Tag Archives: microbiology

Happy 27th birthday to the LTEE!

The title says it all:  today is the 27th birthday of the long-term evolution experiment (LTEE) with E. coli.

Well, the title doesn’t really say everything. I also want to give thanks to the many people—not to mention the trillions of bacteria—who have made it possible for the LTEE to keep on going and giving.

So thank you to all of the students, postdocs, and colleagues with whom I’ve collaborated on this project. There are too many to list here, but you will find their names on the papers that have come from the LTEE. I’ll call out just two, on this occasion, for special thanks. Dom Schneider has been an amazingly talented and generous collaborator for so many years—in fact, our first co-authored paper on the LTEE dates back to 1999. And Neerja Hajela has worked with me for 20 years now, and she is the most organized, dedicated, and all-around wonderful technician and lab manager that one could ever have.

Special thanks, too, to Madeleine Lenski, who has tolerated my long-term affair with the LTEE, and who wisely advised me to keep it going on one or two occasions when I was looking in other directions.

[The image below shows the abstract from the first paper on the LTEE, which appeared in The American Naturalist in 1991. It is reproduced here under the doctrine of fair use.  Some of the conclusions have changed a bit as the LTEE has had more time and we’ve gathered more data—that’s science!]

Abstract 1991 LTEE

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The LTEE as meta-experiment: Questions from Jeremy Fox about the LTEE, part 3

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

~~~~~

This is the 3rd installment in my responses to Jeremy Fox’s questions about the LTEE (my lab’s long-term evolution experiment with E. coli), which he asked at the Dynamic Ecology blog. This response addresses his 2nd and 7th questions, which I’ve copied below. I like all of Jeremy’s questions, but I especially like his 2nd one because it forced me—and many readers, I hope—to think carefully about what experiments are and why we do them.

