Tag Archives: ecology

On the Evolution of Citrate Use

Those who follow the long-term evolution experiment (LTEE) with E. coli know that the most dramatic change we have observed to date is the origin of the new ability to grow on citrate. It’s dramatic for several reasons including the fact (external to the LTEE) that E. coli has been historically defined as a species based in part on its inability to grow on citrate in oxic environments and the fact (internal to the LTEE) that it was so difficult for the bacteria to evolve this ability that only one of the populations did so, and that it took over 30,000 generations even though an abundance of citrate has been present in the medium throughout the LTEE. Even after 64,000 generations, only the Ara–3 population has evolved that new ability.

Zachary Blount, formerly a graduate student and now a postdoc in my lab, has spent the last decade studying the evolution of this population and its new ability. His two first-authored papers in PNAS (2008) and Nature (2012) demonstrated, respectively, that (i) the origin of the ability to grow on citrate in the LTEE was contingent on one or more “potentiating” mutations that happened before the “actualizing” mutation that conferred the new function first appeared, and (ii) the actualizing mutation was a physical rearrangement of the DNA that brought together a structural gene, citT, that encodes a transporter and a previously unconnected regulatory region to generate a new module that caused the phenotypic transition to Cit+. These papers presented and discussed much more than these two points, of course, but they are the key findings. More recently, Zack was a coauthor on a paper in eLife (2015) by Erik Quandt, Jeff Barrick, and others that identified two mutations in the gene for citrate synthase—one that potentiated the evolution of citrate utilization, and another that subsequently refined that new function.

So we were keenly interested when we saw a new paper titled “Rapid evolution of citrate utilization by Escherichia coli by direct selection requires citT and dctA” by Dustin Van Hofwegen, Carolyn Hovde, and Scott Minnich. The paper is posted online as an accepted manuscript by the Journal of Bacteriology. What follows here are some overall impressions of their paper that Zack and I put together. We may follow these impressions later with some further analysis and comments.

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Let’s begin by saying that it’s great to see other groups working on interesting systems and problems like the evolution of citrate utilization in E. coli.

Moreover, the actual science that was done and reported looks fine and interesting, though we have a few quibbles with some details that we will overlook for now. By and large, the work confirms many of the findings that were reported in our papers cited above:

(i) the ability to grow on citrate in the presence of oxygen can and does evolve in E. coli (Blount et al., 2008);

(ii) when aerobic growth on citrate evolves, it does not do so quickly and easily (Blount et al., 2008) but instead takes weeks or longer—more on that below;

(iii) all strains that have evolved this new ability have physical rearrangements that involve the citT gene and appear also to involve a so-called “promoter capture” whereby a copy of this transporter-encoding gene acquires a new upstream regulatory region (Blount et al., 2012); and

(iv) genetic context matters—the strain one uses affects the likelihood of evolving the Cit+ function (Blount et al., 2008) and the resulting ability to grow on citrate (Blount et al., 2012; Quandt et al., 2015).

The problem, then, is not with the experiments and data. Rather, the problem is that the results are wrapped in interpretations that are, in our view, flawed and fallacious.

“No new genetic information”

The authors assert repeatedly (last sentence of their Importance statement, and first and last paragraphs of their Discussion) that “no new genetic information evolved.” However, that statement flatly contradicts the fact that in their experiments, and ours, E. coli gained the new ability to grow on citrate in the presence of oxygen. We would further add (which we have not emphasized before) that these Cit+ strains can grow on citrate as a sole carbon source—when E. coli grows anaerobically on citrate, it requires a second substrate for growth in order to use the citrate (a phenomenon called “co-metabolism”).

The claim that “no new genetic information evolved” is based on the fact that the bacteria gained this new ability by rearranging existing structural and regulatory genetic elements. But that’s like saying a new book—say, Darwin’s Origin of Species when it first appeared in 1859—contains no new information, because the text has the same old letters and words that are found in other books.

In an evolutionary context, a genome encodes not just proteins and patterns of expression, but information about the environments where an organism’s ancestors have lived and how to survive and reproduce in those environments by having useful proteins, expressing them under appropriate conditions (but not others), and so on. So when natural selection—that is, differential survival and reproduction—favors bacteria whose genomes have mutations that enable them to grow on citrate, those mutations most certainly provide new and useful information to the bacteria.

That’s how evolution works—it’s not as though new genes and functions somehow appear out of thin air. As the bacterial geneticist and Nobel laureate François Jacob wrote (Science, 1977): “[N]atural selection does not work as an engineer works. It works like a tinkerer—a tinkerer who does not know exactly what he is going to produce but uses whatever he finds around him, whether it be pieces of string, fragments of wood, or old cardboards; in short, it works like a tinkerer who uses everything at his disposal to produce some kind of workable object.”

