Category Archives: Science

Hat Trick

This past weekend Madeleine and I attended the annual meeting of the American Academy of Arts & Sciences in Cambridge, Massachusetts. The Academy was founded in 1780 by John Adams and several dozen other scholar-patriots “to cultivate every art and science which may tend to advance the interest, honor, dignity, and happiness of a free, independent, and virtuous people.”

It was an especially exciting meeting for us because we got to see three of our dear friends inducted as new members:  Paul Turner and, as International Honorary Members, Valeria Souza and Sebastian Bonhoeffer.  All three of them signed the membership book, putting their “John Hancocks” alongside those of distinguished artists, scientists, scholars, and leaders (including the John Hancock) from the past 239 years, while Madeleine and I celebrated with their spouses and families.

I’ve known Paul, Valeria, and Sebastian for decades.  Paul was a graduate student in my group at UC-Irvine, and then he moved with me to MSU, receiving his PhD in 1995. For his dissertation, Paul studied issues related to vertical and horizontal transmission in bacteria, including the roles of density- and frequency-dependent selection. Paul is a professor at Yale University, where he and his team study the evolution and ecology of viruses, including some that can specifically target antibiotic-resistant bacteria and have been used to cure life-threatening infections.

Valeria was a postdoc in my group, also first at UCI and then again at MSU. She worked with Paul on a fascinating, but challenging, experiment to investigate the effects of horizontal gene transfer on the speed of adaptive evolution in bacteria. Valeria is a professor at Universidad Nacional Autonoma de Mexico, where she and her group conduct research and work with the local community, governmental agencies, and nonprofits to conserve the Cuatro Cienegas basin, a biologically unique and fragile system of oases in the Chihuahuan Desert.

Sebastian and I met at Oxford University in 1993, where he was a graduate student and I was on sabbatical. We collaborated on a theory project that examined the hypothesis that pathogens with long-lived propagules would evolve to be more virulent. More recently we’ve taught together in the Guarda (Switzerland) summer course on evolutionary biology. Sebastian is a professor at ETH Zurich, where he and his team construct and analyze mathematical models of population dynamics to understand, for example, the pathogenesis and spread of HIV and other viruses.

Besides being creative and talented scientists, Paul, Valeria, and Sebastian are three of the nicest people around. I’ve been incredibly fortunate not only to work with them, but also to know them as close friends.

And there were so many other outstanding inductees, some of whom I’ve also known for many years including microbial ecologist Jo Handelsman (University of Wisconsin), theoretical ecologist Mercedes Pascual (University of Chicago), evolutionary biologist Mark Rausher (Duke University), plant biologist Detlef Weigel (MPI Tubingen), and evolutionary biologist Kelly Zamudio (Cornell University).

Each of the 5 “classes” had a speaker give a short talk to all the inductees and their families. Representing the Biological Sciences, Jo Handelsman gave an impassioned talk “On the importance of soil.” It’s something almost everyone takes for granted, and yet fertile topsoil is incredibly valuable, it’s disappearing in many areas, but it can be preserved and even enhanced with improved agricultural practices. Representing Public Affairs, Business, and Administration, Sherrilyn Ifill (NAACP Legal Defense Fund) gave a clarion call to fix American democracy.

The evening before the induction ceremony there were artistic performances and presentations by several new members including jazz pianist, composer, and singer Patricia Barber.  The morning after the induction included a performance by, and discussion with, the incredible playwright, filmmaker, and actress Anna Deavere Smith, who performed and described how she constructs her amazing one-woman shows.

Throughout all the events, the staff of the American Academy of Arts & Sciences were superbly organized and warmly welcoming.

[Paul Turner’s family in the theater just before he signs his name into the book of members of the American Academy of Arts & Sciences]

Paul's family at AAAS 2019

[Valeria Souza signing the book, with Paul Turner just behind her and waiting his turn]

Valeria signing, Paul just behind, AAA&S 2019

[With Valeria Souza and her family following the induction ceremony]

Valeria and family at AAAS 2019

[Sebastian Bonhoeffer and his wife Hanna (next to me) with Madeleine (next to Sebastian) and me the evening before the induction ceremony]

bonhoeffers-and-lenski-aaas-2019.jpg

[Yours truly along with Mercedes Pascual, Paul Turner, and Sebastian Bonhoeffer]

AAAS 2019 Mercedes, Paul, Sebastian and me

[Sebastian, Paul, Valeria, me, and Luis Eguiarte (Valeria’s husband, and also a superb evolutionary plant biologist]

Sebastian, Paul, Valeria, me, Luis AAA&S 2019

[Paul and I compare our biologically themed ties.]

