I’m happy now to announce what has become of these papers. After positive reviews and revisions, the editor at Science asked the two teams to combine their papers into a single long-format Research Article. Both teams agreed to do so, but it was a lot of work, requiring further reviews and revisions. At last, the combined paper was accepted, and it appears in the latest issue of Science. Hooray!

I think it’s fair to say that the paper provides an unprecedented description and analysis of how the fitness effects of mutations change over time, even under the constant conditions of the LTEE, and even as the overall shape of the DFE was little changed. As a matter of practicality for the high-throughput analyses, the paper relied on generating libraries of insertion mutations. That approach means that the new study focuses on loss-of-function mutations, which are undoubtedly important in the LTEE, but which means that mutations that refine existing functions or even generate new functions are substantially under-represented. Perhaps some future methodology will allow an even more comprehensive analysis, but the new paper provides a great start.

I want to provide a little background, not to the science, but to the interaction among the wonderful people who did the lab work—Alejandro (Alex) Couce and Anurag Limdi along with Melanie Magnan, Siân Owen, and Cristina Herren—and who led the two teams—Michael Baym and Olivier Tenaillon. When I first realized that two teams were preparing papers about the evolution of the DFEs in the LTEE, I had an immediate pang of guilt. When people ask me for LTEE strains, I ask them why they want the samples. That way, I can let them know about potential work that others are doing, which might cause someone’s work to get ‘scooped’ by another team. However, I don’t always remember all the projects, or I might fail to see the connection in the words people use to describe their plans, and people’s projects may also change after they start their work. In any case, thanks to Tanush Jagdish for letting us know that the two teams were pursuing related projects.

After that, the two teams soon began having zoom meetings to openly share and explain their questions, hypotheses, methods, and findings. There was some overlap, of course, but I think that’s valuable in science, because it indicates broad interest in the topic and, moreover, shows that the results are robust. Thus, the decision was made to submit two separate papers as joint submissions to the same journal.  In a thread on social media, Michael nicely described the situation and the resulting comradery (my emphasis added):

As we were working on this we discovered that another group led by Alex Couce and Olivier Tenaillon were working on something very similar.  Instead of competing we decided to talk to one another. While either group could have scooped one another, what we did instead was have some of the most fun scientific calls I’ve ever been part of, in which we discussed our work and our results, and even cross-reviewed each other’s manuscripts. We found that while we were superficially overlapping (TnSeq on the LTEE) the experiments and the questions we asked were complementary. While we’d focused on detrimental mutations after long periods, they looked at early beneficial mutations. Yet we’d found similar trends! In the end, Alex and Olivier’s insights and perspective gave us both a much deeper understanding of the science, and a lot more confidence in the results. As our papers are complementary and speak to one another, we coordinated preprinting and will be co-submitting.

More recently, as we combined the two papers into one, we wanted to leave a ‘footprint’ that would reveal the origins of the integrated study. At the start of the Discussion, we wrote:

This paper began as two separate projects performed by two different teams, using similar but not identical methods. As we discussed our findings together, we discovered that each project reinforced and complemented the other. They reinforce one another by finding the same evolution of the overall form of the DFE; they are complementary because one project delved deeply into the fine-scale genetic changes in the deleterious tail while the other did so for the beneficial tail.

We wondered if the editor might reduce or eliminate this passage, since a paper’s history doesn’t bear directly on its results. We were delighted, therefore, when we saw that the passage remained, because it sheds light on how civility, cooperation, and collaboration can succeed even in the highly competitive world of science.

@article{Couce2024,

title = {Changing fitness effects of mutations through long-term bacterial evolution},

author = {Alejandro Couce and Anurag Limdi and Melanie Magnan and Siân V. Owen and Cristina M. Herren and Richard E. Lenski and Olivier Tenaillon and Michael Baym},

url = {https://www.science.org/doi/10.1126/science.add1417},

doi = {10.1126/science.add1417},

year  = {2024},

date = {2024-01-26},

urldate = {2024-01-26},

journal = {Science},

volume = {383},

number = {6681},

pages = {eadd1417},

abstract = {The distribution of fitness effects of new mutations shapes evolution, but it is challenging to observe how it changes as organisms adapt. Using \textit{Escherichia coli} lineages spanning 50,000 generations of evolution, we quantify the fitness effects of insertion mutations in every gene. Macroscopically, the fraction of deleterious mutations changed little over time whereas the beneficial tail declined sharply, approaching an exponential distribution. Microscopically, changes in individual gene essentiality and deleterious effects often occurred in parallel; altered essentiality is only partly explained by structural variation. The identity and effect sizes of beneficial mutations changed rapidly over time, but many targets of selection remained predictable because of the importance of loss-of-function mutations. Taken together, these results reveal the dynamic—but statistically predictable—nature of mutational fitness effects.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

Check out the video and the article if you want to learn about the LTEE or if you are considering starting your own microbial evolution experiment. The video is also embedded in this post below.