To say there’s no new genetic information when a new function has evolved (or even when an existing function has improved) is a red herring that is promulgated by the opponents of evolutionary science. In this regard, it seems relevant to point out that the corresponding author, Scott Minnich, is a fellow of the Discovery Institute and was an expert witness for the losing side that wanted to allow the teaching of “intelligent design” as an alternative to evolution in public schools in the landmark Kitzmiller v. Dover case.

“Rapid evolution of citrate utilization”

In the title of their paper and throughout, Van Hofwegen et al. emphasize that, in their experiments, E. coli evolved the ability to grow aerobically on citrate much faster than the 30,000 generations and ~15 years that it took in the LTEE. That’s true, but it also obscures three points. First, we already demonstrated in replay experiments that, in the right genetic background and by plating on minimal-citrate agar, Cit+ mutants sometimes arose in a matter of weeks (Blount et al. 2008). Second, rapid evolution of citrate utilization—or any evolution of that function—was not a goal of the LTEE. So while it is interesting that Van Hofwegen et al. have identified genetic contexts and ecological conditions that accelerate the emergence of citrate utilization (as did Blount et al., 2008), that in no way undermines the slowness and rarity of the evolution of this function in the context of the LTEE (or, for that matter, the rarity of Cit+ E. coli in nature and in the lab prior to our work). Third, the fastest time that Van Hofwegen et al. saw for the Cit+ function to emerge was 19 days (from their Table 1), and in most cases it took a month or two. While that’s a lot faster than 15 years, it’s still much longer than typical “direct selections” used by microbiologists where a readily accessible mutation might confer, for example, resistance to an antibiotic after a day or two.

So while we commend the authors’ patience, we do not think the fact that their experiments produced Cit+ bacteria faster than did the LTEE is particularly important, especially since that was not a goal of the LTEE (and since we also produced them much faster in replay experiments). However, in a manner that again suggests an ulterior nonscientific motive, they try to undermine the LTEE as an exemplar of evolution. The final sentence of their paper reads: “A more accurate, albeit controversial, interpretation of the LTEE is that E. coli’s capacity to evolve is more limited than currently assumed.” Alas, their conclusion makes no logical sense. If under the right circumstances the evolution of citrate utilization is more rapid than it is in the LTEE, then that means that E. coli’s capacity to evolve is more powerful—not more limited—than assumed.

“Speciation Event”

To us, one of the most interesting facets of the evolution of the citrate-using E. coli in the LTEE is its implications for our understanding of the evolutionary processes by which new species arise. Part of the reason for this interest—and the one that’s most easily stated in a popular context—is that the inability to grow on citrate is part of the historical definition for E. coli as a species, going back almost a century. But the deeper interest to us lies not in labeling a new species or debating where to draw the line between species—various criteria are used by different scientists, and inevitably there are many cases that lie in grey areas. Rather, as evolutionary biologists, we are most interested in the process of speciation—the ecological and genetic dynamics that lead to changing biological forms that, over time, are more and more like a new species until, eventually, perhaps far in the future, there is no doubt that a new species has evolved.

In short, speciation is not an event. As Ptacek and Hankison (2009, in Evolution: The First Four Billion Years) put it, “[S]peciation is a series of processes, with a beginning stage of initial divergence, a middle stage wherein species-specific characteristics are refined by various forces of evolution, and an end point at which a new species becomes a completely separate evolutionary lineage on its own trajectory of evolutionary change with the potential for extinction or further diversification into new lineages.” We realize that scientists (ourselves included) often use shorthand and jargon instead of writing more carefully and precisely. We have no doubt that one can find solid scientific papers that talk about speciation events; but except for cases that involve hybridization leading to polyploids that are reproductively isolated in a single generation (as sometimes occurs in plants), this is simply an imprecise shorthand.

In our first paper on the citrate-using E. coli that arose in the LTEE, we clearly emphasized that becoming Cit+ was only a first step on the road to possible speciation (Blount et al., 2008). One criterion that many biologists would apply to investigate speciation is whether a later form merely replaced an earlier form (evolution without speciation) or, alternatively, one lineage split into two lineages that then coexisted (incipient speciation). In fact, we showed that, after the new function evolved, the Cit+ and Cit lineages coexisted (and their coexistence was confirmed using genomic data in Blount et al., 2012). We concluded the 2008 paper by asking explicitly: “Will the Cit+ and Cit– lineages eventually become distinct species?” (emphasis added) and discussing how we might assess their ongoing divergence.

By contrast, Van Hofwegen et al. dismiss the idea of speciation out of hand, not only by calling it an event but by treating the issue as though it hinges, literally, on the individual mutations that produced a Cit+ cell. For example, they write: “[B]ecause this adaptation did not generate any new genetic information … generation of E. coli Cit+ phenotypes in our estimation do not warrant consideration as a speciation event.” And in the penultimate sentence of their paper, they say: “[W]e argue that this is not speciation any more than any other regulatory mutant of E. coli.” (We also note that this is a rather bizarre generalization, as though the gain of function that gave access to a new resource is equal in regards to its speciation potential to, say, the loss of regulation of a function that is no longer used by a lineage in its current environment. Both might well be adaptations, but one seems much more likely to begin the process of speciation.)

