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Multiple Mutations Needed for E. Coli

3:35 PM PDT, June 6, 2008
Dear Readers,

An interesting paper has just appeared in the Proceedings of the National Academy of Sciences, “Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli.” (1) It is the “inaugural article” of Richard Lenski, who was recently elected to the National Academy. Lenski, of course, is well known for conducting the longest, most detailed “lab evolution” experiment in history, growing the bacterium E. coli continuously for about twenty years in his Michigan State lab. For the fast-growing bug, that’s over 40,000 generations!

I discuss Lenski’s fascinating work in Chapter 7 of The Edge of Evolution, pointing out that all of the beneficial mutations identified from the studies so far seem to have been degradative ones, where functioning genes are knocked out or rendered less active. So random mutation much more easily breaks genes than builds them, even when it helps an organism to survive. That’s a very important point. A process which breaks genes so easily is not one that is going to build up complex coherent molecular systems of many proteins, which fill the cell.

In his new paper Lenski reports that, after 30,000 generations, one of his lines of cells has developed the ability to utilize citrate as a food source in the presence of oxygen. (E. coli in the wild can’t do that.) Now, wild E. coli already has a number of enzymes that normally use citrate and can digest it (it’s not some exotic chemical the bacterium has never seen before). However, the wild bacterium lacks an enzyme called a “citrate permease” which can transport citrate from outside the cell through the cell’s membrane into its interior. So all the bacterium needed to do to use citrate was to find a way to get it into the cell. The rest of the machinery for its metabolism was already there. As Lenski put it, “The only known barrier to aerobic growth on citrate is its inability to transport citrate under oxic conditions.” (1)

Other workers (cited by Lenski) in the past several decades have also identified mutant E. coli that could use citrate as a food source. In one instance the mutation wasn’t tracked down. (2) In another instance a protein coded by a gene called citT, which normally transports citrate in the absence of oxygen, was overexpressed. (3) The overexpressed protein allowed E. coli to grow on citrate in the presence of oxygen. It seems likely that Lenski’s mutant will turn out to be either this gene or another of the bacterium’s citrate-using genes, tweaked a bit to allow it to transport citrate in the presence of oxygen. (He hasn’t yet tracked down the mutation.)

The major point Lenski emphasizes in the paper is the historical contingency of the new ability. It took trillions of cells and 30,000 generations to develop it, and only one of a dozen lines of cells did so. What’s more, Lenski carefully went back to cells from the same line he had frozen away after evolving for fewer generations and showed that, for the most part, only cells that had evolved at least 20,000 generations could give rise to the citrate-using mutation. From this he deduced that a previous, lucky mutation had arisen in the one line, a mutation which was needed before a second mutation could give rise to the new ability. The other lines of cells hadn’t acquired the first, necessary, lucky, “potentiating” (1) mutation, so they couldn’t go on to develop the second mutation that allows citrate use. Lenski argues this supports the view of the late Steven Jay Gould that evolution is quirky and full of contingency. Chance mutations can push the path of evolution one way or another, and if the “tape of life” on earth were re-wound, it’s very likely evolution would take a completely different path than it has.

I think the results fit a lot more easily into the viewpoint of The Edge of Evolution. One of the major points of the book was that if only one mutation is needed to confer some ability, then Darwinian evolution has little problem finding it. But if more than one is needed, the probability of getting all the right ones grows exponentially worse. “If two mutations have to occur before there is a net beneficial effect — if an intermediate state is harmful, or less fit than the starting state — then there is already a big evolutionary problem.” (4) And what if more than two are needed? The task quickly gets out of reach of random mutation.

To get a feel for the clumsy ineffectiveness of random mutation and selection, consider that the workers in Lenski’s lab had routinely been growing E. coli all these years in a soup that contained a small amount of the sugar glucose (which they digest easily), plus about ten times as much citrate. Like so many cellular versions of Tantalus, for tens of thousands of generations trillions of cells were bathed in a solution with an abundance of food — citrate — that was just beyond their reach, outside the cell. Instead of using the unreachable food, however, the cells were condemned to starve after metabolizing the tiny bit of glucose in the medium — until an improbable series of mutations apparently occurred. As Lenski and co-workers observe: (1)

Such a low rate suggests that the final mutation to Cit+ is not a point mutation but instead involves some rarer class of mutation or perhaps multiple mutations. The possibility of multiple mutations is especially relevant, given our evidence that the emergence of Cit+ colonies on MC plates involved events both during the growth of cultures before plating and during prolonged incubation on the plates.

In The Edge of Evolution I had argued that the extreme rarity of the development of chloroquine resistance in malaria was likely the result of the need for several mutations to occur before the trait appeared. Even though the evolutionary literature contains discussions of multiple mutations (5), Darwinian reviewers drew back in horror, acted as if I had blasphemed, and argued desperately that a series of single beneficial mutations certainly could do the trick. Now here we have Richard Lenski affirming that the evolution of some pretty simple cellular features likely requires multiple mutations.

If the development of many of the features of the cell required multiple mutations during the course of evolution, then the cell is beyond Darwinian explanation. I show in The Edge of Evolution that it is very reasonable to conclude they did.


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

2. Hall, B.G. 1982. Chromosomal mutation for citrate utilization by Escherichia coli K-12. J. Bacteriol. 151:269-273.

3. Pos, K.M., Dimroth, P., and Bott, M. 1998. The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts. J. Bacteriol. 180:4160-4165.

4. Behe, M.J. 2007. The Edge of Evolution: the search for the limits of Darwinism. Free Press: New York, p. 106.

5. Orr, H.A. 2003. A minimum on the mean number of steps taken in adaptive walks. J. Theor. Biol. 220:241-247.
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I am Professor of Biological Sciences at Lehigh University in Pennsylvania. I received my Ph.D. in Biochemistry from the University of Pennsylvania in 1978. My current research involves delineation of design and natural selection in protein structures. In addition to teaching and research I work as a senior fellow with the Discovery Institutes Center for Science & Culture.

In addition to publishing over 35 articles in refereed biochemical journals, I have also written editorial features in Boston Review, American Spectator, and The New York Times. My book, Darwin's Black Box, discusses the implications for neo-Darwinism of what I call "irreducibly complex" biochemical systems and has sold over 250,000 copies. The book was internationally reviewed in over one hundred publications and recently named by National Review and World magazine as one of the 100 most important books of the 20th century.

I have presented and debated my work at major universities throughout North America and England.

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