Climbing Mount Citrate
As we discussed yesterday, the most dramatic innovation yet observed in the E. coli Long Term Evolution Experiment (LTEE) was the ability, acquired by one of the twelve cultures, to use citrate as a carbon source under aerobic conditions. When we last discussed the LTEE in 2011, we noted what was known then about the mutations that eventually combined to produce the Cit+ trait:
Tracking down the nature of this dramatic change led to some interesting findings. The ability to use citrate as a food source did not arise in a single step, but rather as a series of steps, some of which are separated by thousands of generations:
- The first step is a mutation that arose at around generation 20,000. This mutation on its own does not allow the bacteria to use citrate, but without this mutation in place, later generations cannot evolve the ability to use citrate. Lenski and colleagues were careful to determine that this mutation is not simply a mutation that increases the background mutation rate. In other words, a portion of what later becomes “specified information for using citrate” arises thousands of generations before citrate is ever used.
- The earliest mutants that can use citrate as a food source do so very, very poorly – once they use up the available glucose, they take a long time to switch over to using citrate. These “early adopters” are a tiny fraction of the overall population. The “specified information for using citrate” at this stage is pretty poor.
- Once the (poor) ability to use citrate shows up, other mutations arise that greatly improve this new ability. Soon, bacteria that use citrate dominate the population. The “specified information for using citrate” has now been honed by further mutation and natural selection.
- Despite the “takeover”, a fraction of the population unable to use citrate persists as a minority. These cells eke out a living by being “glucose specialists” – they are better at using up glucose rapidly and then going into stasis before the slightly slower citrate-eaters catch up. So, new “specified information to get the glucose quickly before those pesky citrate-eaters do” allows these bacteria to survive. As such, the two lineages in this population have partitioned the available resources and now occupy two different ecological niches in the same environment. As such, they are well on their way to becoming different bacterial species.
As such, we noted three distinct steps observed by the Lenski group: steps they call potentiation, actualization, and refinement. Potentiation mutations do not themselves result in the ability to use citrate under aerobic conditions, but they are necessary for it to appear later. Actualization is the mutation that first brings about the Cit+ trait, though, as we noted, this step produced only a very weak Cit+ effect. This nascent ability, however, then undergoes refinement through additional mutations and selection to give the final, robust Cit+ trait observed in the culture.
While some things were known about these steps when the Lenski group last published on this topic (in 2008), the precise details remained unclear. What was needed was a complete characterization of the Cit+ bacteria through whole-genome sequencing to help indentify the changes. These long-awaited results are now available in a new paper published last month by the Lenski group, and they shed light on all three stages of the process.
Lights, camera, actualization
The key step - and the one of greatest interest - is of course actualization: the mutation that converted a Cit- cell to a Cit+ one. This is also one of the easiest steps to study, since the mutation provides the cell with a new feature that can be detected experimentally. Though E. coli cannot use citrate as a carbon source in the presence of oxygen, they are capable of using citrate in anoxic conditions (i.e. when oxygen is absent). To do so, they employ a protein that imports citrate in to the cell while at the same time exporting a compound called succinate. Since this protein is already present in the E. coli genome, it was long suspected that a genetic regulatory change that turned on its production in the presence of oxygen could be the key innovation that produced the first Cit+ bacterium in the culture. As we discussed yesterday, Behe notes that this change could result from a loss-of-FCT or a gain-of-FCT mutation:
“If the phenotype of the Lenski Cit+ strain is caused by the loss of the activity of a normal genetic regulatory element, such as a repressor binding site or other FCT, it will, of course, be a loss-of-FCT mutation, despite its highly adaptive effects in the presence of citrate. If the phenotype is due to one or more mutations that result in, for example, the addition of a novel genetic regulatory element, gene duplication with sequence divergence, or the gain of a new binding site, then it will be a noteworthy gain-of-FCT mutation.”
Interestingly, the actualization mutation was indeed a change of regulation of the anoxic citrate / succinate transporter, and it arose through a gain-of-FCT mutation. The mutation turned out to be a side-by-side duplication of the citrate / succinate transporter gene, as well as portions of two genes on either side of it. This imprecise duplication placed a partial fusion of these flanking genes next door to one of the copies of the citrate / succinate transporter gene. This brought the copy under the control of promoter sequences derived from of one of its neighbors, a gene that is active when oxygen is present. The resulting product was a copy of the citrate / succinate transporter gene that was now very weakly expressed in aerobic conditions. Since this is an example of a mutation that duplicates a gene and simultaneously creates a new regulatory element for it (causing significant sequence divergence), this is a clear-cut example of a gain-of-FCT mutation.
Responding to the data
While Behe has not yet, to my knowledge commented on this particular development within the LTEE, one of his colleagues in the Intelligent Design Movement (IDM), microbiologist Ann Gauger, has offered her thoughts. Two themes emerge in her commentary: that the Cit+ trait is “not new”, and that the number of mutations it required were within the bounds set out by Behe and another member of the IDM, structural biologist Douglas Axe:
When is an innovation not an innovation? If by innovation you mean the evolution of something new, a feature not present before, then it would be stretching it to call the trait described by Blount et al. in "Genomic analysis of a key innovation in an experimental Escherichia coli population" an innovation [...]
The total number of mutations postulated for this adaptation is two or three, within the limits proposed for complex adaptations by Axe (2010) and Behe in Edge of Evolution. Because the enabling pre-adaptive mutations could not be identified, though, we don't know whether this was one mutation, a simple step-wise series of adaptive mutations, or a complex adaptation requiring one or two pre-adaptations before the big event.
But does this adaptation constitute a genuine innovation? That depends on the definition of innovation you use. It certainly is an example of reusing existing information in a new context, thus producing a new niche for E. coli in lab cultures. But if the definition of innovation is something genuinely new, such as a new transport molecule or a new enzyme, then no, this adaptation falls short as an innovation. And no one should be surprised.
While Gauger does not speak to the tension between her description of the Cit+ mutation as “not genuinely new” and Behe’s criteria that this should be classified as a gain-of-FCT mutation, it is clear that she views this event as within Behe’s “edge” – i.e. within the bounds of “what Darwinism can do.” Additionally, she sees it as falling within the scope of what is evolutionarily possible as proposed by Axe’s work. In the next installment of this series, we’ll revisit how Behe defines his (claimed) limit of what evolutionary processes can accomplish, with this new evidence in hand. In doing so, a careful examination of the potentiation and refinement phases of the Cit+ transition will be informative.
For further reading:
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.
Michael J. Behe, The Edge of Evolution: The Search for the Limits of Darwinism (New York: Free Press, 2007).
Michael J. Behe (2010). Experimental evolution, loss-of-function mutations, and “The first rule of adaptive evolution”. The Quarterly Review of Biology 85(4); 419-445.