We Interrupt this Nasty Virus with Some Good News about Bacteria

Today is the 32nd birthday of the E. coli long-term evolution experiment (LTEE).  I started it on February 24th, 1988, when I was at the University of Califonia, Irvine.

It also happens to be daily transfer number 11,000 for the experiment.  But wait, you ask: Is 365 x 32 really equal to 11,000?  (Not to mention the complication of leap years.)

No!  365 x 32 = 11,680.  We’re almost 2 years behind perfection!  Over the years, we missed daily transfers for various reasons including the fact the experiment was frozen for several months around the time of my move from Irvine to Michigan State University, as well as some missed transfers and various mishaps (including contamination) along the way that have led us to restart the experiment from frozen samples.

Luckily, we don’t have to go back to the beginning–the LTEE wouldn’t have survived if we did. We freeze whole-population samples every 75 days, and those provide the backups that keep us going when needed.

So the LTEE is 32 years old today.  The evolving bacteria lineages, though, are younger, at a little over 30 years (11,000 / 365).  I prefer to think of them as timeless, though … having survived in and adapted to their tiny flask worlds for more than 73,000 generations.

Here’s grad student and lab manager Devin Lake doing today’s transfer.

And here’s Devin & me with the lab notebook. Devin is pointing to today’s entries.

And here’s what we wrote:

For those with pathogens on their mind (and that’s a lot of us, with the new coronavirus spreading), you might wonder: Aren’t E. coli dangerous?  The short answer is only rarely. All of us have harmless or even beneficial strains of E. coli and many other bacterial species in our GI tract. The LTEE uses one of these harmless strains, one that has been studied in many labs for close to a century without problems. There are some strains of E. coli, though, that are nasty, and which are usually acquired by eating contaminated foods.  So wash your raw fruits and vegetables, cook your meats, and don’t worry about the LTEE bacteria … Just wish them a happy birthday today, and many more years of scientific discovery.

Coach Izzo and me

Chalk up another great year for the Michigan State men’s basketball team and coach Tom Izzo. The Spartans were co-champions of the Big10 and won the conference’s grueling tournament. And in the NCAA’s March Madness, they made it all the way to the Final Four, knocking out the top-seeded team in the process.

Being a fan of this team got me thinking: Coach and I have a lot in common. We’ve both been doing our jobs, mostly at MSU, for a long time. Coach Izzo came here as a part-time assistant in 1983, becoming head coach in 1995. I was on the faculty at UC-Irvine starting in 1985, before moving here in 1991.

But the real similarities are deeper and more important:

First and foremost, we’ve both been fortunate to be surrounded by talented and hard-working students who listen to our ideas, experiment with them, develop them in their own ways, and translate them into meaningful outcomes—winning big games and making new discoveries.

That’s not to say there aren’t frustrations along the way: games lost, grants and papers rejected, grinding practice on the court and repetition in the lab, and even occasional conflicts. But our students are usually resilient—they overcome those setbacks and frustrations, and they go on to productive lives as players and coaches, researchers and teachers, and other careers as well.

We also both had mentors who helped us start our own careers. In Coach Izzo’s case, one mentor was Jud Heathcote, the previous head coach who hired him as an assistant. My mentors included my doctoral advisor, Nelson Hairston, and my postdoctoral supervisor, Bruce Levin. Coach Izzo and I also had friends who helped shape our careers early on: Steve Mariucci, who went on to become an NFL coach; and Phil Service, who did important work on life-history evolution.

Coach Izzo and I also both benefitted, I think, from early successes—again, largely due to our students—that helped establish our reputations, allowing us to retain our jobs and thrive by recruiting more talented, hard-working students. For Tom Izzo, it was players like Mateen Cleaves, Charlie Bell, and Mo Peterson who took the Spartans to the Sweet 16 in his 3^rd year as head coach and to the Final Four the next year, and who won the 1999-2000 National Championship. For me, the early students included Judy Bouma, Felisa Smith, John Mittler, Mike Travisano, Paul Turner, and Farida Vasi, and postdocs Toai Nguyen and Valeria Souza.