~~~~~

  • Is the LTEE actually an experiment, and wouldn’t it have been even better if it was? It’s just one “treatment”–12 replicates of a single set of conditions. Wouldn’t it have been even more interesting to have, say, two treatments? Two different culture conditions, two different founding genotypes, two different founding species…?
  • Is the LTEE itself now a “model system”? Model systems in biology–systems in which it’s tractable to ask a given question–often are systems that we know a lot about. We can leverage that background knowledge to ask questions that otherwise wouldn’t be tractable. coli of course is a model organism for many purposes, because we know so much about it. But is the LTEE itself now a model system?

 ~~~~~

You’re certainly right, Jeremy, that experiments in the fields of ecology and evolutionary biology typically have two or more treatments. But it’s not an essential part of the definition of an experiment that it has that sort of structure. It would have been nice, perhaps, if the LTEE did have two or more environments and/or two or more ancestors, as you suggest—in fact, we’ve run several of those types of experiments over the years in my lab, and I’ll mention a few of them below.

The reason I didn’t do that with the LTEE, though, was because one of my core motivating questions (see part 2 of my response) concerned the repeatability of evolutionary dynamics across replicate populations. That’s a question about the trajectory of variances over time, which is challenging statistically because estimates of variances have large uncertainties. So if the LTEE had two treatments, I might have been able to say something meaningful about the differences between them, but I would have had less power to say anything about the among-replicate variances for either treatment. In other words, with respect to that motivating question, going from 12 replicate populations down to 6 replicates would have been risky.

It certainly would be nice to have more total populations, say, 24 or even more; and nowadays many labs use 96-well plates for evolution experiments, with each well a replicate population and liquid-handling robots to automate the transfers. When I started the LTEE, though, we worked with flasks (albeit small ones); 12 may not seem like too many, but when we run the competition assays to measure fitness, we then have replicate assays for each population and we analyze multiple generations simultaneously, so the students and postdocs running these assays are handling many dozens or even hundreds of flasks.

The LTEE as a meta-experiment

Stepping back a bit, I’d like to suggest that the LTEE is a sort of meta-experiment, to coin a term. (This idea echoes the question where you suggested that the LTEE has itself become a model system.) By “meta” I mean the LTEE transcends—goes above and beyond—what one usually considers an experiment because the LTEE enables experimentation at several levels.

Level 1: The LTEE as an experiment

First, it is an experiment in the sense that it set out to measure, under defined conditions and with replication, certain specific quantities, such as fitness trajectories. It may not be typical in having a single treatment, but the temporal dimension coupled with being able to analyze multiple time points simultaneously—that is, the “time travel” enabled by the frozen samples across the generations, including the use of the ancestral strain as an internal control in fitness assays—functions in much the same way from an analysis standpoint.

Level 2: The LTEE as a generator of new questions and experiments to answer them

Second, the LTEE has generated a number of new questions and hypotheses that are themselves amenable to structurally independent follow-on experiments. Let me give two examples. We observed fairly early on that several populations had evolved changes in their DNA metabolism and repair that caused their mutation rates to increase by roughly 100-fold (Sniegowski et al. 1997). Such “mutator” mutations can arise by hitchhiking, albeit only occasionally and stochastically, with beneficial mutations that they cause (Lenski 2004, see pp. 246-251). It wasn’t clear, though, whether they would necessarily increase the rate of fitness improvement, given the large populations and correspondingly large potential supply of beneficial mutations in the LTEE. So we designed a separate, shorter-duration experiment with some 48 populations where we varied the mutation rate, population size, and initial fitness of the founding ancestor, and assessed the resulting fitness gains over 1,000 generations (de Visser et al. 1999).

Another case is the “replay” experiments that Zachary Blount ran after one lineage evolved the ability to grow on citrate in the presence of oxygen, which E. coli generally cannot do (Blount et al. 2008). Zack ran thousands of populations that started from genotypes isolated at different times from the population that eventually evolved this new function, in order to test whether it could have arisen at any time by an appropriate mutation or, alternatively, whether it required first evolving a “potentiated” genetic background, or context, in which the “actualizing” mutation would then confer the citrate-using phenotype.

In both of these examples, the subsequent experiments, though separate and distinct from the LTEE, nonetheless emerged from the LTEE. That is, the questions and hypotheses tested in these later experiments were motivated by observations we had made in the LTEE itself.

