Readers of my posts know that I’m a big fan of Professor Richard Lenski, a microbiologist at Michigan State University and member of the National Academy of Sciences. For the past few decades he has been conducting the largest laboratory evolution experiment ever attempted. Growing E. coli in flasks continuously, he has been following evolutionary changes in the bacterium for over 50,000 generations (which is equivalent to roughly a million years for large animals). Although Lenski is decidedly not an intelligent design proponent, his work enables us to see what evolution actually does when it has the resources of a large number of organisms over a substantial number of generations. Rather than speculate, Lenski and his coworkers have observed the workings of mutation and selection. For this, we ID proponents should be very grateful.
In a manuscript published a few years ago in the Quarterly Review of Biology (Behe 2010), I discussed laboratory evolution results from the past four decades up to that point, including Lenski’s. His laboratory had shown clearly that random mutation and selection improved the bacterium with time, as measured by the number of progeny it could produce in a given time. He demonstrated without doubt that beneficial mutations exist and can spread quickly in a population of organisms. However, once Lenski’s lab eventually identified the mutations at the DNA level (a difficult task), many of the beneficial mutations turned out to be, surprisingly, degradative ones. In other words, breaking or deleting some pre-existing genes or genetic regulatory elements so that they no longer worked actually helped the organism under the conditions in which it was grown. Other beneficial mutations altered pre-existing genes or regulatory elements somewhat.
What conspicuously was not seen in his work were beneficial mutations that resulted from building what I dubbed new Functional Coded elemenTs, or “FCTs.” Roughly, a FCT is a sequence of DNA that affects the production or processing of a gene or gene product (see my review for a more rigorous definition). In short, improvements had been made by breaking existing genes, or fiddling with them in minor ways, but not by making new genes or regulatory elements. From this information I formulated “The First Rule of Adaptive Evolution“: Break or blunt any functional coded element whose loss would yield a net fitness gain. To say the least, the First Rule is not what you would expect from a process, such as Darwinian evolution, which is touted as being able to build amazingly sophisticated molecular machinery.
Before my review was published, the Lenski lab observed a mutant strain in their experiments that could metabolize citrate in the presence of oxygen, which unmutated E. coli cannot do. (Blount et al. 2008) (Importantly, however, the bacterium can metabolize citrate in the absence of oxygen.) This allowed the mutated bacterium to outcompete its relatives, because the growth medium contained a lot of citrate, as well as oxygen. It was an intriguing result, and was touted as a major innovation, but at the time Lenski’s lab was unable to track down at the DNA level the exact mutations that caused the change.
Now they have. In a recent publication in Nature (Blount et al. 2012) they report the multiple mutations that confer and increase the ability to transport citrate in an atmosphere containing oxygen. They divide the mutations conceptually into three categories: 1) potentiation; 2) actualization; and 3) refinement. “Actualization” is the name they give to the mutation that first confers a weak ability to transport citrate into the laboratory E. coli. (It turns out that the bacterium is lacking only a protein to transport citrate into the cell in the presence of oxygen; all other enzymes needed to further metabolize citrate are already present.) The gene for the citrate transporter, citT, that works in the absence of oxygen is directly upstream from the genes for two other proteins that have promoters that are active in the presence of oxygen. A duplication of a segment of this region serendipitously placed the citT gene next to one of these promoters, so the citT gene could then be expressed in the presence of oxygen. Gene duplication is a type of mutation that is known to be fairly common, so this result, although requiring a great deal of careful research to pin down, is unsurprising.
Over time the mutant got better at utilizing citrate, which the authors called “refinement.” Further work showed this was due to multiple duplications of the mutant gene region, up to 3-9 copies. Again, gene duplication is a fairly common process, so again it is unsurprising. In another experiment Lenski and co-workers showed that increasing the concentration of the citrate transporter gene was sufficient in itself to account for the greater ability of E. coli to grow on citrate. No other mutations were needed.
A more mysterious part of the whole process is what the group called “potentiation.” It turns out that the original E. coli they began with decades ago could not benefit from the gene duplication that brought together a citT gene with an oxygen-tolerant promoter. Before it could benefit, a preliminary mutation had to occur in the bacterium somewhere other than the region containing the citrate-metabolism genes. Exactly what that mutation was, Lenski and coworkers were not able to determine. However, they examined the bacterium for mutations that may contribute to potentiation, and speculated that “A mutation in arcB, which encodes a histidine kinase, is noteworthy because disabling that gene upregulates the tricarboxylic acid cycle.” (They tried, but were unable to test this hypothesis.) In other words, the “potentiation” may involve degradation of an unrelated gene.
Lenski’s lab did an immense amount of careful work and deserves much praise. Yet the entirely separate, $64,000 question is, what do the results show about the power of the Darwinian mechanism? The answer is, they do not show it to be capable of anything more than what was already known. For example, in my review of lab evolution experiments I discussed the work of Zinser et al. (2003) where a sequence rearrangement brought a promoter close to a gene that had lacked one. I also discussed experiments such as Licis and van Duin (2006) where multiple sequential mutations increased the ability of a FCT. Despite Lenski’s visually startling result — where a usually clear flask became cloudy with the overgrowth of bacteria on citrate — at the molecular level nothing novel occurred.
Another person who follows Lenski’s results closely is Dennis Venema, chair of the Biology Department at Trinity Western University and contributor to the BioLogos website. Founded by Francis Collins, BioLogos defends the compatibility of Darwinian science and Christian theology. I agree that the Darwinian mechanism (rightly understood) is theoretically compatible with Christian theology. However, I also think Darwinism is grossly inadequate on scientific grounds. A number of BioLogos writers think it is adequate, and attempt to defend it against skeptics of Darwinism, most especially against proponents of intelligent design such as myself.