Coach Izzo has also had assistant coaches and staff, who I imagine do a lot of the heavy lifting. While some might eventually become head coaches of their own teams, many others labor in relative obscurity. In a similar vein, I’ve had outstanding lab managers including Sue Simpson, Lynette Ekunwe, and—for over 20 years, before retiring last year—Neerja Hajela.

Coach Izzo and I have both had deep benches—students who helped the team succeed without being in the limelight themselves. For Coach Izzo, they include the walk-ons and others who see limited action in games, but who compete against the starters every day in practice, helping everyone become even better. I think of three undergraduates who joined my lab when it was just getting started in Irvine (all Vietnamese refugees, by the way) who asked if they could work in my lab. Trinh Nguyen, Quang Phan, and Loan Duong prepared media and performed experiments like some incredible three-brained, six-handed machine, setting a high standard for everyone who followed in their footsteps.

Coach Izzo and I are nearly the same age. Retirement might be easier, but neither of us is ready for that. It’s too much fun when you’ve got talent to encourage and guide like Cassius Winston, Joshua Langford, Nick Ward, Xavier Tillman, and Aaron Henry—and on my team Jay Bundy, Kyle Card, Nkrumah Grant, Minako Izutsu, and Devin Lake.

Of course, there’s more that Coach Izzo and I have in common—we were lucky to be born into circumstances that allowed us to pursue our dreams without the obstacles that many others face.

Last but not least, Coach Izzo and I have had supportive partners who’ve accepted our peculiar obsessions and the long hours and frequent travel that our work entails.

Go Green! Go Students!!

Thank you, Neerja

Neerja Hajela has worked with me for over 22 years as a technician and lab manager. She is extremely skillful, diligent, organized, and dedicated in her work. On top of all that, she is a wonderfully kind and warm person. Now, this is her last week before she retires.

It’s impossible to put into words all that Neerja has done for me, for everyone in the lab, for the LTEE, and for my collaborators. But let me mention a few of the things she has done.

By keeping the lab running in a smooth and orderly fashion, Neerja has enabled me to spend more of my time thinking about science, writing papers, giving talks, etc., etc. We scientists sometimes complain that we have too much to do, and so we joke that we want to clone ourselves. Well, I’ve done better—I’ve had Neerja.

Those of us with labs know that our institutions take laboratory safety very seriously, as well they should. Neerja runs such a tight ship that, on many occasions after inspecting our lab, the safety officers have made comments to the effect that they wish all labs were as neat, clean, organized, and safety-conscious as ours.

One of the challenges of the long-term evolution experiment (LTEE) with E. coli is freezer management. We now have over 30 years of samples, spread over half a dozen freezers, which provide a record of past evolution. Neerja has overseen this ever-growing collection with extraordinary care and dedication. The samples provide critical backups that allow us to restart the LTEE from a recent milestone when mishaps occur, and they provide unique research materials such as when new technologies emerge. A case in point: Michael Desai wrote me a few years ago with a request. In essence, he wanted all of the LTEE samples for metagenomic sequencing. All of them—from each population and every generation with saved samples. Since I started the LTEE in 1988, we’ve always saved duplicate samples, with one of them being a backup to be opened only in an emergency. I could send Michael the backups, perhaps, but that didn’t seem like a good idea. So I decided we should make additional sets by going into the ~1500 key samples spread over several freezers; taking a subsample of each and culturing it to produce a larger sample; splitting the new culture into ten sub-cultures; and freezing those to provide a new set for Michael as well as other sets for collaborators and institutions. It took Neerja many months to accomplish all of this, but as always, she did it with great skill and care. (Oh, and you can read about the results of Michael’s request here.)

Last, but surely not least, Neerja has done more of the daily transfers of the LTEE than anyone else. She performed her first LTEE transfer on February 5, 1996, and since then she has done well over 4,200 daily transfers. (Thanks to Zachary Blount, who went through the LTEE lab notebooks for its 30^th birthday.) And when Neerja hasn’t done the transfers herself, she has organized who else is responsible for each and every day’s transfers.

Thank you, Neerja, for all that you have done for me, for everyone in the lab, for the LTEE, and for science. Everyone in the lab joins me in wishing you and Ravindra all the very best in your retirement and new home!

