E-coli mutation and evolution
E-coli is a single celled very common bacteria (Figure 1 and 2) and normally not harmful. Each of us has about a billion e-coli in our intestinal system – there is a symbiotic relationship between these bacteria and man. Some members of the family are pathogenic to humans of course and that is why sanitation is important. E-coli motor around using more than one flagellum. The E-coli genome consists of 4.6 million nucleotide pairs making it about one tenth of one percent of the size of the human genome. There are 4377 genes in the most common type and fully 98% of them encode for proteins and the other 88 genes code for RNA. There are no non-coding DNA sequences in these bacteria despite the evidence for gene duplication and the use of transposons to accomplish their “adaptive” mutations in their genomes. The E-coli genome is simply one closed loop and there is only one replication start and stop unlike most eucaryote genomes that have many separate chromosomes with much longer sequences and many starts and stops. Once the replication of DNA starts, it progresses to completion in one step.
Dr. Jerry Bergman (Bergman) makes the argument that the smallest amount of information necessary for non-parasitic life is probably close to the size and complexity of E-coli though there are simpler procaryotes with smaller genomes. That means of course, that inorganic molecules had to jump across the gap from non-life to life producing an organism that could replicate itself forever (until extinction) in one incredibly complex step.
Cyanobacteria is purported to have evolved 3.6 billion years ago. E-coli are usually depicted in the evolutionary tree of life as descending from the same common ancestor as cyanobacteria and of similar age. As far as anyone knows, E-coli are the same organism today as it was when life began. There is no detectible change. It continues to do the work in our digestive system that it has done since man arrived on the earth. Yet many believe that by artificial breeding in the laboratory, they can evolve E-coli to something not yet provided by nature and demonstrate the mechanism for evolution in the process. This is akin to the breeding of dogs that look different but they are all still just dogs – some with a loss of genetic information, but none with a gain over the wolves found in the wild.
The Lenski experiments
Dr. Richard Lenski (Blount, Borland and Lenski) at Michigan State University has been experimenting with E. coli bacteria in the lab for over 20 years starting in 1988. He has 12 separate populations (or segregated tribes) reproducing from an original single strain and he continuously observes their behavior looking for changes that will contribute to understanding the mechanisms for evolution. If successful, he will become quite famous but so far, his observations have led to a number of discoveries about the uniquely designed capabilities of bacteria but he has not accomplished the larger mission of explaining how evolution can produce the orders and families that we see in nature. There are now well over 44,000 generations and after 31,500 generations, Lenski observed that one of his populations could now grow in citrate and use it as food. This was considered outside the bounds of E. coli as a species whose normal diet is high in glucose.
The culture was a mixture of glucose and citrate with 10 times more citrate than glucose which caused the quick consumption of glucose and starvation after that. Having saved specimens from every 500th generation, Lenski went back to the 20,000th generation and found that only by beginning again at or after that population, could he reproduce this same capability after 31,500 generations. Apparently there was a mutation at generation 20,000 that opened the door for a second mutation at generation 31,500. Note that E-coli reproduce once in every 20 to 40 minutes and produce over 50 new generations per day. One E-coli cell can produce several billion descendants per day. The storage of a frozen flask after every 500 generations creates a fossil record of the genome over time. Over 10 trillion E-coli have been produced over the 22 year old experiment and the number of generations is equivalent to 1 million years of human evolution. As of this time (March 2010), the actual mutations have not been identified by the researchers in the literature and the latest publication found at their website (Lenski) does not report anything particularly noteworthy in this regard (Barrick, Kauth and Strelioff).
The research paper (Blount, Borland and Lenski) describing these results seems to have created some real excitement in the evolution community. In fact the author of the summary article in New Scientist (Holmes), Bob Holmes quotes an evolutionary biologist at the University of Chicago who says: “Lenski’s experiment is also yet another poke in the eye for anti-evolutionists, notes Jerry Coyne…‘The thing I like most is it says you can get these complex traits evolving by a combination of unlikely events. That’s just what creationists say can’t happen.’ This “poke is the eye” caused a number of intelligent design and creation scientists to respond (Batten), (Purdom), (Behe, 2008).