In conclusion, Van Hofwegen, Hovde, and Minnich have done some interesting experiments that shed further light on the nature of the mutations and ecological conditions that allow E. coli cells to evolve the ability to grow aerobically on citrate, a function that this species cannot ordinarily perform. However, they misunderstand and/or misrepresent the relevance of this system for evolutionary biology in several important respects. 

And the meaning of historical contingency

The paper by Hofwegen et al. is accompanied by a commentary by John Roth and Sophie Maisnier-Patin. Their abstract begins: “Van Hofwegen et al. demonstrate that E. coli rapidly evolves ability to use citrate when long selective periods are provided. This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of ‘historical contingency’ may require reinterpretation.”

Historical contingency is a complicated notion, but it essentially means that history matters. In Blount et al. (2008), we made it clear what we mean by historical contingency in the context of the evolution of the Cit+ lineage in one of the LTEE populations. Was this an extremely rare event that could have happened at any time? Or did it instead depend on the occurrence of a sequence of events, a particular history, whereby an altered genetic context evolved—a potentiated background—in which this new function could now evolve?

Roth and Maisnier-Patin’s suggestion that our idea of “historical contingency” may require reinterpretation reflects a false dichotomy between historical contingency, on the one hand, and the effects of different selection schemes, on the other. The fact that evolution might be fast and not contingent on genetic background (though the evidence of Van Hofwegen et al. is, at best, ambiguous in this regard) in one set of circumstances has no bearing on whether it is contingent in another set of circumstances. The historical contingency of Cit+ evolution is not mere conjecture. We showed that the evolution of this new function in the LTEE was contingent. In replay experiments, Blount et al. (2008) showed that that the Cit+ trait arises more often in later-generation genetic backgrounds than in the ancestor or early-generation backgrounds. Moreover, Blount et al. (2012) performed genetic manipulations and showed that a high-copy-number plasmid carrying the evolved module that confers the Cit+ function had very different phenotypic effects when put in a Cit clone from the lineage within which Cit+ evolved than when placed in the ancestor or even other late-generation lineages not on the line of descent leading to the emergence of the Cit+ bacteria. In the clone on the line of descent, this module conferred strong, immediate, and consistent growth on citrate. In the other genetic backgrounds, growth on citrate was weak, delayed, and/or inconsistent.

The hypothesis of historical contingency is not mutually exclusive with respect to causal factors of an ecological or genetic nature—it simply says that factors that changed over time were important for the eventual emergence of Cit+. Moreover, historical contingency was invoked and demonstrated in a specific context, namely that of the emergence of Cit+ in the LTEE—it does not mean that the emergence of Cit+ is historically contingent in other experimental contexts, nor for that matter that other changes in the LTEE are historically contingent—in fact, some other evolved changes in the LTEE have been highly predictable and not (or at least not obviously) contingent on prior mutations in the populations (e.g., Woods et al., PNAS, 2006). [For more on historical contingency and the LTEE, you can download a preprint of Zack’s latest paper from his website: Blount, Z. D. A Case Study in Evolutionary Contingency. Studies in the History and Philosophy of Biology and Biomedical Sciences.]

Erik Quandt offers this analogy to illustrate our point that contingency depends on context: “It’s kind of like the difference between being an average person attempting to dunk a basketball when all by yourself, with unlimited time, and maybe even with a trampoline versus having to get to the rim in a game with LeBron James and the Cavs playing defense. Just because you can do it by yourself under optimal conditions, does this negate the difficulty of doing it in an NBA game or say anything about the kind of history (training and/or genetics) that you would need for that situation?”

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LTEE lines centered on citrate #11


Filed under Education, Science

Bacterial Niche Finally Defined

The following scholarly contribution comes from my wife Madeleine Lenski after conversing with her “sister” (my former postdoc) Valeria Souza.

For those with an itch for criteria,

Scratch this: What’s a niche for bacteria?

Don’t take me to task

If I answer “a flask” –

It’s a bitch from warm broth to Siberia.


January 19, 2016 · 9:08 pm

Thirty Years

No, the LTEE did not suddenly jump forward by almost 3 years. That milestone will be reached on February 24, 2018.

Next Friday is the end of the semester at MSU and, for me, it will mark 30 years that I’ve been on the faculty: six at UC-Irvine, and 24 here at MSU. (I also taught for one semester at Dartmouth as a sabbatical replacement, while I was doing a postdoc at UMass.)

Holy cow: 30 years. Where did all that time go?

Well, a lot of it was spent advising, supervising, and mentoring graduate students. And those have been some of the most interesting, enjoyable, and rewarding professional experiences that I can imagine.