Level 3: The LTEE-derived strains as useful ancestors for a variety of experiments meant to address existing questions

The third level of the meta-experiment involves questions that arise outside of the LTEE, but for which the LTEE generates a set of materials—specifically, strains—that are especially useful for experiments to address those questions. Again, I’ll give a couple of examples.

Many ecologists, physiologists, and others are interested in studying adaptation to specific environmental factors—such as resource availability, temperature, etc.—as well as examining possible tradeoffs associated with adaptation to those factors. One difficulty, though, is that by moving organisms from nature into the lab and allowing them to evolve under, say, different temperature regimes, adaptation to the shared features of the lab environments may well outweigh adaptation to the specific variable of interest. If so, that would interfere with one’s ability to identify the mutations and adaptations most relevant to the factor of interest, and it could also obscure tradeoffs that might be important if populations were already well adapted to the other aspects of the environment. With these considerations in mind, Albert Bennett and I took a strain from the LTEE that had evolved in and adapted to those conditions—the resources, pH, absence of predators, etc.—and we used it as the ancestor for a new evolution experiment where 6 replicate populations evolved under each of 4 different thermal regimes: 32C, 37C (the same as in the LTEE), 42C, and daily alternations between 32C and 42C (Bennett et al. 1992, Bennett and Lenski 1993). In that way, we could focus attention on temperature-specific adaptations, which were Al’s main interest, rather than having such changes overwhelmed by adaptation to the lab environment.

My second example where LTEE-derived strains were ancestors for an experiment meant to address an extrinsic question is one of an abstract nature. In this study, we quantitatively partitioned the effects of adaptation, history, and chance on phenotypic evolution by founding 3 replicate populations from 12 different ancestors—each one a genotype sampled from a different one of the LTEE populations—and we then let these 36 populations evolve in a new environment, where we changed the identity of the limiting nutrient (Travisano et al. 1995). By measuring the fitness of the 12 ancestors and 36 derived lines in the changed environment, we were able to disentangle and quantify the relative contributions of adaptation, history, and chance to the observed outcomes (see figure below). That is, adaptation measured the mean tendency for fitness to increase, history reflected the effect of the different starting genotypes on the fitness achieved, and chance the variation in the resulting fitness among the replicates that started from the same ancestor.

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.

Lenski, R. E. 2004. Phenotypic and genomic evolution during a 20,000-generation experiment with the bacterium Escherichia coli. Plant Breeding Reviews 24:225-265.

De Visser, J. A. G. M., C. W. Zeyl, P. J. Gerrish, J. L. Blanchard, and R. E. Lenski. 1999. Diminishing returns from mutation supply rate in asexual populations. Science 283:404-406.

Blount, Z. D., C. Z. Borland, and R. E. Lenski. 2008. Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc. Natl. Acad. Sci. USA 105:7899-7906.

Bennett, A. F., R. E. Lenski, and J. E. Mittler. 1992. Evolutionary adaptation to temperature. I. Fitness responses of Escherichia coli to changes in its thermal environment. Evolution 46:16-30.

Bennett, A. F., and R. E. Lenski. 1993. Evolutionary adaptation to temperature. II. Thermal niches of experimental lines of Escherichia coli. Evolution 47:1-12.

Travisano, M., J. A. Mongold, A. F. Bennett, and R. E. Lenski. 1995. Experimental tests of the roles of adaptation, chance, and history in evolution. Science 267:87-90.

[The figure below appeared in Science (Travisano et al. 1995), and it is reproduced here under the doctrine of fair use.]

Adaptation, chance, history image

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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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Infectiously Fun Science

Science is sometimes frustrating. The work is often repetitive and even tedious. It can be hard to explain to our friends and families—and sometimes even to peers—what we’re doing and why we think it’s important and interesting. The current state of the academic job market is terrible.

But science is also often fun. There’s the joy of discovery, which grows out of the quieter excitement of seeing data come together to support or refute an existing idea and, perhaps, to generate a brand-new idea. If we’re lucky, we enjoy the recognition of our peers that comes when a paper is accepted, a grant funded, or a talk well received.

For those of us who study evolution, the frustrations can be magnified by critics and trolls who aren’t interested in evidence or reason, having already closed their minds to even the idea of evolution based on their narrow, literal reading—or, more often, someone else’s reading—of texts written in other languages long before science provided an evidence-based way to understand the world in which we live.