I’m looking forward to sharing these materials with new researchers joining my group who are learning to help maintain the LTEE or beginning to study it, and also with researchers we send these ever-evolving E. coli.

Some highlights:

• We discuss aspects of the design of the LTEE that make it simple and sustainable.
• We document all kinds of expected results: how the flasks appear after growth and the optical densities the evolved E. coli populations reach in different media; photos of the colonies formed by the evolved LTEE populations on various agar plates; and agar plates showing E. coli that form red and white colonies competing for dominance over several transfers and what the changes mean in terms of relative fitness values.
• We created and an R package called fitnessR (available on GitHub) and an Excel spreadsheet (available as a supplemental file) for calculating relative fitness from co-culture competition experiment results.
• We describe alternative procedures for performing competition assays that can be used when the fitnesses of the two competitors are very similar or very different.
• We discuss some ways that the methods of the LTEE have been and are still being updated as technology has changed, and the potential advantages and disadvantages of other evolution experiment setups.

See also: If you are interested in LTEE methods, check out our protocols.io group which has media recipes and will gradually be populated with additional protocols. Rich also has a recent paper out that discusses the design of the LTEE, both in terms of procedures and as an experiment designed to answer questions about evolution (link).

@article{Barrick2023,

title = {Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli},

author = {Jeffrey E. Barrick and Zachary D. Blount and Devin M. Lake and Jack H. Dwenger and Jesus E. Chavarria-Palma and Minako Izutsu and Michael J. Wiser},

doi = {10.3791/65342},

issn = {1940-087X},

year  = {2023},

date = {2023-08-18},

urldate = {2023-08-18},

journal = {Journal of Visualized Experiments},

volume = {198},

pages = {e65342},

abstract = {The Long-Term Evolution Experiment (LTEE) has followed twelve populations of \textit{Escherichia coli} as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions. },

keywords = {},

pubstate = {published},

tppubtype = {article}

}

It was an occasion that called for some decorations! The colors of the balloons and streamers draw from the red, pink, and white of the colonies that E. coli from the LTEE form when plated on TA agar, which is one way that we monitor their evolution.

Over the past several months Devin Lake and others in Richard Lenski’s lab at Michigan State University brought the last few “straggler” populations that had fallen behind for various reasons all up to 75,000 generations so they would be in sync for the handoff. Then, the E. coli were shipped to Texas and spent a few weeks in cryopreservation while we double-checked our supplies, procedures, and equipment. Once the LTEE starts, it needs attention daily.

We did a test run of reviving the LTEE populations last week, including plating them on various media on which they form colonies with different colors and morphologies. Everything checked out as expected.

On June 21st, it was time for reviving cultures that we will continue propagating in the days to come as the official LTEE populations. All the steps went smoothly: from taking the cultures out of the freezer, to inoculating them into media in flasks, to placing them carefully in the incubator. (Even with the added pressure of some cameras in the background documenting things.)

The first entry in LTEE Notebook #13 in the Barrick lab draws from a classic American novel that’s one of my favorites. It’s a quote (of a quote) from Moby Dick that, like much of the book, can be read as being about more than whales, perhaps even the LTEE. That the passage is in a prologue section titled “Extracts. (Supplied by a Sub-Sub-Librarian)” also seems somehow fitting for continuing this (hopefully) never-ending experiment.

We’ll be doing some additional plating and genetic checks over the next few weeks in addition to training new researchers in my lab to do the LTEE transfers. I bet the E. coli won’t even notice that they aren’t in Michigan anymore.

Evolve, E. coli, evolve!

Link: https://www.youtube.com/watch?v=w4sLAQvEH-M&t=1s

Through the Wormhole (Science Channel)
Season 6 Episode 3: Are we here for a reason?
Original Airdate May 13th, 2015
Link: https://www.youtube.com/show/SCVRl6Rj03lfF8v5nX7eJwyA?season=6

Link: https://doi.org/10.1126/science.342.6160.790

Link: https://www.npr.org/2012/11/23/165030844/experiments-that-keep-going-and-going-and-going

Link: https://www.discovermagazine.com/planet-earth/the-birth-of-the-new-the-rewiring-of-the-old

Link: https://www.youtube.com/watch?v=PQr8ldEeO04

Link: https://www.nationalgeographic.com/science/article/replaying-evolution-reveals-the-benefits-of-being-slow-and-steady