In several posts at BioLogos, Professor Venema compared the results of Lenski’s current citrate work to arguments I had made in my QRB review and in my 2007 book, The Edge of Evolution. Whereas I had argued that there was a limit to the number of unselected (either detrimental or neutral) mutations we could reasonably expect an undirected, Darwinian process to have at its disposal in building a complex system, Venema thought Lenski’s recent work showed that limit to be exceeded. Furthermore, while I had pointed out that none of the mutations seen in Lenski’s work up to the date of the review was a gain-of-FCT, Venema wrote the newly published citrate mutations constituted such a feature.
I disagree on both counts. The gene duplication which brought an oxygen-tolerant promoter near to the citT gene did not make any new functional element. Rather, it simply duplicated existing features. The two FCTs comprising the oxygen tolerant citrate transporter locus — the promoter and the gene — were functional before the duplication and functional after. I had written in my review that one type of mutation that could be categorized as a gain-of-FCT was gene duplication with subsequent sequence modification, to allow the gene to specialize in some task. Venema thinks the mutation observed by Lenski is such an event. He has overlooked the fact that there was no subsequent sequence modification; a segment of DNA simply tandemly duplicated, bringing together two pre-existing FCTs. (It is true that the sequence of the protein coded by the duplicated gene includes a fragment from one of the nearby genes, but there is no evidence nor reason to think that the fused fragment is necessary for the activity of the protein.) In my review I classify that as a modification-of-function event. An example of a true gain-of-FCT by duplication cited in my review was the work of Olsthoorn and van Duin (1996) where a 14-nucleotide duplication led to the formation of new functional coded elements (it did not simply repeat pre-existing elements), so it is not just a modification-of-function mutation. The citrate mutation did nothing like that.
Venema counts the number of mutations needed to get a fully functioning citrate-importing function in Lenski’s work, and arrives at roughly a half-dozen. Unfortunately, several of those are tandem duplications of the weak citT transporter, which clearly are selectable, beneficial mutations. In arriving at the limits to Darwinism, I emphasized that that mechanism would certainly work if gradually-increasing, serial, beneficial mutations could do the job. Thus such mutations do not count in estimating the limit. Only required deleterious and neutral mutations count against the limit to Darwinian evolution. Venema argues that perhaps all of complex functional biology could be reached by gradual, beneficial mutations. Well, bless his optimistic heart, but the data give us no reason to think that, because gradually increasing one protein’s total cellular activity by sequential gene duplication is successively beneficial, all routes to complex systems involving multiple distinct elements will be. Quite the opposite, as I have often argued.
Professor Venema also counts several “potentiating” mutations as contributing to the system. Unfortunately, whatever those mutations are, they are not part of the citrate metabolic system itself. Rather, they are at most part of the genetic background. If Lenski and co-workers’ speculations are correct (Blount et al. 2012), at least one of the potentiating mutations degrades an unrelated gene, and thus itself counts as a loss-of-FCT mutation. When counting the mutations contributing to the edge of evolution for building a feature, only the ones directly involved in the feature are counted, not ones that indirectly contribute to a receptive genetic background (which are legion). Thus, unlike Venema, I count perhaps three to four mutations — the original duplication placing the oxygen-tolerant promoter near the citT gene, plus several rounds of duplication of that region. All of the mutations are modification-of function ones in the classification system I described. I should add that there is no reason to think that Darwinian processes cannot produce gain-of-FCT mutations, and I reviewed several such events. But they are greatly outnumbered by loss-of-FCT and modification-of-function beneficial mutations.
In my own view, in retrospect, the most surprising aspect of the oxygen-tolerant citT mutation was that it proved so difficult to achieve. If, before Lenski’s work was done, someone had sketched for me a cartoon of the original duplication that produced the metabolic change, I would have assumed that would be sufficient — that a single step could achieve it. The fact that it was considerably more difficult than that goes to show that even skeptics like myself overestimate the power of the Darwinian mechanism.
Barrick,J.E., Yu,D.S., Yoon,S.H., Jeong,H., Oh,T.K., Schneider,D., Lenski,R.E., and Kim,J.F. 2009. Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461:1243-1247.
Behe,M.J. 2010. Experimental Evolution, Loss-of-function Mutations, and “The First Rule of Adaptive Evolution.” Q. Rev. Biol. 85:1-27.
Behe,M.J. 2007. The Edge of Evolution: the Search for the Limits of Darwinism. Free Press: New York.
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.
Blount,Z.D., Barrick,J.E., Davidson,C.J., and Lenski,R.E. 2012. Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489:513-518.
Licis,N. and van,D.J. 2006. Structural constraints and mutational bias in the evolutionary restoration of a severe deletion in RNA phage MS2. J. Mol. Evol. 63:314-329.
Olsthoorn,R.C. and van Duin,D.J. 1996. Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus. Proc. Natl. Acad. Sci. U. S. A 93:12256-12261.
Venema,D. 2012. Behe, Lenski and the “Edge” of Evolution, Part 1: Just the FCTs, Please. The Biologos Forum. http://biologos.org/blog/behe-lenski-and-the-edge-of-evolution-part-1.
Zinser,E.R., Schneider,D., Blot,M., and Kolter,R. 2003. Bacterial evolution through the selective loss of beneficial Genes. Trade-offs in expression involving two loci. Genetics 164:1271-1277.
Image credit: Nina H./Flickr.