Paul and me at AAA&S 2019

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Freezer Burn

One of the more challenging aspects of running a microbiology lab, in my opinion, is freezer management.  There’s a lot to keep track of, both in terms of quantity and quality.  My lab team and I take great pride in the quality control of our work that has allowed us, for example, to keep the LTEE running for over 30 years and 70,000 generations without contamination.  Or rather, as I’ve posted before, we’ve had occasional accidents including cross-contamination of the replicate lines, but we’ve caught those mistakes and, using frozen samples, restarted as needed to keep things going smoothly and cleanly.

With my lab group now running for ~34 years (I started at UCI in 1985), and with so many hard-working students and postdocs, we’ve filled up lots of –80C freezers.  And that’s despite shipping many strains to scientific collaborators and former lab members who’ve continued to work on the various projects—the LTEE is only one (albeit the longest) of the many projects we’ve done in my lab.  Adding to the storage challenge, we’ve got duplicates of most samples in case we have a problem with the primary sample (say, someone drops a vial on the floor).  Also, to avoid compromising our primary or backup samples, I ask that everyone who plans to use any sample (usually a set of many samples) more than once make his or her own working copies of the samples.

And freezers sometimes fail, despite our best efforts to maintain them in tip-top shape.  So over the years, I’ve always tried to keep a freezer’s worth of spare capacity across our multiple freezers, so when one fails, everything can be moved into a functioning freezer.

On Sunday, one of our workhorse freezers failed. Most of our freezers have alarms that send out an email alert to members of the lab that something is amiss.  This one did not (oops!), but fortunately undergraduate Jessica Baxter (working hard even on the weekend), noticed that it had “warmed up” to –40C or so.  I was off visiting grandkids, but Jessica was able to reach Devin Lake, who manages the lab’s operations extremely well, even as he does double-duty as a grad student.  Devin and Jessica were able to find enough spare capacity to get everything into one of the surviving freezers, so nothing was lost.

But that meant we had no more spare capacity.  We can buy a new freezer, although my experience (and hearing about many other failures) is that they don’t make them like they used to.  And what if another freezer were to fail before we got a new one?

I knew we had many freezer racks full of now-unimportant samples—working copies made by people who’ve left the lab, as well as samples from abandoned experiments and various long-ago projects that won’t be revisited.  So I asked Devin to look through the freezers for the identifiers on various racks (besides the LTEE and any associated with current lab members) that would give me ideas of what we could discard to free up some space that we will need for ongoing projects … as well as the possibility of another freezer failure.  (But please not that!  I’m not trying to tempt fate—I just want to be prepared.)  It turns out there were lots of possibilities, so Devin and I spent a couple of hours checking boxes and then removing about 20 freezer racks, most holding 6 to 10 boxes, and most of those with dozens of small vials, each holding many millions or even billions of bacterial cells.  Seeing the names of former lab members on the boxes, and the numbers on all those vials, was a humbling reminder of all the hard work that so many have done over the years.  Devin carted three loads of discards down to one of our workrooms, where hardworking tech John Baltusis emptied each box and prepared the vials for the sterilization (autoclaving at high temperature) that’s required before they can be discarded.

Thanks to the hard work of Jessica, Devin, and John, the lab avoided any setback. In fact, our freezer collection is now a little more manageable than it was before.

[Devin Lake, in front, with one of three cartloads of samples to discard, while John Baltusis removes the samples from one box before autoclaving.]

Devin, John, and freezer mess

 

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Half Life

Today marks a unique day for the LTEE and me.

The LTEE started on February 17, 1988.  That was 11,517 days ago.

I was born on August 13, 1956.  That was 23,034 days ago.

That means that the LTEE is now half as old as I am.

To put it another way, I’ve spent half a lifetime on the LTEE.

Well, that’s not quite the right way to put it, since I’ve done a few other things during that time. Like raising a family—with a lot of help.  And a lot of other science, also with a lot of help, not to mention all the work of so many students and collaborators on the LTEE itself.

And unlike a radioactive isotope, the bacteria haven’t been decaying—they’ve been getting better and better at living in their flask-worlds.

My hope is that this long-term evolution experiment will continue for a long time. A very long time. For a lot longer than my own lifetime.

Here are a couple of photos from around the time the LTEE started. The first one shows Madeleine and me camping near Joshua Tree National Park in the summer of 1987, at the annual retreat of the UC-Irvine EEB department, and only a couple months before the birth of our youngest. The next one shows me snuggling with my three kids in early 1989.

june-1987-desert-x-with-mjan-1989-with-3-kiddos

How time flies. Luckily, though, I get to snuggle with my three grandkids now.

Bacterial generations. Human generations. Growing, evolving, and learning.

 

 

 

 

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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 3rd 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!!