In fact, this afternoon Caroline Turner defended her dissertation – congratulations Dr. Turner! Her dissertation is titled “Experimental evolution and ecological consequences: new niches and changing stoichiometry.” It contains four fascinating and meaty chapters, two on the interplay between evolutionary and ecological processes in the LTEE population that evolved the ability to grow on citrate, and two on evolved changes in the elemental stoichiometry of bacterial cells over experimental time scales.

Caroline is the 20th student to complete her Ph.D. with me serving as the advisor or co-advisor. Here they all are, with links to their professional pages or related sites.

  1. Felisa Smith, Ph.D. in 1991 from UC-Irvine.
  2. John Mittler, Ph.D. in 1992 from UC-Irvine.
  3. Mike Travisano, Ph.D. in 1993 from MSU.
  4. Paul Turner, Ph.D. in 1995 from MSU.
  5. Greg Velicer, Ph.D. in 1997 from MSU.
  6. Brendan Bohannan, Ph.D. in 1997 from MSU.
  7. Phil Gerrish, Ph.D. in 1998 from MSU.
  8. Farida Vasi, Ph.D. in 2000 from MSU.
  9. Vaughn Cooper, Ph.D. in 2000 from MSU.
  10. Danny Rozen, Ph.D. in 2000 from MSU.
  11. Kristina Hillesland, Ph.D. in 2004 from MSU.
  12. Elizabeth Ostrowski, Ph.D. in 2005 from MSU.
  13. Bob Woods, Ph.D. in 2005 from MSU.
  14. Dule Misevic, Ph.D. in 2006 from MSU.
  15. Gabe Yedid, Ph.D. in 2007 from MSU.
  16. Sean Sleight, Ph.D. in 2007 from MSU.
  17. Zack Blount, Ph.D. in 2011 from MSU.
  18. Justin Meyer, Ph.D. in 2012 from MSU.
  19. Luis Zaman, Ph.D. in 2014 from MSU. (Charles Ofria was the primary advisor.)
  20. Caroline Turner, Ph.D. in 2015 from MSU.

There are also 8 doctoral students at various stages currently in my group at MSU including Brian Wade (Ph.D. candidate), Mike Wiser (Ph.D. candidate), Rohan Maddamsetti (Ph.D. candidate), Alita Burmeister (Ph.D. candidate), Elizabeth Baird, Jay Bundy, Nkrumah Grant, and Kyle Card.

My own advisor – the late, great Nelson Hairston, Sr. – said that he expected his graduate students to shed sweat and maybe even occasional tears, but not blood. I would imagine the same has been true for my students.

Thirty years, holy cow. Time flies when you’re working hard and having fun!

Added November 4, 2015:  And now #21 in my 31st year, as  Mike Wiser successfully defended his dissertation today!


Filed under Education, Science

Questions from Jeremy Fox about the LTEE, part 1

EDIT (23 June 2015): PLOS Biology has published a condensed version of this blog-conversation.


Over at the Dynamic Ecology blog, Jeremy Fox asked me some interesting questions about the history, philosophy, and science of the E. coli long-term evolution experiment. Perhaps mistakenly—in terms of time management, not my interest!—I agreed to try to answer them … though over what time frame, I’m not sure. Anyhow, here is Jeremy’s first question followed by my (very) short and (too) long answers.


  • When you first started the LTEE, did you consider it to be a low risk or high risk experiment? Because I could see arguing both ways. In some ways, it’s low risk, because one can imagine lots of different possible outcomes, all of which would be interesting if they occurred. But in other ways, it’s high risk–I imagine that many of the interesting outcomes (including those that actually occurred!) would’ve seemed unlikely, if indeed they’d even occurred to you at all. Or did you not worry much about the range of possible outcomes because the experiment was basically a lottery ticket? “This’ll be cheap and not much work, let’s just do it and see what happens. Something really cool might happen, but if it turns out boring that’s ok because it wasn’t a big investment.”


The short answer: Life was good, and I wasn’t thinking about risk. Or as they say about investing: it’s better to be lucky than smart!

The long, non-linear* answer: I’d already had success with some shorter duration, more traditionally designed experiments (e.g., Lenski, 1988), and so it wasn’t a total shot in the dark—that is, I knew the LTEE would yield data. I also knew, though, that it was an unusually abstract, open-ended, and non-traditional experiment, and that it might not appeal to some people for those reasons. But I loved (and still do) the seemingly simple (but in reality complex) questions, issues, and hypotheses that motivated the LTEE.

