it deserved the nickname “the long-term evolution experiment.” I had no idea it would go as long as it actually lasted – and hopefully it will last much longer.

Why did you go on – and go and go?

Lenski: It’s actually a very easy experience to maintain. The amount of work for an individual in a typical day is perhaps half an hour. It’s 365 days a year, at least in principle, but the amount of work per day is not huge.

And then, of course, the bacteria were doing some very interesting things over time. New technologies have emerged, such as the ability to sequence genomes at lower cost. People like Jeff joined the lab and brought new ideas and questions. And the bacteria kept doing interesting things. I realized this had to go on for as long as humanly possible.

How many generations had passed when Jeff started working on the experiment?

Lenski: Did you join the lab around 2007? It was probably around 40,000 generations.

Jeffrey Barrick: Seems fair to me. I knew less about the history of the experiment – perhaps less than many of Rich’s post-docs and graduate students. But I was at a point in my career where I was studying evolution at the molecular level, looking at all kinds of bacterial genomes. I wanted to study the evolution of whole organisms in the laboratory and be able to observe evolution. It’s just something that has fascinated me since I was a student.

Was it ever difficult to continue the experience?

Barrick: Like Rich said, it’s pretty simple. There are major snowstorms and other things that happen, and other infrastructure anomalies that can sometimes make it difficult, but you can always go back to the freezer, which is a very good thing about of the experiment and makes it much more doable than other experiments. People have tried long-term experiments with mice and flies and other organisms where it’s really hard if something goes wrong.

Lenski: One of the benefits of long-term experience is that everything is so simple. We’re working with a chemically defined medium, we can freeze strains and revive them to knock on wood, I think that should continue pretty well in the new home.

What has experience taught us about the repeatability of evolution?

Lenski: My bias at the beginning of the experiment was that all the strains would go in very different directions. I thought the roles of chance and contingency in evolution would have been more important than they were. And over the years, we’ve actually seen striking amounts of reproducibility. So, although a typical line has improved its relative fitness over the ancestor by perhaps 70% or 80%, the variance in competitive fitness between most lines is more like a few percent. So they all increased tremendously, but in very similar ways to each other.

But then, over the years, we’ve also seen some pretty striking discrepancies between the lines. Thirty thousand generations after the experiment began, one of the 12 lineages developed the ability to consume citrate, instead of just glucose. And it has attracted a lot of attention, and even, shall we say, hostility from some people skeptical of the power of evolution. And after 75,000 generations, it’s still the only one of the 12 lines that has developed this ability.

Are there any big questions about evolution that you hope to answer by digging deeper?

Barrick: For many bacteria that ended up in simple, constant environments — especially simple endosymbionts that live inside insect cells — their genomes gradually shrink over time. And I would say that one of the most surprising things for me is that these E.coli have been in a very constant environment, but their genomes haven’t shrunk much.

Lenski: I think part of the problem with genome shrinking is that it’s a slow process. Thirty years and 75,000 generations is a drop in the bucket in evolution. So I guess if we could come back – in a million years or whatever, the bacterium would probably have extremely reduced genomes. This is a reason to continue.

Photograph of Richard Lenski's long-term evolution experiment with E. coli taken June 25, 2008.

THE. coli has now spread some 75,000 generations.Credit: Brian Baer and Neerja Hajela via Wikimedia Commons (CC BY-SA 1.0)

Why did you decide to pass the torch of LTEE?

Lenski: I’m not going to stay here forever. I think it’s best to do these things now, to plan them carefully and thoughtfully. So that made sense. I’m 65, and while I don’t plan on retiring in at least the next few years, the lab is getting smaller and smaller. And one of the important things to maintain long-term lines is this daily rhythm. I think a lab that has half a dozen or more people is perfectly suited to the weekend and holiday coverage that the experiment enjoys.

So I asked Jeff, maybe in 2018 or 2019. I have a grant from the National Science Foundation to conduct the experiment, and Jeff is now co-principal investigator on it. And when we wrote the most recent renewal, we laid out this plan for the transfer and that it would take place around now.

Why did Jeff take it?

Barrick: I’m a big believer in open science. It’s a great resource that I want to support, share and continue. It has become a kind of common touchstone for many stories about bacterial evolution. And something that people can take in so many directions. I am happy to support the community.

Are you anxious about taking responsibility for such a long experience?

Barrick: As Rich said, it’s not very difficult to pursue the experiment. What gets difficult is organizing all the strains in the freezer, making sure you can send them to people, and all the paperwork related to that kind of stuff.

I don’t want to have the experience where the experience comes into my lab and then gets contaminated, and I put it back two or three years. I want to make sure I keep things going. But a lot of the pressure is off, because it’s frozen, not only in Rich’s lab and in my lab, but also in labs in France and elsewhere. It relieves the pressure that I could cause an irreparable problem. So I’m more excited about educating my colleagues about the experience.

Rich, what’s your advice for Jeff?

Lenski: Keep calm and carry on. Frustrating things will happen. But the experience is quite forgiving. As long as my brain is working, I’ll be really excited to see what new spin-off experiences he and his collaborators generate, what new analytics he and the wider community generate to make sense of what’s going on with the long lines. Probably the most important thing Jeff will have to think about in 20 or 30 years is who’s next?

Barrick: Your work is not done, Richard. You’re still the best science communicator and the best person to bring the long-term evolution experience to people and build that community. It’s actually the most intimidating thing that anyone could replace right now. Experimenting is very easy.

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