At the same time—and perhaps driven in part by the controversy surrounding evolution and religion—the field of evolution has long been blessed with great writers and speakers who are willing and able to engage the public. Twenty years before he published On the Origin of Species, Charles Darwin had already cemented his place in the public eye with his travelogue The Voyage of the Beagle. As a result, the Origin was an instant best seller on both sides of the Atlantic. And while Darwin shied away from speaking in public about his discoveries, Thomas Henry Huxley was a gifted orator who became “Darwin’s Bulldog” in public lectures and debates.

That tradition continues to this day. Some of my favorites include The Selfish Gene by Richard Dawkins, Wonderful Life by the late Stephen Jay Gould, Darwin’s Dangerous Idea by Daniel Dennett, and Your Inner Fish by Neil Shubin. Experts argue about scientific issues, minor and even major, contained in these books. But it’s hard for me to imagine an open-minded reader, someone interested in science and evolution, who would not find these books highly stimulating—even infectious in the sense of wanting to share them and the ideas they contain with others.

And speaking of infectious, new ways of communicating science have burst onto the scene since the printing press. For example …

Baba Brinkman is a rapper who raps about science, literature, public policy, and more. For your scientific enjoyment, here are three of my favorites from The Rap Guide to Evolution:

Performance, Feedback, Revision

Creationist Cousins

I’m A African

Here’s another from The Rap Guide to Human Nature:

Short Term Mating Dance

And here’s a brand-new one—on microbiology and disease—with a cameo appearance by yours truly and three students who work in my lab:

So Infectious

Whether you’re a scientist or not, I hope you’ll agree that these are worth sharing with your students, friends, and families!

[Image source: music.bababrinkman.com/album/the-rap-guide-to-evolution]

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On Time and Space

The long-term evolution experiment (LTEE) began in 1988, and the E. coli populations are approaching 60,000 generations.  That’s a long time for an experiment, and I hope it continues for much, much longer.

But when I give talks about the LTEE, I also try to remind people that 26 years is only a drop in the proverbial bucket of evolutionary time.  If you were to add these experimental populations to the tree of life—or even to a tree showing only other E. coli strains—they would not be visible to the eye because the branches they represent—tiny twigs, really—would be so short (in time) and so close (in genetic distance) to their ancestors.

On Time and the LTEE

Life has existed on Earth for roughly 3.5 to 4 billion years.  That’s about 140 million times longer than the LTEE has existed.  Expressed the other way around, this experiment has been running for about 0.0000007% of the time that life has been evolving on our planet.

As I said, a mere drop in the bucket of time …

That’s a somewhat mixed metaphor, though, with “a drop in the bucket” being a statement about space and relative volumes, not about time.  And that got me wondering about the spatial scale of the LTEE relative to the spatial scale of the biosphere.

If the LTEE is just 0.0000007% as old as life on Earth, what fraction of the space—of the total biovolume—of life on our planet exists in the confines of the LTEE?

On Space and the LTEE

That’s a harder a question to answer.  We know the volume of the LTEE:  there are 12 flasks, one for each of the evolving populations, and each flask contains 10 milliliters (mL) of liquid medium.  (In medicine, by the way, a drop has been defined as 1/20th of a mL, so each flask in the LTEE contains 200 drops.)  If we sum across the populations, then the LTEE occupies 120 mL.

Before you read further:  What’s your quick intuition?  Is the LTEE larger on this spatial scale than on the temporal scale?  Or is the LTEE smaller?

Volumes and Numbers

How should we estimate the volume of Earth’s biosphere?  Here are three back-of-the-envelope approaches to get a rough sense of the scale.

1)  Most of the Earth is covered by its oceans, which are full of life.  While life is not equally abundant throughout the oceans, none of that space is entirely devoid of life.  The total volume of Earth’s oceans is about 1.3 billion cubic km.  That’s a lot of mL!  A mL is a cubic centimeter, or cc, and that’s 1/(100^3) = 1 millionth of a cubic meter.  A cubic meter is 1/(1000^3) = 1 billionth of a cubic kilometer, and the oceans contain over a billion of those cubic kilometers.

So the 120 mL in the LTEE correspond to 120 / (1.3 x 10^9 x 10^9 x 10^6), or about 9 x 10^-22 of what  the oceans contain.  That’s just 0.000000000000000000009% of the volume of the oceans.

By this calculation, then, the temporal scale of the LTEE is ~75 trillion times greater than its spatial scale, when both are expressed relative to nature.  If the LTEE is “a drop in the bucket” with respect to time, then that drop has to be diluted by a factor of 75 trillion with respect to the oceans.

2)  Let’s try another quick-and-dirty calculation.  Most life, in the oceans and on land, is near the Earth’s surface.  The surface area of our planet is about 510 million square kilometers.  If we take just the top meter, that’s equivalent to 510/1000  = 0.51 million cubic kilometers.  That’s about 1/2600 of the volume of the ocean.  But even this conservative estimate of the volume of the biosphere makes the relative scaling of the LTEE with respect to time and space differ by a factor of 30 billion.