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Evolution goes viral! (And how real science works)

This is the fourth in a series of posts about a new book by Michael Behe, Darwin Devolves. Behe is a leading proponent of intelligent-design creationism (IDC), which asserts that known processes cannot adequately account for evolution and, therefore, some intelligent agent must be involved in the process. Behe is a professor of biochemistry, which gives him knowledge and credentials that most IDC advocates do not have. However, my posts explain why I think his logic is unsound and his evidence weak and biased.

In brief, Behe argues that random mutation and natural selection are almost entirely degradative forces that break or blunt the various functions encoded by genes, producing short-term advantages that are so pervasive that they prevent constructive adaptations, which he claims are very unlikely to emerge in the way that evolutionary biologists have proposed. Unlike young-Earth creationists, Behe accepts the descent of living species from common ancestors over billions of years. To reconcile these seemingly conflicting views, Behe invokes that an intelligent agent (presumably God, though IDC proponents avoid that word so that their ideas might appear to be scientific) has purposefully guided evolution over its long history by somehow inserting new genetic information into chosen lineages along the way. To make his strange argument, Behe works very, very hard to convince readers that standard evolutionary processes are (i) really, really good at degrading functions, and (ii) really, really bad at producing anything new.

In my first post, I explained that Behe’s arguments confuse and conflate what is easy and commonplace over the short run (i.e., mutations that break or blunt functional genes) with the lasting impacts of less frequent but constructive adaptations (i.e., new functions and subsequent diversification) over the long haul of evolution. My second post examined a case involving polar bears, which Behe highlighted as a compelling example of degradative evolution, but where a careful review of the science suggests that gene function improved. Behe also highlighted results from my lab’s long-term evolution experiment with bacteria, but in my third post I explained that he overstates his case by downplaying or dismissing evidence that runs counter to his argument.

In this post, I’ll discuss an experiment that Behe ignores in Darwin Devolves. (Behe clearly knows the work, because he wrote about it on the Discovery Institute’s anti-evolution blog. But as usual, he spun the story to obscure the problems for his arguments, all the while accusing the scientists who collect data to test hypotheses of spinning the story.) In fact, as I’ll explain, the results also undermine the claims in Behe’s two previous books, Darwin’s Black Box and The Edge of Evolution, about the supposed shortcomings of evolution.

(Before presenting this experiment, I want to mention briefly two other papers that readers interested in what else Behe missed or downplayed might want to read. First, Rees Kassen posted a preprint of a paper on “Experimental evolution of innovation and novelty.” He reviews empirical evidence and discusses conceptual issues bearing on the origin of new functional abilities observed in many experiments with bacteria and other microbes. Second, Chris Adami, Charles Ofria, Rob Pennock, and I published a paper over 15 years ago that demonstrated the logical fallacy of Behe’s assertions about irreducible complexity. Behe mentions that paper derisively, without addressing its substance in Darwin Devolves, as follows: “A computer simulation of computer program development that ignores biology entirely.” A more accurate statement would have been: “Computer programs can evolve by random mutation and natural selection the ability to perform complex functions that show the concept of irreducible complexity is total nonsense.” The rest of this post is longer than I planned, because I want to provide background for readers who aren’t microbiologists, and because—like so much of science—it’s an interesting story with unexpected twists and turns along the way.)

IV. Phage lambda evolves a new capability without breaking anything

There are a lot of viruses in the world. Fortunately, most of them don’t infect humans. Many of them infect bacteria, as it so happens. In fact, before antibiotics were used as therapeutic agents, there was hope that bacteriophages (“bacteria eaters”), or phages for short, would be useful in treating diseases. And now, with the evolution of pathogenic bacteria that are resistant to many or all available drugs, researchers are reconsidering the possibility of using phages to treat some infections.

My lab is best known for the long-term evolution experiment (LTEE) with E. coli bacteria. But over the years, my students have also performed other experiments with a variety of microbes, including some viruses that infect E. coli. One of those viruses is called lambda. For decades, lambda was probably the most intensively studied virus on the planet—just as E. coli was a model for understanding bacterial genetics and physiology, lambda became a model for understanding viral genetics and infection.

One reason lambda became a hit was because it has an interesting life cycle. After lambda enters a bacterial cell (and assuming the cell lacks some internal defenses), the virus can do one of two things. It can commandeer the host, hijacking the cellular machinery to produce a hundred or so progeny before bursting the host cell and releasing its “babies” to find new cells to infect. Alternatively, the virus’s DNA may be integrated into the host’s chromosome, hiding out and being replicated alongside the host’s genes—though the virus may later exit the chromosome and reactivate its lethal program. (Pretty neat, and a bit scary, right?)

