‘Evolution under a microscope’ going strong at MSU by Bethany Mauger

P.S. The sign is viewed backwards in the photo, it is 82,007 from the other side!

Time flies, and Devin Lake just froze down the 82,500-generation samples last week.

Evolve, E. coli, evolve!

Episode: 12 Tiny Worlds

To recognize everyone’s efforts, we’ve created the LTEE Leaderboard! (Follow the link for the interactive version.)

If we zoom out, the most overwhelming signal is that Lenski lab manager Neerja Hajela is the all-time LTEE champion with 4349 transfers. That’s more than 1/3 of all LTEE transfers to date, a feat than is unlikely to ever be equaled! As we can see from the plot over time, Neerja did the transfers on most weekdays from 1996 to 2018.

There were other historical epochs marked by other Lenski lab managers: Lynette Ekunwe (1427 transfers), Sue Simpson (950 transfers), and Devin Lake (838 transfers). So far, only one member of the Barrick lab, Jack Dwenger (407 transfers), cracks the top ten at 7th. The plot also shows some gaps (when the experiment was paused for a move or pandemic) and a plateau (when we allowed populations that were behind to catch up).

We can also use the LTEE Leaderboard to slice and dice the data. For example, what does the leaderboard look like when we restrict it to transfers that were done on the weekend (Saturday or Sunday)?

Longtime LTEE researcher Zack Blount wins this category and we see some new graduate students and postdocs appear in this top 10 list.

You can even use the LTEE Leaderboard to figure out who did a transfer on a specific day in history. Darwin Day (February 12th) in 1993? Ryszard Korona.The Ides of March (March 15th) in 2009? Zack Blount.

Our plan is to continue to periodically update the LTEE Leaderboard, at least once a year when it is time to determine bragging rights for our annual Barrick lab Year in Review group meeting.

P.S. Your humbled-by-his-small-contribution author only clocks in at 26th on the all-time list with 78 transfers.

Data Acknowledgements and Availability

Thanks especially to Zack Blount and Devin Lake for digging through many years of LTEE notebooks and compiling the spreadsheet that made creating the LTEE Leaderboard possible. Code for the LTEE Shiny app and a CSV file of the underlying data are available on GitHub (https://github.com/barricklab/LTEE-leaderboard).

The LTEE moved to the Barrick Lab at UT Austin in 2022, and when I was hired as lab manager in July 2024, the proverbial torch (Bunsen burner?) of day-to-day LTEE operations was passed on to me. More specifically, I’m responsible for the daily population transfers, and I plate the populations every 500 generations to check for contamination or changes in phenotype as new samples are archived in the frozen ‘fossil’ record. When I plated the populations at 80,500 generations, however, I noticed something odd. Ara+3 had grown both red and white colonies on TA agar in approximately equal amounts. Though the REL606 and REL607 ancestors form colonies after only 24 hours on TA agar, many evolved populations do not grow as well on these plates because they are unlike the LTEE environment. It took ~48 hours for both red and white colonies to grow in this case.

My first thought was that there had been cross-contamination by an Ara− strain either during transfers or plating. I tested this hypothesis via diagnostic PCR on three red and three white colonies, targeting the rbs operon which contains a deletion that varies in size between the twelve populations. The predicted product size for Ara+3 was 863 bp, and all six of my samples (both red and white) produced a clear band about halfway between 700 and 1000 bp, suggesting that the red colonies might genuinely be from Ara+3. In parallel, I re-plated Ara+3 at 80,540 generations (no picture) and observed the same colony dimorphism, although surprisingly the red colonies had gone on to vastly outnumber the white. I also looked back at photos from the previous plating at 80,000 generations and noticed that Ara+3 on TA had just one red colony among a hundred or so white colonies. The obvious next step was to sequence the genomes of a red colony and a white colony to figure out exactly what was going on.

The sequencing results from my run on our lab’s MiniON revealed that the red colonies shared nearly all of their mutations with the white colonies and clones sequenced from population Ara+3 at earlier generations. Everything was fine—no contamination! Our ‘Red Scare’ was over. But what caused it?

I was stunned when Prof. Barrick announced that the change in phenotype from white to red was caused by the araA mutation G92D (GGC→GAC), an exact nucleotide-for-nucleotide reversion of the mutation that separated REL607 from REL606 decades ago—something which has never been observed in the LTEE’s 30+ years. Nearby mutations in the red and white colony genome sequences are consistent both with each other and with previous generations of Ara+3, making it unlikely that the mutation resulted from horizontal gene transfer by a rogue Ara− contaminant.

This mutation is consistent with the hypermutator signature of a mutS defect that evolved in this population, but as Prof. Barrick put it, it’s a little ‘spooky’ that the change was caused by this exact mutation when many others could also lead to a loss of araA function. It’s possible that this site is some kind of ‘hotspot’ in the gene that mutates at an especially high rate or that other loss-of-function mutations in AraA are deleterious in a way that this particular mutation is not, perhaps because they lead to defective proteins that misfold or have other side-effects.