3)  Here’s one more approach—it’s based not on the volume of the physical environment but, instead, on the number of organisms in the LTEE and in the biosphere.  When grown to stationary-phase density in the LTEE environment (i.e., when the limiting resource, glucose, is depleted), the ancestral bacteria could achieve a maximum density of ~5 x 10^7 cells per mL.  Most populations have evolved so that they now produce slightly fewer, but larger, cells; and one population has evolved the ability to use the citrate that is also in the medium, and it now reaches a density that is several times greater than the other populations.  In any case, given 10 mL of medium for each population, and 12 populations, the total population size across the LTEE is on the order of 10^10 cells.

And how many cells exist in the Earth’s biosphere?  Whitman et al. (1998, PNAS) estimated that there are more than 10^30 prokaryotes—bacteria and archaea combined—in the biosphere, and they make up the great majority of all living things.

So by this approach, using the number of cells as a proxy for the spatial scale, the size of the biosphere is over 10^20 (a hundred-million-trillion) times larger than the LTEE.  We’re back into the trillions in terms of the relative scaling of the temporal and spatial scales of the LTEE.

On Time, Space, and the LTEE

By all three approaches, then, the LTEE is vastly older with respect to the history of life on Earth than it is large with respect to the size of Earth’s biosphere.

The LTEE really is a long-running experiment, as experiments go.

But the LTEE is a “drop in the bucket” with respect to how long life has been evolving on Earth.  And it is a vastly more miniscule “drop in the bucket” when compared to the spatial extent and number of living organisms on our planet.

Maybe I should give the LTEE a new name—the “incredibly tiny but relatively long-term evolution experiment.”

[Photo of a water drop on a leaf taken by tanakawho and shared on Wikipedia (en.wikipedia.org/wiki/File:Water_drop_on_a_leaf.jpg).]

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An Absence of Posts, an Abundance of Talks, and More

Dear Reader:  No, I have not given up on this blog.  But I’ve been busy, busy, busy!

In the last four weeks alone, I have traveled to the University of Arizona, Harvard University, Duquesne University, and Princeton University.  Besides giving talks at each place (two public lectures and two academic seminars, with cumulative audiences of well over a thousand people), I have met with dozens and dozens of amazing scientists, from graduate students and postdocs to faculty both young and old.  It’s been a blast:  an exhausting blast, but a blast all the same!

And next week?  I’m hosting four terrific colleagues from two continents who will work with me to begin making sense of hundreds of newly sequenced genomes from the LTEE.

Oh, and we have some more job searches starting next week.

And did I mention?  We just had a fascinating (if I may so myself) and complex paper come out today in Science (on-line express for now) on the most deeply divergent (i.e., oldest sustained polymorphism) of the 12 LTEE populations.  And no, it’s not about the citrate eaters from population Ara–3.

Plucain, J., T. Hindré, M. Le Gac, O. Tenaillon, S. Cruveiller, C. Médigue, N. Leiby, W. R. Harcombe, C. J. Marx, R. E. Lenski, D. Schneider.  2014.  Epistasis and allele specificity in the emergence of a stable polymorphism in Escherichia coli.  Science.

It’s population Ara–2 instead, where two lineages—dubbed the Larges (L) and Smalls (S)—have coexisted for several tens of thousands of generations.  In superb research led by Dr. Jessica Plucain that she did in the lab of my long-time collaborator (and dear friend!) Prof. Dom Schneider (Grenoble, France), Jessica led the work to identify—out of hundreds of mutations—three that are sufficient to allow a “constructed” S ecotype (i.e., the ancestor plus three derived alleles) to invade and stably coexist with the evolved L ecotype.  Ecological context and specific genetic interactions are key to establishing this “half” of the polymorphism … and the other “half” of the story— what makes the L ecotype special—might well turn out to be just as complex, or perhaps even more so.

The S and L types are especially challenging (even painful!) to work with because this population became a mutator very early on—before the two lineages diverged—and so there are many, many mutations to contend with; moreover, they make colonies on agar plates that are quite challenging to score and count.  So congratulations to Jessica, Dom, and other members of Dom’s lab for their perseverance in studying this extremely interesting population.

Also on the list of authors are Prof. Chris Marx and two members of his lab.  They performed metabolic analyses showing how the carbon fluxes through the central metabolism of the S ecotype have diverged from both the ancestor and the L ecotype.  Chris was a postdoc in my lab almost a decade ago, but most of his work (then and since) has been on experimental evolution using Methylobacterium, and so this is the first paper we’ve co-authored.

There was a production error, though, in the on-line version of our paper; the final sentence of the abstract was dropped (except for one word).  The abstract, in total, should read as follows:

“Ecological opportunities promote population divergence into coexisting lineages. However, the genetic mechanisms that enable new lineages to exploit these opportunities are poorly understood except in cases of single mutations. We examined how two Escherichia coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.”

[Edited on 07-Mar-2014:  The on-line PDF at Science Express now has the complete abstract.]