Well, as cool as that is, it’s not what my student Justin Meyer (now on the faculty at UCSD) was studying. He was using a strain of lambda that can’t integrate into the bacterial cell’s chromosome—a successful infection takes only the first route, killing the cell in the course of making more viruses. Justin was studying this simpler virus because we were interested in whether the evolution of the bacterial hosts in response to the presence of lambda virus might depend on what food we gave the bacteria.

Let’s back up and explain why that might matter. Viruses like lambda don’t just glom onto any part of a bacterial cell; instead, they adsorb to specific receptors on the cell’s surface, with a successful attachment triggering the injection of their DNA into the cell. Lambda recognizes a particular cell-surface protein called LamB. (Despite decades of study of the interaction between lambda and E. coli, including experiments that specifically sought to see whether mutants could exploit other receptors, no one had ever seen lambda use any other receptor.) Of course, E. coli doesn’t make LamB for the sake of the virus. The LamB protein is one of several “porin” proteins that E. coli produces, and which serve as channels to allow molecules, like sugars, to cross the outer cell envelope. (Other proteins transport sugars across the inner cell membrane.) LamB, in particular, is a fairly large channel that allows the sugars maltose and maltotriose to enter the cell. Maltose and maltotriose are made of two and three linked glucose molecules, respectively. Glucose, being smaller, can readily enter a cell via smaller channels. When growing on glucose, E. coli cells don’t bother to produce much LamB protein. However, when cells sense that maltose or maltotriose, but not glucose, is present they activate the gene that encodes LamB. In doing so, however, the cells become more vulnerable to lambda, because that protein serves not only to transport these larger sugars but also as the receptor for the virus.

Coming back to Justin Meyer’s research, we wanted to see how different sugars affected the bacteria’s evolutionary response to lambda. (Justin and I have a paper in press comparing outcomes across the glucose, maltose, and maltotriose environments.) We reasoned that, if the bacteria were fed glucose, they could damage or delete the lamB gene that encodes the LamB protein. If the bacteria mutated the LamB protein, then the virus might counter with a mutation that restored their affinity for the mutated protein; but if the bacteria deleted or otherwise destroyed the LamB protein, we reasoned the virus would go extinct.

However, the first experiment using only the glucose treatment played out differently than what we expected—that’s science, and that’s why you do experiments—and it set Justin’s research off in a new direction. Instead of mutating the lamB gene, the bacteria evolved resistance to the virus by mutating another gene, called malT, that encodes a protein that activates the production of LamB. The viruses didn’t go extinct, however, because there was some residual, low-level expression of the LamB protein. That was enough to keep the viruses going, which also meant they could keep evolving.

To make a long story short, after just 8 days, one of six lambda populations evolved the ability to infect malT-mutated cells by attaching to a different surface protein, one called OmpF (short for outer membrane protein F). This evolved lambda virus could now infect E. coli cells through the original receptor, LamB, or this new one, OmpF. It had gained a new functional capability.

To understand this change, Justin sequenced the genome of this virus. He found a total of 5 mutations compared to the lambda virus with which he had begun. All 5 mutations were in the same gene, one that encodes the J protein in the “tail” of the virus that interacts with the cell surface. He also sequenced the J gene for some other viruses isolated from the same population. He found one virus that had 4 of these 5 mutations, but which could not infect cells via the OmpF receptor. Did that mean that only one of the 5 mutations was necessary to evolve this new function?

As it turns out, the answer is no. To better understand what had happened, Justin scaled up his experiments and ran an additional 96 replicates with lambda, E. coli, and glucose. In 24 cases, the viruses evolved the new mode of infection within three weeks. Justin sequenced the J gene from the viruses able to target OmpF in those 24 cases, and in 24 other cases where the virus could still use only the LamB receptor. He found that all 24 with the new capability had at least 4 mutations; these included 2 changes that were identical in all 24 lines, a third that further mutated one of the same codons (sets of 3 DNA bases that specify a particular amino acid to be incorporated into a protein), and another mutation that was always within a span of 11 codons. All of these mutations cause amino-acid substitutions near the end of the J protein, which is known to interact with the LamB receptor. The J protein is over 1100 amino acids in length, and so this concentration and parallelism (repeatability across lineages) is striking and strongly implies that natural selection favored these mutations.

Remember, too, that nothing is broken. These viruses can now use both the original LamB receptor and the alternative OmpF receptor. (This fact was demonstrated by showing that the viruses can grow on two different constructed hosts genotypes, one completing lacking LamB and the other completely lacking OmpF.)

None of the 24 viruses that had not evolved the ability to use the OmpF receptor had all 4 of these mutations. However, three of them shared 3 of the 4 mutations with viruses that had acquired that new ability. And yet, none of those had any capacity to grow on cells that lacked the LamB receptor. In other words, the set of all 4 of these mutations was needed to produce this new ability—no subset could do the job. (We initially lacked one of the four possible viral genotypes having each subset of 3 mutations. Later work confirmed that all four mutations are required.)