 

[Neerja Hajela]

[Here’s Neerja doing yesterday’s LTEE transfer]

[Neerja pointing at two of her LTEE entries: her first transfer on February 5, 1996, and the one from yesterday July 30, 2018. The lab notebooks in the background record the daily transfers since she joined the lab.]

[Close-up of Neerja’s first entry.]

[Neerja’s protocols for the LTEE, mounted in the lab, so nobody makes a mistake]

[And in case that doesn’t work, here’s Neerja enforcing lab discipline]

[The LTEE transfer board from earlier this year]

[Horsing around Tanush’s plate tower]

[Neerja making copies of freezer samples]

[Neerja and me]

You gotta know when to hold ‘em

I was honored and humbled to speak at the Doctoral Hooding Ceremony last weekend at the University of North Carolina at Chapel Hill. I received my Ph.D. there in 1982. It was great to be back in Chapel Hill, seeing some old friends and making many new ones.

There was also one of those interesting small-world connections: UNC Chancellor Carol Folt is an ecologist. I first met Carol when she was an assistant professor at Dartmouth and I was commuting from Amherst, where I was a postdoc, to Dartmouth, to teach evolution as a sabbatical replacement for one semester. Carol is such a positive person, always smiling, and an energetic chancellor.

Anyhow, I had never given a talk like this before, so it was a challenge to prepare. Here’s what I had to say to new doctorates; maybe some of you will find it useful as well.

~~~ ~~~ ~~~

Let me begin by congratulating all of the new PhDs and recipients of other doctoral degrees. Each of you climbed a mountain that no one before you had ever climbed. That’s what made it a doctorate — your original research leading to new knowledge.

My remarks today are about constancy versus change, and about luck versus skill. They turn out to be core themes in the research I do, and they also have a lot to do with life, including the decisions we make in our professional careers.

Speaking of constancy, some things hardly seem to change. I got my degree here in 1982. And who won the NCAA men’s basketball title that year? Yep, it was the Tar Heels, just like this year.

Of course, there have also been a lot of changes since I was a student. Music, for example. When we went to the bar, we had these awesome communal listening devices, called jukeboxes. You didn’t even need headphones to hear the music.

Kool & the Gang’s “Celebration” was hot then — and it’s still a great song if you’ve got a party tonight! Cross-over country music was big, too.

Kenny Rogers had a hit called “The Gambler”, about advice from an old poker player. You’ve probably heard it. It goes like this:

“You got to know when to hold ‘em, Know when to fold ‘em, Know when to walk away, Know when to run.”

Of course, the song is about life, using poker as a metaphor. Just as in our careers and lives, poker requires making decisions in the face of uncertainty.

I had a lot of very good luck at Carolina. I went to a party where I happened to meet Madeleine, a graduate student in the School of Public Health, who is now my wife.

However, I also faced some difficulties, and while I managed to get through them, they led me to change the direction of my research.

I came to UNC to study ecology, which focuses on species and their interactions in nature. I got interested in biology when I took a non-majors course as an undergraduate at Oberlin College, and I saw the sweep of discoveries from molecular biology to vertebrate evolution.

As I contemplated graduate school, I focused on ecology because it was filled with interesting and unanswered questions that, to my naïve self back then, seemed like they wouldn’t be too hard to study.

Many ecologists are superb naturalists, including Nelson Hairston, my advisor here at Carolina, who loved the salamanders he studied, and who knew their biology inside and out.

Or Charles Darwin, who was fond of beetles. On a collecting trip, he already had two beetles he wanted, one in each hand, when he came upon a third that he also wanted to keep. He was so in love with his beetles that he popped one into his mouth to free up a hand. Well, it turns out that the one he put in his mouth was a bombardier beetle. To escape predators, they combine and squirt out two chemicals in an explosive exothermic reaction. Needless to say, Darwin lost all three of those beetles.*

As a kid, I loved being outdoors, hiking and playing sports. But I wasn’t a naturalist; I didn’t know very much about any particular group of animals or plants. At least partly because of that lack of familiarity with organisms in the wild, my first efforts at doing ecological research were failures.

Let me give one example, because it’s kind of funny — at least in hindsight. I tried to do a field experiment using praying mantises. I reared batches of them in the lab from egg cases, and then released them on small plots with two treatments. I had painstakingly cleared the vegetation around each plot by hand to keep the mantises where I put them. Well, the next time I went to see how they were doing, I couldn’t find a single one! Maybe some birds were watching me when I released the mantises, wondering: “What is this crazy guy doing?” before gobbling them up. I have no idea what happened, but that experiment was a total bust.