~~~

The picture below shows Dom Schneider and Richard Lenski in Paris in 2013.  They are holding a petri dish that Jessica Plucain made to celebrate the 25th birthday of the LTEE.

Dom and Rich, Paris, 2013

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Zachary Blount on “Ham on Nye” Debate, Follow-up #3

I’m very pleased to present this guest post written by Dr. Zachary Blount, aka Dr. Citrate, as an in-depth follow-up to the “Ham on Nye” science versus creation debate.  Zack did his undergraduate studies at Georgia Tech, and then obtained a masters degree from the University of Cincinnati.  After that, he came to MSU, where he completed his Ph.D. in 2011.  With his doctoral work generating so many interesting results and new questions, Zack stayed on here as a postdoctoral researcher.  His current research is funded by a grant from the John Templeton Foundation Program on Foundational Questions in Evolutionary Biology.

Zack has devoted years to studying the evolution of the ability to grow on citrate that occurred in one of the 12 populations from the long-term evolution experiment (LTEE) with E. coli.  We still don’t fully understand all of the steps involved.  But a simple “on/off” switch it is not!

However, even if it had been a single, simple mutation that allowed the cells to grow on citrate, that still would have been evolution … it still would have been a beneficial mutation in the context of the experiment … and it would still have demonstrated the acquisition of new information encoded in the genomes of the bacteria that fits them to their environment.  Of course, if it were so easy as a single, simple mutation, then we would have seen that capability evolve in many or all of the populations. But after almost 60,000 generations to date, only one population has evolved that ability.

You can read about the technical details of our findings in two papers here and here, as well as in a recent paper from a team at the University of Texas, Austin.

In what follows, the nomenclature Cit+ refers to the bacteria that evolved the ability to grow on citrate in the presence of oxygen, while Cit refers to the bacteria – their ancestors and other E. coli – that lack that ability.

 — Richard Lenski

* * * * *

My work on the evolution of aerobic growth on citrate in one of the LTEE populations has received a fair amount of attention over the years.  (Sometimes there is a bit of a dream-like quality to it all.  I still have a hard time conceiving that people unknown to me know about what this here kid from north Georgia has done.)  The attention is rather gratifying because I’ve spent many years and a great deal of effort in school and in the lab to become an evolutionary biologist. But why did I do all that in the first place? Because I find evolution to be endlessly fascinating, beautiful, and even inspiring.

It means a lot to know that the work I spent several thousands of hours toiling away on has made a contribution to science.  Even more satisfying is that my work has come to be viewed as a go-to example of evolution in action that may, perhaps, inspire in others some of the same feelings that have motived me.

Of course, this attention has also been a bit troubling because it has led to repeated disparagement, dismissal, distortion, and misrepresentation of my work by both professional and amateur creationists.  These creationists often get entirely wrong the work my colleagues and I toiled long and hard to do, likely because they haven’t bothered to read our papers, learn the details and methods, or think much about the results.  (I suspect some duplicity is in there, too.)  Reflexive, unthinking dismissal bothers me – maybe because my parents and devoutly Southern Baptist Granny told me when I was a child that this is something that civilized folk simply should not do.

This brings me to the recent debate between the legendary science educator Bill Nye and the legendary obfuscator and anti-science showman Ken Ham.  It was the standard sort of set-up, with Nye defending evolution and science against creationism, and Ken Ham, well, doing what Ken Ham does.