At first glance, it seems like none of the viral lineages should have been able to acquire all 4 mutations, at least if you accept the flawed reasoning from Behe’s previous book on The Edge of Evolution. If you need all 4 mutations for the new function, so the thinking goes, and if none of them provide any degree of that function, then you would need all 4 mutations to occur in one lineage by chance, which is extremely unlikely. (How unlikely is difficult to calculate precisely. To get some inkling, none of the 48 sequenced J genes—including both those that did and did not evolve the new capability—had even one synonymous mutation. Synonymous mutations don’t change the amino-acid sequence of an encoded protein, and so they provide a benchmark for the accumulation of selectively neutral mutations.)

And yet, 24 of the 96 lineages did just that—they evolved the new ability, and in just a few weeks time. If you’re into intelligent design, then I guess you’d have to conclude that some purposeful agent was pretty darn interested in helping the viruses vanquish the bacteria. If you’re a scientist, though, you’re trained to think more carefully and look for natural explanations—ones that you can actually test.

So how could 4 mutations arise so quickly in the same lineage? Natural selection. But wait, didn’t Justin find that all 4 of those mutations were required for the virus to exploit the new OmpF receptor? Yes, he did.

Our hypothesis was that the mutations that set the stage for the virus to evolve the ability to target OmpF were beneficial because they improved lambda’s ability to use its original LamB receptor. But wait, that’s the receptor they’ve always used. Shouldn’t they already be perfectly adapted to using that receptor? How can there be room for improvement?

If you’ve read my posts on polar bears and bacteria, you’ve probably got the idea. When the environment changes, all bets are off as to whether a function is optimally tuned to the new conditions. Lambda did not evolve in the same medium where Justin ran his experiments; and while lambda certainly encountered E. coli and the LamB receptor in its history, the cell surfaces the virus had to navigate in nature were more heterogeneous than what they encountered in the lab. In other words, there might well be scope for the viral J protein to become better at targeting the LamB receptor under the new conditions.

To an evolutionary biologist, this hypothesis is so obvious, and the data on the evolution of the J protein sequence so compelling, that it scarcely needs testing. Nonetheless, it’s always good to check one’s reasoning by collecting new data, and another talented student joined the project who did just that. Alita Burmeister (now a postdoc at Yale) competed lambda strains with some (but not all) of the mutations needed to use OmpF against a lambda strain that had none of those mutations. She studied six “intermediate” viruses, each of them isolated from an independent population that later evolved the ability to use OmpF.

Alita ran two sets of competitions between the evolved and ancestral viruses. In one set, the viruses fought over the ancestral bacterial strain; in the other set, they competed for a bacterial strain that had previously coevolved with lambda and become more resistant to infection. Four of the six evolved intermediate viruses outcompeted their ancestor for the naïve bacteria, and all six prevailed when competing for the tough-to-infect coevolved host cells. Alita ran additional experiments showing that the intermediates were better than the ancestral virus at adsorbing to bacterial cells—the precise molecular function that the J protein serves. These results clearly support the hypothesis that the first few mutations in the evolving virus populations improved their ability to infect cells via the LamB receptor.

Natural selection did its thing, in other words, discovering mutations that provided an advantage to the viruses. Some of the resulting viruses—those with certain combinations of three mutations—just happened to be poised in the space of possible genotypes such that a fourth mutation gave them the new capacity to use OmpF.

Now let’s step back and think about what this case says about the validity of the arguments that Behe has made in his three books.

Anybody remember Behe’s first book, Darwin’s Black Box, published in 1996? There, Behe claimed evolution doesn’t work because biological systems exhibit so-called “irreducible complexity,” which he defined as “… a single system composed of several well-matched, interacting parts that contribute to the basic function, wherein the removal of any one of the parts causes the system to effectively cease functioning.” Evolution can’t explain these functions, according to Behe, because you need everything in place for the system to work. Strike one! Lambda’s J protein required several well-matched, interacting amino acids to enable infection via the host’s OmpF receptor. Removing any one of them leaves the virus unable to perform that function. (Alas, Behe’s argument wasn’t merely mistaken, it also wasn’t new—since Darwin, and as explained in increasing detail by later biologists, we’ve known that new functions evolve by coopting and modifying genes, proteins, and other structures that previously served one function to perform a new function.)