I never thought of the LTEE project as a “lottery ticket”, but some follow-up work that grew out of it had that feel.** And, oddly enough, there was one lottery-ticket aspect of the research early on, although that reflected a lack of preparation rather than a well-conceived feature.***

Maybe I was overly confident, but I’d also say that I was pretty sure the outcomes—whatever they might be—would be “cool.” The questions were intriguing, and there hadn’t been many, if any, previous attempts to answer them quite so directly. Data would be forthcoming, and even if the results weren’t definitive, I felt there would be some interest in trying to interpret whatever data emerged.**** Plus, I knew enough about what would happen—based on the experiments I had already done—that I was confident that the data and analyses would be informative with respect to at least some of my questions. Also, the use of microbes to study evolution in action was still uncommon, so the novelty of the approach would ensure some interest among my colleagues—although let me emphasize that Lin Chao, Dan Dykhuizen, Barry Hall, and Bruce Levin, among others, had already demonstrated the power of using microbes for experimental studies of evolutionary questions.*****

I should also say, in case it’s not obvious, that I had no idea or intention that the experiment would continue for anywhere near as long as it has lasted—nor that it might, I now hope, be running long after I’m gone. I had previously performed some experiments that lasted several hundred generations, and as I saw the dynamics and thought about the math behind the dynamics, I realized that over those time scales I might be seeing the effects of only one or two beneficial mutations as they swept to fixation. That hardly seemed satisfactory for experiments to explore the structure of the fitness landscape. So I decided the experiment should run for 2,000 generations, over which time I expected there would be at least several fixations of beneficial mutations in each population (and I was right), and that would deserve calling it long-term. That would take a little less than a year, given the 100-fold dilution and 6.6 generations of re-growth each day.

Of course, propagating the lines for 2,000 generations was one thing—running the competitions to measure fitness, analyzing the data, writing the paper, responding to reviews, all that took longer. So while the experiment began in February 1988, the first paper (Lenski et al., 1991) was not submitted until August 1989, resubmitted September 1990, accepted that November, and finally published in December 1991. Meanwhile, the LTEE itself continued and the generations ticked by. The baseline work of keeping the populations going is not that onerous—yes, somebody has to attend to the transfers every day, but once a lab team reaches a moderate size, it’s not too hard to arrange. And I lived next to the campus in Irvine, so it wasn’t hard for me to come in on the weekends and holidays … and my wife still loves me, and my kids recognized my face ;>)

You also wondered whether some of the interesting possible and actual outcomes had occurred to me when I started. Definitely not! I had made a strategic decision to make the environment of the LTEE very simple in order to eliminate, or at least reduce, certain complications (especially frequency-dependent interactions and clonal interference). And while I think my planning kept these complications from getting out of hand, the tension between the simplicity of the experimental design and all the complications has definitely been part of its interest. That tension, along with time, the evolutionary potential of the bacteria, and the smart, talented, creative** and hard-working students and colleagues have made the LTEE what I call “the experiment that keeps on giving.”


*Hey, that’s what footnotes are for, right?

**I’ve thought that way about some follow-on work that uses the LTEE lines, but not about the project as a whole. Here are a couple of examples of “lottery tickets” that people suggested to me, and that won big. A former postdoc Paul Sniegowski, now on the faculty at Penn, wanted to know whether the actual mutation rate itself might be evolving in the LTEE populations. Bingo! Several lines evolved hypermutability and so, curiously enough, the first mutations we ever mapped affected the mutation rate itself (Sniegowski et al., 1997). Another example: Dominique Schneider is a molecular microbiologist in Grenoble, and we’ve collaborated for over 15 years. He thought we should look at whether DNA topology—the physical supercoiling inside the cell—might have changed in the LTEE lines. Well, I thought to myself, why would it change? But Dom’s lab will do all the work, so sure, why not look? And it turns out, sure enough, that DNA supercoiling changed repeatedly in the LTEE lines (Crozat et al., 2005), and it even led us to discover a gene not previously known to affect supercoiling (Crozat et al., 2010). There’s a lesson here, by the way—work with people who are smarter, who have different interests, and who have different skills than oneself.

***I actually started two versions of the LTEE—not one experiment with two proper treatments, but two separate experiments that differed in terms of both the starting strain and the environment. Unlike the successful LTEE, I hadn’t done any previous evolution experiments with the other ancestral strain and environment. Anyhow, I soon stopped the other version when the populations evolved a phenotype that made it very difficult to work with them. In brief, the populations evolved to make pinprick-sized colonies that were next-to-impossible to count in the assays we use to measure fitness. Who needed that headache! So, in a way, I guess I had two lottery tickets: I hadn’t done the relevant prior work for one of them, whereas the one that paid off was actually a pretty strategic gamble.

****I was at UC Irvine when I started the LTEE, and Michael Rose was one of my colleagues there. His work on the evolution of aging—postponed senescence—in fruit flies (e.g., Rose 1984) was an inspiration in terms of the importance and power of long experiments. We also spent a lot of time discussing fitness landscapes, the alternative perspectives of Sewall Wight and R. A. Fisher about the dynamics on those landscapes, and what experiments might tell us. Michael didn’t design, direct, or do the lab work for the first LTEE paper, but he helped me clarify my thinking and write the first paper on the LTEE (Lenski et al., 1991). Perhaps more importantly, his interest in the questions and issues made me realize that other smart people would also be interested.

