A Switch is Born

i-fe5c7bfd51abfe457b966f67ced9c816-escherichia.jpgThe language of DNA is written in a four-letter alphabet. The four different chemical units of DNA (called nucleotides) create an incomprehenisbly vast range of possibility codes. Consider a short sequence of 41 nucleotides. There are over 4.8 trillion trillion possible sequences it could take. In this vast universe of possibilities, how can natural selection hit on new DNA sequences that help life survive?

All living things have genes. Enzymes read those genes and produce a copy of their code, which a cell can then use to build a protein. But in order to read a gene, the enzymes must first lock onto a distinctive segment of DNA near the gene, known as a promoter. Promoters act like switches, which a cell can use to turn genes on and off. Different genes carry different promoters, so that they can be switched on under different conditions.

Scientists have studied the promoters of the bacteria Escherichia coli more closely than those of any other species, and they've identified some of its switching patterns. When Escherichia coli is growing quickly, it produces a lot of gene-reading enzymes factors called sigma 70. Sigma 70 can switch on several hundred genes that allow the microbe to feed and build up its biomass and reproduce. If Escherichia coli begins to starve, it slips into a sort of suspended animation, and produces a different enzyme factor called sigma S. Sigma S recognizes a different set of genes that begin to make the proteins necessary for shutting the microbe's operations down.

Here we have a wonderfully precise system for controlling genes. Now imagine that Escherichia coli acquires a gene with no promoter at all--just a random sequence of DNA next to the gene, 41 nucleotides long. Imagine that this DNA starts going through cycles of mutation and natural selection. Would it be possible for a random sequence to change into one Sigma 70 could grab? Could it go from nothing to a promoter?

The answer is yes. How long would it take? According to some recent experiments, two days. Two.

I came across these experiments this week during my research for my next book on Escherichia coli. I've been learning about the long tradition of evolutionary experiments on this marvelous bug. This new promoter experiment was carried out by Shumo Liu of NEC Laboratories and Albert Libchaber of Rockefeller University and published in the Journal of Molecular Evolution.

The gene they studied provides resistance to the antibiotic chloramphenicol. The researchers inserted the gene onto a small loop of DNA called a plasmid. Bacteria often carry plasmids along with their chromosome, and they sometimes donate a plasmid to another microbe. Like other genes, genes on plasmids typically come with promoters. But Liu and Libchaber engineered their plasmids so that in the place of a promoter, the resistance genes had a random, nonfunctional sequence of DNA.

The researchers then put this DNA through some of the essential steps of evolution: mutation, selection, and replication. They made copies of the DNA and introduced mutations into some of them at random. They ran the experiment several times with different mutation rates, ranging from 18% down to .4%. They inserted the mutated DNA back into the plasmids and inserted the plasmids into Escherichia coli. The microbes were allowed to grow rapidly for 12 hours, producing a lot of sigma 70. The scientists then added a poison pill to their feast: a dose of chloramphenicol. Later, Liu and Libchaber scraped off some of the surviving bacteria from the dishes and extracted their plasmids. They introduced more mutations into the DNA, and then repeated the cycle.

Natural selection favored mutants that had stretches of DNA that sigma 70 could grab onto, even if the fit was lousy. If a microbe could produce even a little resistance to the antibiotic, it could survive better than ones that could not. Mutations that produced a better fit would switch on the resistance gene more and give microbes an even bigger evolutionary edge over other microbes.

The scientists were surprised to find that even at the lowest rate of mutation (.4%), it took only three cycles for turn a non-functional sequence of DNA into a full-blown sigma-70 promoter. The promoter made the bacteria so resistant that most of them could withstand a high dose of chloramphenicol. Liu and Libchaber only need a small population to produce these promoters--about a million strong, a thousandth the size of the population of Escherichia coli found in your own gut. When Liu and Libchaber sequenced these evolved promoters, they discovered that the DNA had converged on many of the same elements found in natural sigma 70 promoters.

The scientists caution that their results do not reproduce the precise details of evolution outside the laboratory....

In general, evolution in nature involves a large population over a long period of time and is constrained by certain historical conditions, many of which may forever be obscure. In contrast, the experimental evolution course is very short, the population size is small, and the mutation frequency is usually elevated in order to accelerate the process. Nevertheless, the experiments capture the essence of evolution; they are an iteration of mutation, selection, and replication. Experimental evolution permits us to adjust individually the parameters and repeat the process under controlled conditions, and thus, it lets us observe some essential features in the process and test certain hypotheses. In this way, molecular evolution is a bridge between the purely mathematical modeling and the ''real world'' in nature.

It also lets us watch life defy the odds, day by day.

Update: Thanks to Dr. Jim Hu for some clarifications in the comments.


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Great find!

You mean random processes combined with deterministic processes can lead to new(at least for those artficial populations) information? Gee, I thought Dembsky said that wasn't possible?

Very cool experiment. I wonder how long it would take and what sort of selection you'd need to evolve a regulator binding site. Maybe cyclic passages with and without chloramphenicol?

Pedantic aside from a molecular microbiologist: sigma factors aren't enzymes...

By Michael Gray (not verified) on 26 Aug 2006 #permalink

It also lets us watch life defy the odds, day by day.

But life doesn't really defy the odds, does it? I thought that we just have an intuitively poor understanding of the odds, which is why we are often surprised by certain things happening when we really shouldn't be.

But...but...but all those IDers keep telling us that no one's ever seen evolution happening, or that only "microevolution" going back and forth between various pre-existing states can occur, that random mutation can't produce new information, only dilute it...