The Edge of Evolution, Behe’s second book, claimed that evolution has a hard time making multiple constructive changes, implying the odds are heavily stacked against this occurring. Strike two!! Lambda required four constructive changes to gain the ability to use OmpF, yet dozens of populations in tiny flasks managed to do this in just a few weeks. That’s because the intermediate steps were strongly beneficial to the virus, so that each step along the way proceeded far faster than by random mutation alone.

Darwin Devolves says that adaptive evolution can occur, but that it does so overwhelmingly by breaking things. Strike three!!! The viruses that can enter the bacterial cells via the OmpF receptor are not broken. They are still able to infect via the LamB receptor and, in fact, they’re better at doing so then their ancestors in the new environment. (In his blog post after our paper was published in Science, Behe used the same sleight of hand he used to downplay the evolution of the new ability to use citrate in one LTEE population. That is, Behe called lambda’s new ability to infect via the OmpF receptor a modification of function, instead of a gain of function, based on his peculiar definition, whereby a gain of function is claimed to occur only if an entirely new gene “poofs” into existence. However, that’s not the definition of gain-of-function that biologists use, which (as the term implies) means that a new function has arisen. That standard definition aligns with how evolution coopts existing genes, proteins, and other structures to perform new functions. Behe’s peculiar definition is a blatant example of “moving the goalposts” to claim victory.)

As Nathan Lents, Joshua Swamidass, and I wrote in our book review, “Ultimately, Darwin Devolves fails to challenge modern evolutionary science because, once again, Behe does not fully engage with it. He misrepresents theory and avoids evidence that challenges him.”

If you’ve followed the logic and evidence in the three systems I’ve written about—polar bears adapting to a new diet, bacteria fine-tuning and even evolving new functions as they adapt to laboratory conditions, and viruses evolving a new port of entry into their hosts—you’ll understand why Behe’s arguments against evolution aren’t taken seriously by the vast majority of biologists. As for Behe’s arguments for intelligent design, they rest on his incredulity about what evolution is able to achieve, and they make no testable predictions about how the designer intervenes in the evolutionary process.

[The images below show infection assays for 4 lambda genotypes on 2 E. coli strains. The dark circles are “plaques”—areas in a dense lawn of bacteria where the cells have been killed by the virus. The viruses (labeled at bottom) include the ancestral lambda virus and 3 evolved genotypes. One bacterial strain expresses the LamB receptor (top row), while the other lacks the gene that encodes LamB (bottom row). All 4 viruses can infect the cells that produce LamB, but only the “EvoC” virus is able to infect the cells without that receptor. Images from Meyer et al., 2012, Science paper.]

Lambda plaque assays

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Is the LTEE breaking bad?

Michael Behe has written a third book, Darwin Devolves, that continues his quixotic effort to overturn evolutionary biology. Nathan Lents, Joshua Swamidass, and I wrote a book review for Science. (You can find an open-access copy here.) As our short review states, there are indeed many examples of evolution in which genes and their functions have been degraded, sometimes conferring an advantage to the organism. However, Behe’s book largely ignores the ways by which evolution generates new functions. That’s a severe problem because Behe uses the evidence for the ease of gene degradation to support his claim that our current understanding of the mechanisms of evolution is inadequate.

This is my third in a series of posts delving into various issues where I think Behe’s logic and evidence are weak. These weaknesses undermine his position that the known mechanisms of evolution are inadequate to explain life as we see it in the fossil record and in the diversity of living species. Let me be clear: there is still much to learn about the intricacies of how evolution works, both in terms of a better understanding of the general mechanisms and unraveling all the fascinating particulars of what happened along various lineages. However, I don’t see much chance of future research upending the central role of natural selection—operating over vast time along with mutation, drift, and recombination (including various forms of horizontal gene transfer)—in creating new functions that spark the diversification of life. By contrast, Behe accepts that natural selection occurs, but he treats it almost entirely as a degradative process that weakens and destroys functions. To explain all the new functions that have arisen during evolution (and he accepts the fact that evolution has occurred for billions of years), Behe appeals to an “intelligent agent” who somehow, mysteriously has added new genetic information into evolving lineages.

In my first post, I explained why Behe’s “first rule of adaptive evolution” doesn’t imply what he says it does about evolution writ large. In particular, his overarching thesis confuses frequency over the short run with lasting impact over the long haul of evolution. In my second post, and building on the work of others, I examined a specific case involving polar bears, which Behe argued showed adaptations resulting from degradative evolution. He apparently regarded the case as so compelling that he used it as the lead example in his book, but a careful review of the science suggests an alternative explanation, in which gene function actually improved.

In this post, I examine Behe’s interpretation of findings from a long-term evolution experiment (LTEE) with E. coli bacteria that has been running in my lab for over 30 years. In short, the LTEE represents an ideal system in which to observe degradative evolution, and indeed we’ve seen examples of such changes. However, Behe overstates his case by downplaying or dismissing evidence that runs counter to his thesis.