In general, these responders presented the following arguments:
- E-coli have the ability to import and digest citrate in anaerobic conditions. With oxygen present, the transporter protein is switched off. A dysfunctional mutation jammed the switch in the open position to bring the citrate into the cell under all conditions.
- There is no new functionality discovered or information added to the cell. Rather a loss or gain of function from preexisting information took place
- Lenski’s experiments make no contribution to the explanation of the source of that preexisting information or the transition of one organism to another or the origin of life itself.
- And Behe points out that “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” (Behe, 2008).
Before the article by Lenski and his team of researchers was published in 2008, Dr. Michael Behe had addressed the Lenski work and its significance in his important book entitled The Edge of Evolution (Behe) (The Edge) in 2007. Much of the summary below is based on Dr. Behe’s analysis and secondly on two research papers (Anderson and Purdom, 2008), (Anderson and Purdom, 2008) by Dr. Kevin Anderson and Dr. Georgia Purdom.
Mutation mechanisms in E-coli
The Dawkins discussion on the Lenski experiments and the popularized report in New Scientist mentioned above demonstrate how important it is to understand the actual mechanisms at the molecular level to avoid misunderstanding and unsubstantiated claims. Michael Behe affirms that studying mutation a the molecular level is the only way to understand how change occurs in nature:
- The only way to get a realistic understanding of what random mutation and natural selection can actually do is to follow changes at the molecular level. It is critical to appreciate this: Properly evaluating Darwin’s theory absolutely requires evaluating random mutation and natural selection at the molecular level. Unfortunately, even today such an undertaking is intensely laborious. Yet there is no other way (Behe 10).
Dr. Michael Behe in The Edge discussed the work of Lenski’s research team at length. On page 174 he discusses prior research that sheds light on these latest claims. He starts with the work of two French scientists on understanding the mechanisms involved for proteins to control DNA and this description seems to have direct bearing on the latest claims of Lenski from his E. coli experiments:
- A huge breakthrough in understanding how proteins control DNA and life came with the work of Francois Jacob and Jacques Monod in the 1960s. It was known then that bacteria could digest different types of sugars, including the most common kind, called glucose, as well as another, much less common sugar, called lactose, which is found in milk. Intriguingly, when bacteria were grown in the presence of glucose, they couldn’t use lactose. Only in the absence of glucose and the presence of lactose could they digest the milk sugar. When glucose was missing, the bacteria made proteins that could pull lactose into the cell and metabolize it, but when no lactose was around, the bacteria didn’t make those proteins. This was a very clever trick that made great biological sense, since in normal conditions the bacterium would waste energy if it manufactured proteins that could metabolize only a rarely encountered sugar. The interesting question was, how did the bacteria “know” when to switch on the genes for making the proteins?
- Jacob and Monod discovered a defective mutant bacterium that made lactose-using proteins all the time, even in the absence of lactose. It was lacking a control mechanism. The French scientists reasoned that the bacteria contained another, hidden protein, which they called a “repressor.” They conjectured that the repressor would ordinarily bind to a specific sequence of DNA near the genes that generated the lactose-using proteins, switching them off. In the presence of lactose, the milk sugar would bind to the repressor itself, changing the protein’s shape enough to make it fall off the DNA, switching back on the previously blocked genes. Jacob and Monod surmised that the mutant bacteria had a broken repressor.
- Their model turned out to be exactly correct, earned them a Nobel Prize, and blazed the path for understanding how the genetic program contained in the DNA of all organisms is controlled… (Behe 174)
This work back in 1960 seems very similar to the work of Lenski in 2008 and it indicates that there really is not much new information being presented at least in terms of explaining how evolution can proceed past adaptation to create new information.
As noted above, it is not true that E. coli can’t metabolize citrate. It already had that ability in non-oxygen environments. At this blog site, Behe explains:
- 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.” (Behe)
And it also turns out that Lenski was not the first to observe this citrate digestion with oxygen present as Behe continues:
- 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. In another instance a protein coded by a gene called citT, which normally transports citrate in the absence of oxygen, was overexpressed [constitutively expressed]. 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. (Behe)
Science writer Bob Holmes speculates in New Scientist (Holmes) three possible explanations for the ability of E-coli to digest citrate:
- A rare single mutation [for example, more than one point mutation in the right places in only one generation]
- A rare chromosome inversion (The gene string gets inserted backwards.)