*****I used to complain, mostly in jest, that “Evolutionary biologists say I’m asking the right questions, but studying the wrong organism, and microbiologists tell me I’m studying the right organism but asking the wrong questions.” I got that sort of response occasionally, but many people from both fields were very interested and encouraging. For example, I remember David Wake telling me, after one of my first talks about the LTEE, how much he liked both the questions and the approach.


Lenski, R. E. 1988. Experimental studies of pleiotropy and epistasis in Escherichia coli. II. Compensation for maladaptive pleiotropic effects associated with resistance to virus T4. Evolution 42: 433-440.

Lenski, R. E., M. R. Rose, S. C. Simpson, and S. C. Tadler. 1991. Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations. American Naturalist 138:  1315-1341.

Sniegowski, P. D., P. J. Gerrish, and R. E. Lenski. 1997. Evolution of high mutation rates in experimental populations of Escherichia coli. Nature 387: 703-705.

Crozat, E., N. Philippe, R. E. Lenski, J. Geiselmann, and D. Schneider. 2005. Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection. Genetics 169: 523-532.

Crozat, E., C. Winkworth, J. Gaffé, P. F. Hallin, M. A. Riley, R. E. Lenski, and D. Schneider. 2010. Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli. Molecular Biology and Evolution 27:2113-2128.

Rose, M. R. 1984. Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 38: 1004-1010.


LTEE flasks repeating


Filed under Science

Putting GMOs on a Tight Leash

Two papers appeared in the latest issue of Natureone from Farren Isaacs’ group and the other from George Church and colleagues—that presented, developed, and demonstrated a strategy for limiting the spread of genetically modified organisms, or GMOs, in the event that they are accidentally released or deliberately applied to the environment.

My Involvement with GMO Discussions in the 1980s

I was actively involved in discussions about environmental applications and field testing of genetically engineered organisms back in the 1980s. As a postdoc in 1984, I had a short letter in Nature where I suggested a containment strategy for an early proposed application of genetically modified “ice-minus” bacteria. Later that year I attended a small meeting on environmental applications of GMOs at the Cold Spring Harbor Laboratory, and a short report was published in the Bulletin of the Ecological Society of America. As faculty member at UC Irvine in 1986, I served as a consultant on a report for the Office of Technology Assessment of the US Congress. I also co-organized and moderated a lively public debate on the benefits and risks of genetically engineered organisms between Jerry Caulder, who was CEO of a biotech company, and the distinguished ecologist Daniel Simberloff, an expert on invasive species.

At that time, one of the arguments—the “excess baggage hypothesis”—for the safety of GMOs was that genetically engineered functions would impose a metabolic burden and thereby reduce the fitness of the organisms, so that they wouldn’t be good competitors in nature. While that argument made some sense as a trend or tendency, it didn’t seem likely that it would apply in every possible case given the potential for new environments and/or compensatory adaptations to favor novel functions. In 1988, I wrote a review for Trends in Ecology & Evolution with a postdoc, Toai Nguyen, on the “Stability of recombinant DNA and its effects on fitness” that made these points.

As a result of my interest in and involvement with these issues, I was asked to serve on two expert panels—one convened by the Ecological Society of America (ESA), the other by the National Research Council (NRC) arm of the National Academy of Sciences—that wrote lengthy reports, both published in 1989. In both reports, the committees tried to emphasize that one needed to consider two different issues. (1) What, if any, were the potential problems that might be caused by the release of particular GMO? (2) In the event that some problem actually did arise, would the GMO (or its engineered genes) survive and possibly spread in the environment? Or would the problem be resolved by halting further applications of the GMO, because they would then simply die off?

(These panels were hard work, but through them I met some great scientists, including Jim Tiedje and Rita Colwell among many others.)

After that extensive involvement with this science-policy issue in the 1980s, my research tended toward more basic questions in the years that followed. Meanwhile, of course, there has remained substantial scientific, commercial, and public interest in the methods and applications of genetic engineering. The two recent papers in Nature reflect the latest efforts to ensure the safety of GMOs by putting them on a tight leash.

My Thoughts on the Recent Papers

I was asked to comment on the Nature papers by Malcolm Ritter, a science reporter for the AP, and he briefly (and accurately) quoted me in a short news piece that appeared yesterday. In light of a question about my thoughts on Twitter, I thought I’d share my full remarks here:

Using genetically modified organisms in the environment raises a couple of intersecting issues. One concerns the effects those organisms have. Of course, GMOs are intended to provide some benefit—say, for bioenergy or agriculture—but in some cases the GMOs might have secondary or unanticipated harmful effects. If these harmful effects occur, and if they outweigh the benefits, then one would like to be able to recall the GMOs from the environment—sort of like recalling cars when some problem is discovered after they’ve been sold. The challenge is that GMOs are organisms, they are alive and can reproduce, and so they won’t necessarily just go away if one stops using them. Over the years, different strategies have been proposed to ensure that GMOs will, in fact, just die off after they’ve done their job, but these strategies have had holes, such as the possibility that evolution might break whatever leash the scientists put on the GMOs so that they could be recalled.