III. Evolution of functionality in the LTEE

Recall what Behe calls “the first rule of adaptive evolution: break or blunt any functional gene whose loss would increase the number of a species’ offspring.” In support of that rule, Darwin Devolves pays considerable attention to the LTEE. Behe skillfully uses it to build his case that unguided evolution produces adaptations (almost) exclusively by breaking or blunting functional genes. The implication is that constructive adaptations—those that do not involve breaking or blunting genes—require an “intelligent agent” who has introduced new genetic information, by some mysterious process, into certain lineages over the course of life’s history.

Am I surprised that Behe uses the LTEE as one of the centerpieces of Darwin Devolves? No, not at all. Does the LTEE provide strong support for his argument? No, it does not. The LTEE fits the bill for Behe because it’s just about the best case possible to showcase his rule. But just as loss of sight in cave-dwelling organisms is a special case that won’t tell us how eyes evolved, one must be careful when extrapolating from this experiment to evolution writ large. (I say this even though the LTEE is my scientific “baby” and has been a useful model system for studying some aspects of evolution.)

The LTEE was designed (intelligently, in my opinion!) to be extremely simple in order to address some basic questions about the dynamics and repeatability of evolution, while minimizing complications. It was not intended to mimic the complexities of nature, nor was it meant to be a test-bed for the evolution of new functions. The environment in which the bacteria grow is extremely simple. The temperature is kept constant at 37C, the same as our colons where many E. coli live. The LTEE “host” is an Erlenmeyer flask, not an animal with an immune system and other defenses. There are no antibiotics present, no competing species, and no viruses that plague bacteria in nature. And the culture medium contains a single source of energy that the ancestral bacteria can use, namely the sugar glucose. In contrast, E. coli lineages have endured and adapted over millions of years to countless combinations of resources, competitors, predators, toxins, and temperatures in nature.

Indeed, the LTEE environment is so extremely simple that one might reasonably expect the bacteria would evolve by breaking many existing functions. That is because the cells could, without consequence, lose their abilities to exploit resources not present in the flasks, lose their defenses against absent predators and competitors, and lose their capacities to withstand no-longer-relevant extreme temperatures, bile salts, antibiotics, and more. The bacteria might even gain some advantage by losing these functions, if doing so saved time, energy, or materials that the cells could better use to exploit the limited glucose supply.

And just as one would expect, the bacteria have diminished or lost various abilities during the LTEE. For example, all 12 populations lost the ability to use another sugar, called ribose, and they gained a small but measurable competitive advantage as a result. Similarly, half of the lines evolved defects in one or another of their DNA repair systems, which led to hypermutability. While hypermutability resulted from a loss of function at the molecular level, it produced a slight gain in terms of the rate at which those lineages adapted to their new laboratory environment. There are undoubtedly many functional losses that have occurred during the LTEE, some that have been described and others not.

If that was all there were to the story, I might say that Behe’s portrayal was correct, but that he had missed the point—namely, that of course evolution often involves the loss of functions that are no longer useful to the organism. Biologists have known and understood this since Darwin.

But there is more to evolution than that, not only in nature but, as it turns out, even in the simple world of the LTEE. We’ve discovered cases where beneficial mutations evolved in genes that encode proteins that are essential, not dispensable, including ones involved in synthesis of the cell envelope and in structuring DNA so that it can be copied, transcribed, and packed into the tiny space of a cell. We’ve also found genes in which mutations occur repeatedly near key interfaces of the encoded proteins, in ways that imply the fine-tuning of protein functions to the LTEE environment, rather than degradation or loss of those functions.

In Darwin Devolves, Behe asserts (p. 344) that “it’s very likely that all of the identified beneficial mutations worked by degrading or outright breaking the respective ancestor genes.” He includes a footnote that acknowledges our work that suggests the fine-tuning of some protein functions, but there he writes (p. 609): “More recent investigation by Lenski’s lab suggests that mutations in a small minority (10 of 57) of selected E. coli genes may not completely break them but rather, as they put it, ‘fine-tune’ them (probably by degrading their functions).” Why does Behe assert that fine-tuning of genes occurred “probably by degrading their functions”?