- The accumulation of several mutations in sequence
These molecular mechanisms (or others) may have caused one of the two scenarios described by Behe above which are summarized as:
- The loss of the repressor gene discussed above under the Jacob and Monod work
- And the constitutively expressed (activity is constant) protein possibility just mentioned. Here extra proteins are produced during the copying process that keeps the gene switch turned on in both anaerobic and aerobic environments.
So far we see that these are all known mechanisms which do not prove that evolution in successive mutations can produce anything new - just the loss of the ability to repress a function when not needed. The experiments do display some unique qualities of bacteria while actually demonstrating the limits of random mutation and natural selection. But this citrate digestion mutation is not the breakthrough mechanism that can be extended to macroevolutionary capability. It is not a new discovery and does not justify the excitement expressed in the New Scientist article. But how do these amazing changes actually take place in the cell?
Lenski proposed mechanisms
In the paper summarizing his findings, Lenski is more explicit than the Holmes generalized summary. He observes that given the slow mutation rate of E-coli in normal conditions, the mutations that seem to have evinced would take a very long time. He speculates on the historical contingency of two mutations to eventually yield the ability to transport citrate into the cell for digestion, and the physiological mechanism that was involved. In terms of the history of the population, he postulates two possibilities:
- One mechanism is Epitasis [the effects of one gene are modified by one or several other genes], whereby the functional expression of the mutation that finally yielded the Cit+ phenotype requires interaction with one of more mutations that evolved earlier.
- A second possibility is that the physical production of the mutation that produced the Cit+ phenotype requires some previous mutation that allows the final sequence to be generated. For example, the insertion of a mobile genetic element creates new sequences at its junctures, and one of these new sequences might then undergo a mutation that generates a final sequence that could not have occurred without the insertion. (Blount, Borland and Lenski 7904)
Lenski points out that E-coli should be able to digest citrate but it lacks the transporter protein to carry it through the cell’s membrane. Therefore in terms of the mechanism, he postulates:
- One possibility is that the Cit+ lineage activated a ‘cryptic’ [preexisting but dormant] transporter, that is, some once-functional gene that has been silenced by mutation accumulation…
- A more likely possibility, in our view, is that an existing transporter has been coopted for citrate transport under oxic conditions. This transporter may previously have transported citrate under anoxic conditions or, alternatively, it may have transported another substrate in the presence of oxygen. The evolved changes might involve gene regulation, protein structure, or both. (Blount, Borland and Lenski)
It is important to note that Lenski prefers the second possibility because he says that the first one “seems unlikely to us because the Cit- phenotype is characteristic of the entire species, one that is very diverse and therefore very old. We would expect a cryptic gene to be degraded beyond recovery.” (Blount, Borland and Lenski) This gets back to the speculation about how long the E-coli has existed in its basic genome as we know it today as well as to the capabilities that bacteria have in terms of mechanisms to survive mentioned above – the preference to believe E-coli is old but irrevocably changed rather than say old but unchanged has a paradigm implication.
The mechanisms active in bacteria are summarized by Anderson and Purdom. They first of all point out that the recovery of a cryptic transporter protein is a reversion to a capability that previously existed while the second one would be a loss of specificity for controlling food intake into the cell. In this case the mutation is beneficial but for a different food source, this mutation could severely hamper the competitive ability of the organism. In neither case is there evidence for a new system nor do we see a hint of an explanation for the origin of the preexisting genes and proteins (Anderson and Purdom 78,81,83).
Adaptive mutation and other mechanisms
Anderson and Purdom point out that there are indeed beneficial mutations in the fight for survival in bacteria but they are temporary and limited and they depend on preexisting functionality. Bacteria have numerous mechanisms for inducing variation in their gene expression. Since they multiply rapidly and produce large populations, mutations have a greater opportunity to occur. In addition, the children of bacteria are haploid (except in rare cases) clones of the parents so that there is no second set of genes to use as a backup system to avoid a serious mutation as eucaryotes have to protect them. It is also significant to note that E-coli can have separate strains where 50% of their genes are different but they are still E-coli. Taking all this into account, Anderson and Purdom discuss some important aspects of bacterial mutation:
- Adaptive Mutation and reversion
- Antagonistic Pleiotropy
- The use of transposons and horizontal gene transfer in bacteria
- Starvation as the stressor for rapid change
Behe (Behe, 2008) warns that adaptive mutation is in its early stages of understanding and it would behoove evolutionists not to proclaim it as having abilities past “The Edge”. Adaptive mutation is defined as a collection of growth independent mutations that enhance the cell’s survival and growth when confronted with stressful and growth limiting environments (Anderson and Purdom 75). The mutations seem to arise specifically to the environment and they are non-random and directed. They fit quite well in the intelligent design model. Adaptive mutation in many cases is likely a reversion back to a preexisting prototroph (Anderson and Purdom). In that case, there is nothing remarkable except the adaptation capabilities designed into bacteria.