With hindsight, I was lucky that this project failed right away. The treatment effect I was looking for would probably not have given a significant outcome, even if the mantises had stayed put. So even failures can sometimes be valuable, by keeping us from wasting time—and by forcing us to change direction.

Maybe some of you had failed projects, too, before you found your bearings. It’s a normal part of science and scholarship, though it’s upsetting when it happens.

I had another project that also failed. But this second failure led me to the study system that became my dissertation, which was about the effects of forest cutting and competition on a certain group of insects, called ground beetles.

I loved being outdoors in the mountains of western North Carolina, although the frequent rainstorms often flooded the traps that I used to catch the beetles, drenching both the beetles and me. But this project, at last, was successful, leading to my dissertation and some papers.

But I also had doubts that this line of research was a good fit for my interests and skills. Maybe some of you are at similar points in your career.

I’m sure some of you have found work that you hope to continue for the rest of your life. If so, that’s terrific and more power to you.

Others of you might be pondering or even planning a change—using your degree and experience, but setting off in a new direction. Maybe not right away, but perhaps keeping an eye out for some opportunity that better fits your own skills and interests.

In my case, an exciting opportunity dawned in a graduate reading group, when we read a paper about the coevolution of bacteria and viruses that attack bacteria. Even though I had no experience in microbiology, I wrote the head of that lab with an idea for a project related to the paper, and—lucky for me—he hired me as a postdoc.

Before I started my new position, I was worried about working in an area where, once again, I had no experience. Well, I soon discovered that I enjoyed the work. I wasn’t good at it right away, but I liked the rhythm of a microbiology lab. Unlike praying mantises, the bacteria stayed put in their flasks. Unlike the beetles in the mountains, there weren’t any rainstorms in the lab. And sometimes you could see the results of an experiment the very next day.

Down the road, there were more hurdles. In my first year of looking for a faculty position, I applied for dozens of jobs. I got one interview and no offers. Meanwhile, the grant that funded my research wasn’t renewed, and I had a growing family to support. I even thought about leaving science — and I would have if Lady Luck hadn’t come through for me yet again.

The grant was renewed on the second try, and in my second year on the job market I got two offers. So I headed out to Irvine, California, where I started a project that continues to this day.

The project is an evolution experiment. In fact, the experiment was set up to address the same themes as my talk today—luck and skill, constancy and change—although in a scientific context, rather than a personal one.

In evolution, genetic mutations are random events, while the process that Darwin discovered—adaptation by natural selection, sometimes called “survival of the fittest”—multiplies the best competitors across the generations. I wanted to see how luck and skill—that is, mutation and selection—would play out if we could watch evolution over and over and over.

So I set up 12 populations of E. coli bacteria, all started from the same genetic stock, and I put them in identical flasks, with identical food, the same temperature, etc.

I wanted to know: Would they all change and adapt in the same way, showing the power of natural selection to shape life? Or would each population evolve along a different path, highlighting the importance of random mutation?

One thing that makes bacteria great for this experiment is that we can freeze samples and then later revive them as living cells. In essence, our freezers are time-travel machines for the bacteria, allowing us to directly compare and even compete bacteria that lived at different times.

You’ve all heard about our close relatives, the Neanderthals, who went extinct about 40,000 years ago. Some of you might know that their DNA has been recovered from fossils, allowing their genomes to be analyzed. It’s even been discovered that most of us have stretches of Neanderthal DNA in our own genomes.

But despite these amazing advances, we don’t really know what the Neanderthals were like and how similar they would be to us, if they were raised in our world. How well would they play chess, or music, or basketball? What topics would they choose for their dissertations? What would they talk about if they were at this podium?