I recently plated Ara+3 again at 80,767 generations purely to satisfy my own impatient curiosity. On this occasion, there was not a single white colony on the TA agar plate after 48 hours of incubation, but I counted 54 red colonies. I have yet to double-check the identity of these colonies via diagnostic PCR using the rbs operon, let alone via sequencing, but I hypothesize that they’re genuinely Ara+3 based on previous results.

I’m still struck by the colony ratios from my follow-up plating, as they indicate I may have caught the red subpopulation in the midst of a population sweep when I archived the populations at 80.5k. I also can’t help but wonder about the sheer probability that this exact mutation would become the majority in the population, especially because this mutation in araA is neutral in the experimental environment. That then raises the question of what mutations in the red subpopulation have given it an advantage over the white subpopulation, since this reversion must have ‘hitchhiked’ along with one or more beneficial mutations. I’ll be plating again at 81,000 generations in early January as part of the archiving process, and I’m looking forward to seeing how my new favorite LTEE population has developed!

1. Lenski, R. E., Rose, M. R., Simpson, S. C., & Tadler, S. C. (1991). Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations. The American Naturalist, 138(6), 1315-1341. https://doi.org/10.1086/285289
2. Studier, F. W., Daegelen, P., Lenski, R. E., Maslov, S., & Kim, J. F. (2009). Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21 (DE3) and comparison of the E. coli B and K-12 genomes. Journal of Molecular Biology, 394(4), 653-680. https://doi.org/10.1016/j.jmb.2009.09.021
3. Barrick, J. E., Blount, Z. D., Lake, D. M., Dwenger, J. H., Chavarria-Palma, J. E., Izutsu, M., & Wiser, M. J. (2023). Daily transfers, archiving populations, and measuring fitness in the long-term evolution experiment with Escherichia coli. Journal of Visualized Experiments, (198). https://doi.org/10.3791/65342

It’s been a great team effort keeping the experiment running smoothly. There have been no significant interruptions in the daily transfers or suspected contamination events for over a year now!

Jack Dwenger who “coached” the LTEE from November 2022 to July 2024 has taken a position at Penn State to pursue his interests in chemical ecology. Before Jack left, he trained Alexa Morton so she could step in as their new coach without missing a beat. The LTEE is in good hands. Alexa is a veteran of the 2023 UT Austin iGEM team and a recent Dean’s Honored Graduate.

The Olympics theme has had everyone debating how the 12 different populations of the LTEE would fare against one another in different events. Some are easy to judge: Ara–3 wins the only medal in citrate utilization, and it’s DNF (did not finish) for the other 11 populations. Some events will only be decided after more research: Which population has accumulated the most mutations? Which population has the highest fitness when competing for glucose? Which population has the most complex ecology? Which population has the largest cells?

The 80,000-generation populations are waiting in the freezer to be revived for future experiments that will answer these questions.

*One of the twelve populations (Ara–3) is 2,500 generations behind and another (Ara+6) is 500 generations behind due to past contamination incidents like the one discussed here. The last time we had to roll back any of the populations was October 2023.

Of course, the LTEE E. coli were going about their normal business and replicating during the event, a few hours after their daily transfer. We noted the eclipse in the lab notebook, but just to note the passing of time on human and astronomical scales rather than because it would affect their evolution.

The next total solar eclipse over Austin won’t be for hundreds of years… which is hundreds of thousands of E. coli generations, and also many human ones.

Tyler has a long-running Darwin Day event. Many thanks to my hosts, including Brent Bill at UT Tyler and Danielle Pritchard at TJC, and many others who organized events and offered hospitality. I especially enjoyed discussions with students at the TJC STEM Club and hearing about ongoing research projects from students and faculty at UT Tyler. They even have a new faculty member, Wei-Chin Ho, who works on experimental evolution with E. coli!

Lastly, thanks to Tom Hooten for recording and then assembling this video of the lecture. You can check out his channel on YouTube to see videos from other past Darwin Day speakers that they have hosted.

The Guardian published the following article yesterday on the announcement that Darwin Online, the magnificent online repository of Darwin’s works, has completed its project of cataloguing the full list of titles from Darwin’s extensive library, which he used as references to The Origin and his many other books:

Contents of Charles Darwin’s entire personal library revealed for first time | Charles Darwin | The Guardian

Also, if you want some lessons on the importance of rest and reflection to a productive scientific career from Darwin and others, this is a nice article:

Darwin Was a Slacker and You Should Be Too – Nautilus

Take some time to gaze at Darwin and reflect!

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}

}

Fortunately, you can also learn about the exciting ongoing MuLTEE (multicellular long-term evolution experiment with “snowflake” yeast) at Georgia Tech from Will Ratcliff in this episode. Here, an only slightly more complex environment (allowing cells to settle) has led to the evolution of nascent multicellular forms with interesting properties. You can also muse about the prospects for life (and multicellular life) in the universe with Joe Graves of North Carolina A&T who has used experimental evolution to examine bacterial resistance to metal nanoparticles.

Episode link: https://bigpicturescience.org/episodes/going-multicellular

Photo Credit: Frank Fox (www.mikro-foto.de)