Antagonistic pleiotropy is when an existing system is traded for an altered phenotype that is better suited to survive the specific stressful environment. In this case, the expression of the gene results in multiple competing effects; some beneficial but others detrimental to the organism. Depending on the environment or food source, the alteration may be beneficial to the fitness of the organism.
Transposons are strings of genetic information that can be horizontally transferred from another bacterium. Their primary function is to “float” around the genome providing mutations to adjust to the environment. In other words, they seem to have a designed functionality (Thomas). The transposon normally either knocks out a gene causing it to become silent or it carries a promoter that activates a dormant functionality. It can disrupt regulatory sites on the host chromosome and cause it to lose specificity for example and allow a transport protein to carry more than one kind of sugar through the cell wall.
There is evidence that hypermutation increases the rate of mutation in about 10% of adaptive mutation scenarios (Anderson and Purdom 76). Ironically enough, a mutated gene causes an error prone polymerase protein to replicate the genome with more errors than normal. This hypermutation sometimes allows the bacteria to find a more beneficial gene expression more quickly.
Starvation is frequently a state that bacteria have to endure. In order to survive, they need to have special adaptive capabilities as described in the preceding paragraphs in this section of the paper. Both adaptive mutation and antagonistic pleiotropy come into play.
After describing a number of research experiments using E-coli and other bacteria, Anderson and Purdom sum up the conclusions rather concisely:
- Each of these mutant strains has an antagonistic pleiotropy characteristic. An existing system is traded for an altered phenotype that is better suited to survive the specific stressful environment. Regulation is reduced to enable overexpression. DNA repair and DNA polymerase fidelity are reduced to enable increased mutation rates (increasing the probability of a “beneficial” mutation). A gene is inactivated by a process that concurrently activates a silent gene. Such trade-offs provide a temporary benefit to the bacterium, increasing its chances of surviving specific starvation conditions. However, these mutations do not account for the origin of the silenced genes, as their prior existence is essential for the mutation to be beneficial. (Anderson and Purdom 78)
Bacteria indeed have remarkable capabilities to mutate and to survive as we see with antibiotic resistance. But these capabilities do not apply to multi-celled organisms (Anderson and Purdom 81-83) as a whole nor do they “provide a genetic mechanism that accounts for the origin of biological systems or functions. Rather, they require the prior existence of the targeted cellular systems.” (Anderson and Purdom 83)
What have been the results?
Behe Points out that all of the known mechanisms have limited utility to make new cellular structures: “Nothing – neither point mutation, deletion, insertion, gene duplication, transposition, genome duplication, self-organization, self-engineering, nor any other process as yet undiscovered – was of much use” (Behe 162).
The malaria parasite study is neither a computer simulation with designed conditions that set it up for success nor a lab experiment with limits on its boundaries. It is a real observation in nature covering as much as 10,000 years of true history. It shows what nature can do with these mutational mechanisms and the evidence so far is that they cannot move beyond the level of species adaptation.
