‘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!

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.

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}

}

What contamination checks do we normally perform to ensure the integrity of the LTEE?

First, we look at the flasks each day to be sure that each of the cultures has only a slightly foggy whitish appearance, indicating that roughly the expected number of E. coli cells are present and that there is no obvious contamination with other microbes. Second, when we freeze down the populations every 500 generations to archive them, we plate dilutions on three different types of nutrient agar. We look for some expected patterns of colony growth on these agar plates. Cells from specific LTEE populations either grow or don’t grow on certain plates. There are also some characteristic differences in the morphologies of colonies formed by evolved cells from different LTEE populations that can sometimes be used to recognize which one is which.

These techniques can detect many problems. They have been important for maintaining the integrity of the LTEE over its long history, but they have some limitations.  For example, one can only examine colonies that grow from roughly 100-300 cells in a population on an agar plate, which is a relatively small sample from populations that typically have around 300,000,000 cells after growth each day. It would also be difficult to detect contamination from microbes that don’t form colonies on any of the types of agar plates that we routinely check using these methods.

Using whole-genome DNA sequencing to check the integrity of the LTEE

Recently we started doing nanopore long-read sequencing of experiments in our lab because this lets us assemble the entire genomes of some less-studied microbes we work on (mostly insect symbionts at the moment). We’ve done a lot of short-read genome sequencing in the past, but we tend to do that in large batches to achieve economies of scale, and this means we sometimes don’t get the results for weeks or months after preparing a sample. One of the selling points of nanopore sequencing is that one can do a few samples quickly with a benchtop device. (We have been using an Oxford Nanopore MinION Mk1C.) When we froze down the 76,500-generation LTEE populations for archiving, we experimented with using nanopore sequencing as an additional way of quickly checking their integrity.

We were very glad to have given this a try because we made two important discoveries:

1. Population Switcheroo

The first thing we noticed was that two populations (A–4 and A–5) were in the wrong flasks!

These were both Ara^– populations, which is one reason we didn’t detect differences in their growth on the diagnostic agar plates. The other reason is that we are still getting calibrated in terms of recognizing the colonies formed by the various populations in our lab. If we had been good at this, we probably would have noticed the mix-up sooner. We’ve started taking photos of the agar plates to better document these phenotypes. (More about that in a future post.)

To our relief, we did not detect any contamination of one population with the other in the sequencing results, meaning they were not mixed together. Instead, we must have reversed them when performing the transfers one day. Probably their positions in our tray got swapped after we removed them from the incubator. Then, we didn’t notice that the labels didn’t line up as we were pipetting.

To remedy the situation, we went back to examine earlier samples of these populations. We used PCR to screen for different ribose operon deletion alleles known to be present in all cells in each of the two evolved populations. It was clear from these result that the mix-up had happened sometime between 76,000 and 76,500 generations. We corrected the labels on our frozen samples, and these populations can now continue without interruption.

2. Acinetobacter Interloper

We also noticed a bigger problem. When we sequenced population A–3, we found that there was a sizable amount of DNA from a different bacterium. Remarkably, we got so much sequencing coverage that we were able to fully assemble the genome of the interloper and identify it as a strain of the bacterium Acinetobacter radioresistens.

It happens that we work on Acinetobacter in our lab, but only Acinetobacter baylyi. Acinetobacter radioresistens is quite different, so this wasn’t a case of contamination from a nearby lab bench. It must have randomly gotten into an LTEE flask in a different way. However, we became alarmed when we looked up more about A. radioresistens. It seems a strain of A. radioresistens was found to have survived decontamination procedures used on spacecraft that were intended to prevent us from accidentally contaminating Mars with terrestrial microbes (https://doi.org/10.1046/j.1462-2920.2003.00496.x)! It’s in the name, after all, that it is resistant, not only to radiation but to chemicals and treatments used for sterilization.

When did this contamination occur? Why wasn’t it detected sooner? We went back and looked more carefully at the “fossil record” we have frozen in the freezer and our diagnostic agar plates. We found that a few small colonies with a different appearance grew on minimal glucose plates when we spread dilutions of A–3 populations from 76,000 generations or later (see image). These slow-growing colonies were only visible when we incubated the plates for longer than normal: 48 hours versus 24 hours. Additional Nanopore sequencing of the frozen A–3 population samples found contamination in all samples with cells that formed these colonies. We did not detect any A. radioresistens DNA in the 75,500-generation sample or see any of these colonies when we plated it, so the contamination was introduced after this time point.

The Acinetobacter interloper

Colonies formed by Acinetobacter radioresistens cells in the 76,500-generation sample of the A–3 population can be seen on this minimal glucose agar plate after 48 hours of incubation. They are smaller and appear whiter due to being less translucent than E. coli colonies. There are ~20 A. radioresistens colonies on a plate that is crowded with >600 E. coli colonies due to the high cell density in A–3, the only population that has evolved to use citrate present in the LTEE environment. Photo credit: Jack Dwenger, Barrick Lab, UT Austin)

The solution here was to go back to the freezer and restart the A–3 population from the last “clean” sample we had. We let all of the LTEE populations continue to 77,000 generations, the next time they were set to be archived after we sorted all of this out (which took a while). Leading up to this day, we revived the 75,500 A–3 population from the freezer. Then, we substituted it in for the contaminated A–3 population so it could be in sync with their schedule. The A–3 population is now 1,500 generations behind the other 11 populations, but this isn’t the first time an LTEE population has needed to be rolled back to maintain the integrity of the experiment.

Even though this episode is now over, there are some lingering questions. Do we need to worry about A. radioresistens recontaminating the LTEE or spreading to other populations? Is it endemic in our lab? Why did it apparently invade only A–3 and remain at a low but steady frequency in this population for such a long period of time (at least 1,000 generations). We have no plans to track the answers down – it’s generally a very bad idea to purposefully grow microbes that are good at contaminating your experiments in your lab! — but we can speculate.

We noticed that A. radioresistens grows relatively slowly on minimal glucose agar that mimics the glucose-limited LTEE environment. It grows much faster on rich medium (LB). It may be that it doesn’t stand a chance in competition with most of the glucose-specialist LTEE populations and couldn’t contaminate them even if we spiked it into them. A–3 is a special case because E. coli in this population evolved to use citrate. In the process of metabolizing this abundant nutrient, they secrete various other compounds as byproducts. It’s possible the A. radioresistens persisted (at a low level) by taking over a niche that is peculiar to this population, in which it exploits one or more of these secondary nutrients better than E. coli.

Most likely this episode will remain a mystery and footnote to the LTEE—there are so many other interesting stories that have come out of these flasks in the past and that we and others are working on right now. However, we did preserve the frozen fossil record of the alternative future in which A. radioresistens co-exists with the LTEE E. coli, from sometime between 75,500 and 76,000 generations through 77,000 generations, in case anyone ever wants to study it.

Ever Onwards

The LTEE continues! Soon, we’ll be archiving the 77,500-generation samples (except A–3 will be at 76,000 generations). We will be looking with a more practiced eye at the colonies on our plates to check for possible contamination. We’ll also sequence the LTEE populations again to double-check their integrity and find less obvious problems. It will be nice to have this extra piece of mind each time that we deposit new “strata” of the “fossil record” in the freezer.

Finally, many thanks to Dan Deatherage for handling DNA sequencing and Jack Dwenger for heading up maintenance of the LTEE and taking plate photos.