Back to the experiment with bacteria: We’ve seen many parallel changes in the bacteria across the 12 replicate populations, showing that natural selection can sometimes make evolution predictable, despite the randomness of mutation. But we’ve also seen differences emerge, including in one lineage a surprising new ability to grow on a resource that other E. coli cannot use. And using new technologies that didn’t exist when the experiment was started, we’ve sequenced hundreds of genomes to find the mutations in samples from across the generations and populations, allowing us to test the repeatability of evolution at the level of the DNA itself.

I sometimes call it “the experiment that keeps on giving.” I originally intended the experiment to run for 2,000 generations, which would take about a year. Well, today it’s been running for almost 30 years, and the bacteria have been evolving for 67,000 generations.

This experiment keeps on giving because the bacteria keep evolving in interesting and sometimes unexpected ways, and because students bring new questions and ideas to the project. My hope is that it will continue long after I’m gone.

While the experiment gets a lot of nice press and compliments these days, there have been some obstacles along the way, as there always are in life and science.

When the first paper was submitted, one reviewer was very negative and even hostile. That reviewer wrote: “I feel like a professor giving a poor grade to a good student” — ouch! — without any suggestions for how to improve it. In fact, the reviewer even wrote: “This paper has merit and no errors, but I do not like it.” Well, I wasn’t going to fold — I liked the cards in this hand. So I wrote a rebuttal, and the paper was accepted. In fact, it went on to receive the journal’s award for best paper of the year.

A second obstacle was one of my own making. I came across another experimental system that I found fascinating, and still do — artificial life in the form of computer programs that can replicate themselves and evolve. At the time, I thought maybe the long-term experiment with bacteria had run its course. Well, unlike in poker, when you face important decisions in your research and career, you can ask other people for advice. It’s a good thing, because I was able to have my cake and eat it, too. Everyone told me: “Don’t end the experiment with bacteria. It’s too valuable.” So my lab has kept it going and it has continued to be a scientific gold mine.

Along the way, some creationists have criticized our work. Some don’t believe our results, while others believe us but say: “See, they’re still only bacteria” — as though any scientist would expect to see worms or monkeys or whatever emerge from this experiment.

There can be many reasons for misunderstandings between scientists and the public: problems of education, politics, and communication. The third problem — communication — is one that we can strive to overcome by explaining our work not only to our close colleagues, but also to the general public.

A couple of years ago I had a wonderful opportunity to communicate science to a broad public audience. I was asked by the producer of “Through the Wormhole with Morgan Freeman” to do a segment about our research on bacteria for that show.

One of the scenes had me playing poker with a few of my students. It shows how the effect of a random event—a particular card in a game of poker—depends on the context in which it occurs. The same is true in evolution. A particular mutation that might be advantageous in one species could be detrimental or even lethal in another.

Let’s have a look**:

“When there was a Queen and a King of Hearts on the table and you have the 10 and Ace of Hearts in your hand, you are set up to potentially make a Royal Flush, the most powerful hand in poker. All you need is for the final card to be the Jack of Hearts.”

I’ve been lucky in life. I was born to parents who nurtured me. I was born in a nation dedicated to life, liberty, and the pursuit of happiness. And like those of you receiving your degrees today, I was fortunate to get a superb education here at Carolina.

The French scientist Louis Pasteur — who in the 1800s disproved spontaneous generation, invented what we now call pasteurization, and developed the first rabies vaccine — said: “chance favors the prepared mind.”

Thanks to your Carolina education, and the hard work that brought you here today, you have a prepared mind. You will encounter many uncertainties, probably some obstacles, and hopefully some terrific opportunities as the cards of life are dealt to you.

Play them well: Know when to hold them, know when to fold them. And sometimes you won’t really know what to do, so you’ll just have to give it your best shot.

Thank you, and congratulations again to all of you receiving your doctoral degrees today.

~~~ ~~~ ~~~

*This story is told in the autobiographical chapter of The Life and Letters of Charles Darwin, edited by his son Francis Darwin. I should have checked the source instead of relying on my memory, as Darwin says he lost only two of the three beetles.  The details of the bombardier beetle’s chemical defense system were worked out in the 1960s by Thomas Eisner and others.

**Thanks to Tony Lund, who produced the television show, for also making the short clip that I showed in my talk. You can see a longer clip here.