In fact, Behe explains that like the malaria parasite observations, nothing fundamentally new is being created even after 10 trillion bacteria were produced:
- Nonetheless, the E. coli work has pointed in the same general direction. The lab bacteria performed much like the wild pathogens: A host of incoherent changes have slightly altered pre-existing systems. Nothing fundamentally new has been produced. No new protein-protein interactions, no new molecular machines…One of the most beneficial mutations, seen repeatedly in separate cultures, was the bacterium’s loss of the ability to make a sugar called ribose, which is a component of RNA. Another was a change in a regulatory gene called spoT, which affected en masse how fifty-nine other genes work, either increasing or decreasing their activity. [See Dawkins] Breaking some genes and turning others off, however, won’t make much of anything. After a while, beneficial changes from the experiment petered out. The fact that malaria, with a billion fold more chances, gave a pattern very similar to the more modest studies on E. coli strongly suggests that that’s all Darwinism can do. (Behe 142)
Despite the fascinating insight that the Lenski experiments provide into the adaption of bacteria to their food supply, these experiments have failed to demonstrate the mechanism for evolution. Natural selection is active in nature and mutations explain the adaptation of bacteria to their environment. Bacteria are a different domain of life and they have remarkable capabilities but these mechanisms would largely not apply to higher organisms. Scientists are discovering – an incredible design at the invisible molecular level of life. However, the adaptability of bacteria to starvation does not begin to reveal a mechanism for macroevolution.
Michael Behe in The Edge stressed the importance of understanding mutation and natural selection at the molecular level. Dawkins in his latest book did not get down to these molecular mechanisms and therefore he draws false conclusions as did Darwin. Lenski and his research team observe adaptation or mutational dysfunction at the molecular level and search in vain for a path to macroevolution that does not exist. When we look at the mechanisms and machines of the Lilliputian world in the cell, we see no mechanism to explain life or its macroevolutionary path. We only see a remarkable and undeniable intelligent design.
- Anderson, Kevin L. and Georgia Purdom. "A Creationist Perspective of Beneficial Mutation in Bacteria." Sixth International Conference on Creationism. Pittsburgh, PA: Institute for Creation Research, 2008. 73-85.
- —. "Analysis of Barry Hall's Research of the E. coli ebg Operon: Understanding the Implications for Bacterial Adaptation to Adverse Environments." Sixth International Conference on Creationism. Pittsburgh, PA: Institute for Creation Research, 2008. 149-163.
- Barrick, Jeffrey E., et al. "Excherichia Coli rpoB mutants have increased evolvability in proportion to their fitness defects." Michigan State University, 2010.
- Batten, Don. Bacteria 'evolving in the lab'? 2008. 1 3 2010 <http://creation.com/bacteria-evolving-in-the-lab-lenski-citrate-digesting-e-coli>.
- Behe, Michael. Multiple Mutations Needed for E. Coli. 6 June 2008. 1 3 2010 <http://www.amazon.com/gp/blog/post/PLNK3U696N278Z93O>.
- —. The Edge of Evolution: The Search for the Limits of Darwinism. New York: Free Press, 2007.
- Bergman, Jerry. "Why Abiogenesis Is Impossible." Creation Research Society Quarterly Journal (2000): 1-24.
- Blount, Zachary D., Christina Z. Borland and Richard E. Lenski. "Historical Contingency and the Evolution of a Key Innovation in an Experimental Population of Excherichia coli." PNAS. PNAs, 2008. 7899-7906.
- Dawkins, Richard. The Greatest Show On Earth. New York: Free Press, 2009.
- Holmes, Bob. "Bacteria Make Major Evolutionary Shift in the Lab." New Scientist 9 June 2008.
- Lenski, R.E. (2010). The E. coli long-term experimental evolution project site. http://myxo.css.msu.edu/ecoli
- Purdom, Georgia. A Poke in the Eye? 2008. 1 3 2010 <http://www.answersingenesis.org/articles/aid/v2/n1/a-poke-in-the-eye>.
- Thomas, Brian. "Evolution's Best Argument Has Become Its Worst Nightmare." Acts and Facts March 2010: 16-17.
- Dawkins, 2009, The Greatest Show on Earth. Dawkins uses this 59 gene expression cascade as a proof that natural selection will guide two of the Lenski populations to the same point against great odds. His comments seem disingenuous: “Lenski and a different set of colleagues investigated this phenomenon by taking two of the tribes, called Ara+1 and Ara-1, which seemed, over 20,000 generations, to have followed the same evolutionary trajectory, and looking at their DNA. The astonishing result they found was that 59 genes independently, would completely beggar belief. The odds against its happening by chance are stupefyingly large. This is exactly the kind of thing creationists say cannot happen because they say it can’t happen by chance. Yet it actually happened.” (P. 124)