Twice during the debate, Ham discussed my work with the LTEE population that evolved the capacity to grow aerobically on citrate.  The first time was at about 44 minutes, and included a video clip of Dr. Andrew Fabich, a “Biblical creationist” microbiologist at Liberty University.  [You can read the transcript here.]

The evolution of the new Cit+ function is, and has been discussed as, an instance of evolutionary innovation that arose in a controlled experiment in which we can drill down and figure out how it evolved. Ham and Fabich, however, dismissed Cit+ as an innovation or even an instance of evolution using two arguments suggesting that neither knows the work well at all and likely have not read our papers.  (In Ham’s case, this wouldn’t be surprising, as he has been called “willfully ignorant” even by other creationists, which is a bit like being called unkempt by Pig-Pen or in need of a haircut by Cousin Itt.  In Fabich’s case, however, it would betray a lack of professional courtesy, at best.)

First, Ham repeatedly said that some of the bacteria in the LTEE “seemed” to have developed the ability to grow on citrate.  This wording suggests either stupidity or duplicity on our part, as though we either didn’t check or just lied, but the fact of the matter is that there is no “seem” about it.  The Cit+ bacteria do grow on citrate, and they do so under conditions that E. coli normally does not.  This ability is something that is easy to demonstrate, and which I and my colleagues – not only in the Lenski lab but also other labs that are now working with these bacteria – have documented.  And it’s not as though we don’t have the evidence – as Rich has pointed out to another anti-evolution critic, we have many, many, many vials full of them in our freezers.

The second argument was more direct.  Both Ham and Fabich asserted that the Cit+ function did not evolve because using citrate did not involve “any kind of new information … it’s just a switch that gets turned on and off.”  (Fabich went on to state that this “switch” is what we reported.  That is emphatically not true.  It beggars belief that anyone, much less a trained microbiologist, could actually read our 2012 paper, where we reported the genetic basis of Cit+, and come away thinking this.) Variations on that wording are often used by creationists who discuss the citrate work because it implies that Cit+ arose because of a pre-existing regulatory switch and involved no evolution at all.  But that simply is not the case – that wording, dare I say it, is a lie.

If you take E. coli from a medium in which it is growing on glucose, and move it into a medium where the only thing to eat is something else, like lactose, it turns off the expression of some genes specific to growth on glucose, and it turns on other genes necessary to grow on lactose.  That is what is called gene regulation, and that is what biologists mean when they talk about switching functions on and off – existing genetic circuitry that allows an organism to respond to changes in the external and internal environment.  If you transfer normal, Cit E. coli from a glucose medium to a medium with only citrate to eat, they don’t grow.  They just sit there and starve.  Regular E. coli cells have no existing genetic regulatory circuitry that “flips a switch” to allow them to start growing on citrate in the presence of oxygen.  On the other hand, if you do the same thing with the Cit+ cells that evolved in the long-term experiment, the Cit+ cells will start growing happily on citrate.  This difference is not a matter of gene regulation, but an evolved difference between the ancestral strain and the Cit+ lineage that allows Cit+ cells to grow on citrate.

No, the ability to grow on citrate is not a matter of simply flipping a pre-existing regulatory switch.  Continuing the electrical metaphor, the evolved Cit+ function is instead about rewiring.  My dear little Cit+ cells gained their ability to partake of the previously forbidden citrate by a genetic duplication involving a gene, called citT, which encodes a transporter protein that is used during anaerobic growth on citrate.

This duplication did something very special.  You see, one of the major aspects of gene regulation is that genes have associated regulatory DNA sequences, including what are called promoters that control when genes are expressed.  The citT gene is normally controlled by a promoter that tells the cell to turn it on only when there is no oxygen present.  As shown in the Figure below, the gene duplication put one copy of citT next to, and under the control of, a promoter that normally controls another gene called rnk.  The rnk gene is normally turned on when oxygen is present.  The new association between citT and the rnk promoter – what we call the rnk-citT regulatory module – turns citT on when oxygen is present, and allows Cit+ cells to use citrate under the conditions of the LTEE.  (To really feast on the citrate involved additional evolutionary changes, both before and after this rewiring, but I’ll leave that point aside for this post.)