These two papers, though, point the way towards putting GMOs on a very tight leash, one that is meant to be unbreakable, by changing the genetic code of an organism so that its replication becomes dependent on certain synthetic building blocks—amino acids—that aren’t found in nature. So by applying these molecules along with the GMO in some environment, the GMOs can replicate and do their job. But if the synthetic amino acids aren’t supplied, then the GMOs won’t be able to replicate further after they’ve run out, and so that provides a leash that should rein the GMOs in if there is some problem. Of course, there are a lot of technical challenges to pulling this off, because you can’t make the organisms so weak that they can’t do their intended functions.

And of course, extending this approach from microorganisms—the subject of these papers—to crop plants would raise all sorts of additional questions about nutritional value and safety. Those are different issues and not what these papers are about.

Coda: Does this approach ensure containment of a GMO? Probably not. There aren’t many guarantees in life, and evolution has a history (billions of years, in fact) of finding clever solutions that might not occur to engineers and scientists. Does that mean that we should not use GMOs in nature? Not at all. As our ESA and NRC reports of a quarter-century ago stressed, one should consider both the benefits of a particular environmental application of a GMO and its potential harm if something goes wrong. In those cases where the benefits are great, and the potential for harm is very small (both in likelihood and magnitude), then the issues of containment and recall after a release are less critical. But in those instances where the potential risks of some GMO are substantial—either in terms of the likelihood or the magnitude of adverse effects—then every effort must be made to put the GMOs on a tight leash or, absent that, do not proceed with the proposed application.

[The image below is one part of Figure 1 from the Nature paper, titled “Recoded organisms engineered to depend on synthetic amino acids” and authored by Alexis J. Rovner, Adrian D. Haimovich, Spencer R. Katz, Zhe Li, Michael W. Grome, Brandon M. Gassaway, Miriam Amiram, Jaymin R. Patel, Ryan R. Gallagher, Jesse Rinehart and Farren J. Isaacs.  This image is shown here under the doctrine of fair use.]

Portion Fig 1 from Rovner et al, Nature, 2015

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Valuing Nature

Carl Zimmer has written an excellent piece in the New York Times about a very important study by Robert Costanza et al. on “Changes in the global value of ecosystem services” – in other words, how to place economic value on some of the critical functions that nature provides us for free, and how to quantify the economic fallout when these functions are degraded.

Of course, it’s difficult to put a dollar value on the esthetic aspects of natural ecosystems. And many people see it is a moral imperative to preserve these natural systems for future generations, regardless of their monetary worth.

The Costanza study, though, is based on the fact that natural ecosystems provide us with economic benefits by performing important services that, when diminished, have very real monetary costs in terms of the resulting damages and replacing the services with human-engineered solutions. Alas, many of these ecosystems and services are being rapidly and severely degraded.

Here are three of the several conclusions from Costanza et al., which I’ve taken verbatim from the highlights at the beginning of their article:

  • “Global loss of ecosystem services due to land use change is $US 4.3–20.2 trillion/yr.”
  • “Ecoservices contribute more than twice as much to human well-being as global GDP.”
  • “Ecosystem services are best considered public goods requiring new institutions.”

That last conclusion reminds me of a similar point that was made by the theologian Philip Hefner in his book The Human Factor: Evolution, Culture and Religion.  Hefner says “… in the situation to which biocultural evolution has brought us … the life not only of the human species, but of the entire planetary ecosystem is made to depend on a great wager going well. This wager is that the cultural systems of information that the co-creator [REL: that’s us humans] fashions will interface with the natural systems and with the global human culture so as to promote survival and a wholesome future.”  Hefner then says “… the wager is not going well. The cultural systems of information are not meshing adequately enough with other systems, and calamity is the prospect.” To prevent calamity, Hefner says we need “… revitalization of our mythic and ritual systems [REL: that is, our religious institutions], in tandem with scientific understandings, so as to reorganize the necessary information. This may help us to put our world together …”

I previously posted that, as a scientist, I could not accept Dr. Hefner’s fusion of science and religion. However, I agree with both Dr. Costanza and Dr. Hefner that our political, cultural, and religious institutions must support the natural ecosystems that provide vital services and valuable public goods to ourselves and to future generations.

Link to Carl Zimmer’s article in the New York Times

Link to paper by Robert Costanza et al. in the journal Global Environmental Change

Link to my response to Philip Hefner’s Theological Theory of the Created Co-Creator

[The image below is a photomosaic produced by the NASA Goddard Space Flight Center.]

NASA image of Earth


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Lenski Interview with The Molecular Ecologist

John Stanton-Geddes asked me some great questions for a series on “People Behind the Science” at The Molecular Ecologist blog.  He gave me permission to repost the interview here.