Good thing for all those IDers that this experiment occurred under "artificial," "model" conditions, or they'd be left with very little to continue misleading their incurious followers about.

By Steviepinhead (not verified) on 26 Aug 2006 #permalink


But of course the ID'ers really can't rely on the excuse that the experiment happened under artificial conditions etc...after all, we don't claim that there is really a difference say between natural and artificial electricity, do we? And since Dembsky's ideas are supposedly mathematically based, they ought to apply equally to both natural and artificial situations!

Just wondering if anyone else is reminded of the Luria-Delbruck fluctuation experiments. Hold a gun (Chloramphenicol) to a microbes head and its going to find a way to slip out from under it as soon as it can. If you have a very high selection pressure, things move quickly (and variably).

just wondering how that mutation rate was determined/expressed? 18% of what? 18% substitution per codon per generation? 18%probability of 1 change per genome?.... need info here.

djlactin: In each round of the experiment, the scientists sample bacteria, make copies of their promoter sequences and introduce mutations into those sequences through PCR mutategenesis. The mutation rate refers to the fraction of promoter sequences that mutate in this stage of the cycle.

I wouldn't be surprised if the highly-resistant strains lost some fitness in other respects, and would be outcompeted by normal E coli in a "normal" petri dish environment. That would be pretty close to an Ernst Mayr definition of new species.

Do E. coli exchange DNA sequences amongst themselves? I know some bacteria do that, but I don't know which ones or to what extent. If they did, they would even more closely resemble eukaryotic species.

Carl, if you haven't already, look into the work of Steve Finkel's group at USC. They have been observing the GASP phenotype (basically, succession in old cultures) and the relationship to mutations in sigma factors. Some other big shots in the field are Roberto Kolter (Yale), Richard Lenski (Michigan State?), and Al Bennett (UCI). The switching stuff (stochastic controls on gene expression, single cells) is cool too; Mary Lidstrom is working on that with a neat methanogen system. If you ever look at "monocultures" of environmental bacteria, you know that the uniform colonies on a petri dish are a pure laboratory artifact - bacterial gene expression is heterogenous, and complex environmental cues are responsible, as well as stochastic processes - it's fascinating stuff, and we have the tools to study it now.

By Paul Orwin (not verified) on 28 Aug 2006 #permalink

Minor corrections:

The microbes were allowed to grow rapidly for 12 hours, producing a lot of sigma 70.

The EÏ70 is constant in the host cells in these experiments. The 12 hour growth allows the mutagenized plasmids to proliferate and make whatever chloramphenicol acetyl transferase they can make. There will be some additional mutations from this outgrowth as well.

The mutation rate refers to the fraction of promoter sequences that mutate in this stage of the cycle.

The frequency is expressed per bp sequenced, so it's pretty high. The authors describe their low frequency (not rate) as:

The mutation frequency is 0.4% (13 mutations among 3198 bp examined)

The mutagenized segment is only 41 bp long. I get 13 mutations among 78 promoters sequenced. Although the paper is not totally clear, the correct way to do this is to sequence from the unselected population at each round. It looks like that's what they did, since the sequences shown in Fig 5 have many more than 13 sequence changes.

In their low frequency experiment (Expt. 2), the dominant sequence seems to be derived from something that was not from the PCR mutagenesis (descendents of 2.1.15). The source of this DNA mystifies me...it would help if the papers was written more clearly wrt things like whether they sequenced both strands.

for all those IDers that this experiment occurred under "artificial," "model" conditions, or they'd be left with very little to continue misleading their incurious followers about.
# soapbox
Incurious followers, more like, lack of understanding. Feel sorry for 'em, maybe. Feel sorry for me. I know some in the ID/Evolution circles who purposely trivalize the controversies big mile-long words among and impressing their colleagues, and it alienates curiosity.. #insert mile long stretch of scientific babble-words here /
I've read the article a couple times, and still having a problem understanding, and its not that I don't want to understand, and I certainly don't believe Carl Zimmer is telling lies, however, truthfully, without understanding exactly what the point really is, I would be a liar to state with conviction, "He's telling the truth."

Point blank, it really is so much easier to believe Dembski, when he says "Goddidit"... not that anyone out in the real world really understands (or cares to) any of the science Dembski's babbling about either, but "God", people understand "God".

- "Make everything as simple as possible, but not simpler."
- "Any intelligent fool can make things bigger, more complex, and more violent. It takes a touch of genius -- and a lot of courage -- to move in the opposite direction."
- "I want to know God's thoughts; the rest are details."
- Einstein
- rescomp.stanford.edu/~cheshire/EinsteinQuotes.html

I think the time point of 2 days should be replaced with the number of divisions that the cells might have undergone before getting a promoter sequence. The "2 days" sort of trivialises the fact that it contains roughly a hundred generations in it not to mention the number of organisms that might have been born and died.
Also what I wish to ask is that whether the study says that the promoter sites will spring up right before the first nucleotide that is "read" and copied into a messenger RNA...because there are chances that the promoter might spring up in unlikely locations making it less meaningful for the bacterium.

IDers keep telling us that no one's ever seen evolution happening,

What do they say about a fertilized egg which begins as a single cell (all of them look the same too!) rapidly, blink of an eye in geological time, all evolve into unique species...

Or a frog, starts out like a fish, sprouts legs, and moves on land.

or that only "microevolution" going back and forth between various pre-existing states can occur,

What about humans growing tails count, our ape ancestry, does that qualify as a pre-existing state...