Perhaps it’s because this assertion supports his claim, but more charitably I suspect the underlying reason is similar to the problematic inferences that got Behe into trouble in the case of the polar bear’s genes. That is, if one assumes the ancestral state of a gene is perfect, then there’s no room for improvement in its function, and the only possible functional changes are degradative. In my post on the polar bear case, I explained why the assumption that a gene is perfect (or nearly so) makes sense in certain situations. However, that assumption breaks down when an organism encounters a new environment, where the optimal state of a protein might differ from what it was before. Perhaps, for example, a mutation that would have slightly reduced an essential protein’s activity in the ancestral environment slightly improves its activity in the new environment. As I explained earlier, the LTEE environment differs from the conditions that E. coli experienced before being brought into the lab. It would be surprising if some proteins couldn’t be fine-tuned such that their activities were improved under the particular pH, temperature, osmolarity, and other conditions of the LTEE. It is unreasonable to simply assume that fine-tuning mutations “probably” degrade functions when evolving populations—whether of bacteria or bears—encounter new conditions.

The adaptation in the LTEE that has garnered the most public attention, though, is far less subtle. (The attention grew enormously after I had an email exchange with Andrew Schlafly, who runs the “Conservapedia” website.) After more than 30,000 generations, one of the 12 lines evolved the ability to consume citrate in an oxygen-rich environment—something that E. coli normally cannot do. Citrate, it turns out, has been a potential source of carbon and energy in the culture medium ever since the LTEE started. (The citrate is there, despite the inability of E. coli to import it from the medium, because it chelates iron and, in so doing, makes that micronutrient available to the cells.)

Sequencing the genomes of the citrate-using lineage revealed an unusual mutation—a physical rearrangement that brought together regulatory and protein-coding sequences in a new way—and genetic experiments demonstrated that mutation was responsible for this gain of function. In the line that gained the ability to consume citrate, the rearrangement involved duplicating a particular DNA segment; additional experiments showed that other types of rearrangements could also generate this ability. Even now, after more than 70,000 generations, none of the other LTEE populations has managed to evolve this new ability, despite its great benefit to the bacteria. This difficulty reflects several factors: (i) the low rate of occurrence of the necessary rearrangement mutations; (ii) the fact that efficient use of citrate requires certain additional mutations; and (iii) the absence of other, more highly beneficial mutations that could out-compete early, weakly beneficial citrate-using mutants.

To his credit, Behe does write about the lineage that evolved the ability to consume the citrate. However, he dismisses it as a “sideshow” (p. 365), because he refuses to call this new capability a gain of function. Instead, Behe writes (p. 362) that under his self-fulfilling scheme “the mutation would be counted as modification-of-function—because no new functional coded element was gained or lost, just copied.” In other words, Behe won’t count any newly evolved function as a gain of function unless some entirely new gene or control region “poofs” into existence.

But that’s not how evolution works—unless you believe, as Behe apparently does, that God or some other “intelligent agent” intervened to insert new genetic information into various lineages during the course of history. (Suffice it to say that I don’t regard this as a scientifically useful hypothesis, because I don’t think it can be tested.) Evolutionary biology doesn’t require that new genes poof into existence. Instead, old genes and their products are coopted, modified, and used in new ways—a process called exaptation. For example, crystallin proteins in the lenses of our eyes derive from proteins that performed other functions. At a larger physical scale, the wings of birds and bats derive from the forelimbs of their four-legged ancestors, which in turn derive from fins of fishes.

In short, Darwin Devolves presents a biased picture of the LTEE’s findings. Behe is overly confident in asserting that the vast majority of beneficial mutations have degraded functions, when the functional effects of most of these mutations have not been measured under relevant conditions. In any case, the experiment was designed to address issues other than molecular functionality, with the environment deliberated constructed to be as simple as possible. And yet, having closed the door on nearly all opportunities for new functions to evolve, a striking example arose in a tiny flask after a mere decade or two.

[This image shows some of the LTEE populations in their flasks. The one in the center is more turbid because the bacteria have reached a higher density after they evolved the ability to consume citrate in the culture medium.  Photo credit: Brian Baer and Neerja Hajela.]

LTEE lines centered on citrate #11

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We interrupt this program

We interrupt our irregularly scheduled blogging to wish the long-term evolution experiment a very fit 31st birthday!

Here are two pictures of graduate student Kyle Card doing today’s transfers, and thereby starting off the next year of their evolutionary journey.

Kyle Card setting up LTEE transfers on 31st birthday

Kyle Card transfers LTEE on 31st birthday

Today’s entries in the LTEE notebook are shown below.

LTEE notebook on 31st birthday

We also had a visitor who picked up some strains from the freezer over the weekend, and who left us a note on the lab’s whiteboard.

Zack left note 23-Feb-2019

We ate a Galapagos-themed cake, shown below, a couple of weekends ago when we celebrated the February birthdays of Charles Darwin, Abe Lincoln, and the LTEE.

Darwin cake 2019

Thank you Kyle, and thanks to everyone who has ever performed transfers and/or done research on the LTEE lines.

Last but not least, here’s a lovely post by Roberto Kolter at Small Things Considered wishing the LTEE a happy birthday!

 

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