1) Did you always think you’d become an evolutionary biologist?

No!  I always enjoyed being outdoors (sports and hiking), but I didn’t have any particular interest in biology.  However, my mother (who dropped out of college when she married, but then co-authored a sociology textbook with my father) was very interested in biology.  She would give me articles she had read and enjoyed from Natural History and elsewhere.

I went to Oberlin College, where I thought that I might major in government.  But I disliked my first government class.  I also took a team-taught biology class for non-majors.  All of the instructors spoke on topics about which they cared deeply, and I was hooked!  I took more biology courses, and I was especially drawn to ecology because there were so many ideas and questions.  At that time, I wrongly viewed evolutionary biology as a more descriptive, old-fashioned field with fewer questions that one might still address.  (By the way, several other evolutionary biologists were at Oberlin when I was there including Deborah Gordon, Joe Graves, Kurt Schwenk, and Ruth Shaw. Not bad for a small school!)

I went to graduate school at the University of North Carolina, where Nelson Hairston, Sr., was my advisor.  Nelson was interested in the interface of ecology and evolution, and that opened my eyes.  I was also influenced by Janis Antonovics, then at Duke University.  I took his Ecological Genetics course, and he served on my committee.  Janis had written a paper in which he argued that “The distinction between ‘ecological time’ and ‘evolutionary time’ is artificial and misleading.”  That really got me thinking.  I tried to develop a couple of field-based projects that would address evolutionary questions, but I didn’t know what I was doing and they failed.  In the end, my dissertation project was pure ecology.

By then, though, I knew I wanted to pursue evolutionary biology.  While we were finishing our doctoral projects, a fellow grad student Phil Service and I spent a lot of time discussing model systems for studying evolution.  For his postdoc, Phil chose to work with Drosophila.  I recalled an undergrad course in which we read about elegant experiments with microbes that addressed fundamental questions, such as one by Salvador Luria and Max Delbrück showing that mutations happen at random and not in response to selection.  Meanwhile, in a graduate seminar, we read a paper by Lin Chao and Bruce Levin on the coevolution of bacteria and viruses.  I wrote Bruce to ask if he might have an opening for a postdoc.  Lucky for me, Bruce knew Nelson and invited me for a visit.

2) You’ve described the theme of your research as “the tension between chance and necessity”. Can you comment on how chance and necessity have shaped your career?

The ancient Greek philosopher Democritus said, “Everything existing in the universe is the fruit of chance and necessity.”  In my long-term evolution experiment with E. coli, we can explore the tension between chance and necessity because we have replicate populations started with the same ancestor and evolving under identical conditions, and because we can replay evolution from different points along the way.  But it’s difficult, if not impossible, to tease apart the roles of chance and necessity with a sample size of one, which is the life that each of us has experienced, and without the ability to replay our own lives.  (On that last point, let me recommend Replay, a science-fiction novel by Ken Grimwood.)

I would say, though, that most people who have had some success in their adult lives also started out very lucky.  We were fortunate to be born at times and in places where we had food, familial love, education, and opportunity.

3) Reading your blog it’s clear that you are a student of the philosophy and history of science. Do you think we should include more history and philosophy in scientific training? Any advice on something we should all go out and read?

I do think that the history and philosophy of science deserve more emphasis in science and education than they usually receive.  But I didn’t have any formal education in those areas.  Instead, I became interested in these issues through teachers, mentors, colleagues, and my own explorations.

For something to read in this area, I suggest Darwin’s Century by Loren Eiseley.  (Originally published in 1958, it was republished in 2009 by Barnes & Noble.)  The book discusses the fascinating history of evolutionary thought in the decades before and after the publication of The Origin of Species.  I first read Darwin’s Century in a course at Oberlin taught by James Stewart.

4) If you were starting your career today, what would you study? 

If I were starting today, and at my present age, I might choose to study the history of science, especially evolutionary biology and its antecedents.

But if I were starting out young, as one usually does, I’d like things to unfold as they did.  It might be tempting to skip the rough patches, but dissatisfaction with my early research led me to make the switch to microbial evolution.  Would I have enjoyed this lab-based work as much, if I hadn’t discovered that I was not nearly as good at fieldwork as many of my peers?

5) How close have you come to giving up as a researcher and doing something completely different?

The job market was tough when I was a postdoc, and I had a growing family to support.  So after a slew of applications and rejections, and a period of uncertain funding, I started to think about other possibilities.  Luckily for me, things turned around before I had to make a switch.  (You can read more about it in my blog post, The Good Old Days.)

6) What’s the meaning of life?

I think that some understanding of evolution—at a basic level accessible to anyone with an open mind and a decent education—gives perspective about our place, both as individuals and as a species, in the grand sweep of time and space.  Recognizing the transience of my personal existence fills me with awe and respect for the continuity of life and ideas.  And belonging to a species that is profoundly altering the world that enabled the continuity of life reminds me of our responsibility for ensuring its future.


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