There is a very interesting consequence of how the rnk-citT module originated. While Ham did not make this argument, other creationists have asserted that Cit+ arose simply by a loss of gene regulation, because they have the notion that evolution can only break things.  However, the duplication that gave rise to the rnk-citT module caused no such thing.  There is still a copy of citT that is linked to the same adjacent DNA sequence as before, and there is still a copy of rnk that is under the control of its own promoter.  In other words, the cell got something new without losing anything old.

When they actually bother to explain all of that, creationists still dismiss Cit+ as being an instance of evolutionary innovation because it involved the rearrangement of existing components.  True, the duplication responsible for Cit+ did rearrange components that were already there, but that rearrangement generated a new association between components that did not previously exist, and it produced a new function that also did not previously exist.  To argue that rearrangements cannot produce innovation is akin to arguing that a novelist has done nothing creative in writing her novels because she only used words that already existed.

Ham also made a demand that is common among creationists that betrays a fundamental misunderstanding of evolutionary theory. In the later debate segment [starting at ~2:30], Ham says, “What Bill Nye needs to do for me is to show me example of something…uh, some new function that arose that was not previously possible from the genetic information that was there. And I would claim and challenge you that there is no such example that you can give… you’d have to show an example of brand new function that never previously was possible.  There is no such example, uh, that you can give anywhere in the world.”

According to Ham, evolution cannot be true if this burden can’t be met.  Consider that wording for a moment, though: “… show an example that never previously was possible.”  Not possible?  That’s kind of a high bar given that impossible things don’t happen by definition.  Moreover, it is clear from Ham’s words that he won’t regard any capacity that arises from modification of an existing genome to be an innovation, which means that he must think that evolutionary theory holds that new genes just pop into existence fully formed, without precursor states, like Athena from the head of Zeus.

This goes to the larger problem with how Ham, Ray Comfort, Michael Behe, Georgia Purdom, and others of their ilk approach evolution – they just don’t know much about it, and so what they end up arguing against isn’t the science, but a caricature of the science that exists only in their minds.  Evolutionary novelty does not arise from genes just popping into existence.  That is a silly idea, and one that no evolutionary biologist holds!

Instead, evolution innovates and creates through descent with modification of what already exists, a process that Nobel laureate François Jacob called “evolutionary tinkering”.  This modification arises by random mutations: base changes, deletions, duplications, insertions, and so on – and, depending on the organisms, horizontal genetic exchange and sexual recombination.  Natural selection then preserves and accumulates the useful changes – those that enhance survival and reproduction of the organism in its environment – across the generations.  Often, such innovations are based on just what we see with the Cit+ bacteria – novel rearrangements of old components.  Indeed, Jacob wrote that, “(Evolutionary) novelties come from previously unseen association of old material.  To create is to recombine.”

So Ham and other creationists dismiss how evolutionary theory says evolution works as not being evolution, and then they demand the impossible.  That strikes me as neither fair nor honest.  But in the end, their lies, distortions, misrepresentations, and ignorance don’t matter, just as debates, entertaining though they may be, don’t matter, because nature doesn’t care.  To paraphrase a bumper sticker I once saw, they may not believe in evolution, but nature does!

While they go on cycling through their old and ossified rhetoric according to their fixed and incorrect notions, evolution proceeds, MacGyvering the new from the old. Natural selection can’t do the impossible, but it is pretty darn spiffy at doing the improbable with the rare.

If you are interested in learning more, please visit my website, where you will find my papers available for download.  You can also watch my Ph.D. defense presentation, in which I go into much more detail about the evolution of the Cit+ E. coli.

— Zachary Blount

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The figure below shows schematically the tandem duplication in the population that evolved the new ability to grow on citrate.  This duplication produced the new rnk-citT regulatory module by placing the second copy of the citT gene adjacent to the rnk promoter region.  The figure comes from Blount et al., 2012, Nature; it is shown here under the doctrine of fair use.

tandem duplication

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