This coming Monday, February 24th, will be the 37th birthday of the Long-Term Evolution Experiment (LTEE) with E. coli. Happy birthday to the 12 lines! I hope you will keep on evolving for many, many more years.
I started the LTEE in 1988 while I was on the faculty at UC-Irvine. The LTEE moved with me to MSU in late 1991, where it reached the milestone of 75,000 generations. In May 2022, the LTEE moved to UT-Austin, where it continues in the able hands of Jeff Barrick and his team and has now passed 80,000 generations. I think it’s fair to say that the LTEE has worked out pretty well.
Another evolution experiment that worked well was just published in Science by Michael Barnett, Lena Meister, and Paul Rainey. Titled “Experimental Evolution of Evolvability,” they show that bacteria can evolve to become more adept at adapting to changing conditions.
One way to do that is by increasing a cell’s mutation rate across its entire genome. In fact, that has happened in several LTEE populations, though in most cases the hypermutability was later reduced or even reversed. Genome-wide hypermutability is a double-edged sword, because random mutations may break other important functions before finding a solution to the new circumstances.
A better solution, in some scenarios, would be to mutate only those “local” bits of the genome that encode the functions that must change to fit the new conditions. Localized hypermutability might suggest some foresight, but that’s not really so. If populations of microbes have experienced similar changes repeatedly during their evolutionary history, then a lineage that evolved a more mutable local sequence in a relevant gene could be more likely to persist.
We know from molecular biology that some sequences — for example, homopolymeric runs like AAAAAA — are much more mutable than others. And we know from comparative studies that some microbes possess localized hypermutability in a subset of their genes that are important for dealing with unpredictable aspects of their environment. Imagine, for example, a protein that is required for transmission between hosts, but which makes the cell vulnerable within a host. This scenario would favor a lineage that has the capacity to inactivate that specific protein at a high rate and then to reactivate it at a high rate. The new study by Barnett et al. is the first to demonstrate this process experimentally. They did so with Pseudomonas fluorescens by selecting for “repeated phenotypic transitions between … the mat-forming, cellulose-overproducing CEL+ type and the mat-colonizing, non-cellulose-producing CEL– type.”
This nifty new result reminds me of a conceptually similar experiment that Paul Sniegowski and I did way back in the 1990s, but which did not work out so nicely. Paul was a postdoc in my lab, and he discovered that some of the LTEE populations had evolved genome-wide hypermutability. (Paul later joined the faculty at Penn and, last year, became President of Earlham College.) Paul and I were also examining the evidence concerning the randomness of mutations in light of the possibility of so-called “directed” mutations; and I had recently collaborated with Richard Moxon and Paul Rainey on a review article that discussed the evidence and evolutionary hypothesis for the emergence of localized hypermutability in what we called “contingency genes.” So, Paul Sniegowski and I set out to see if we could evolve a brand-new contingency gene.
It’s been a long time, and I may misremember some details. But to a first approximation, we sought to do the same experiment as Barnett et al., except using E. coli and two alternating environments appropriate to the biology of that species. In particular, in the course of my earlier work on the coevolution of E. coli and phage T4, I had learned that mutations that confer resistance to T4 infection also make the mutants more sensitive to the antibiotic novobiocin. This collateral sensitivity occurs because (i) phage T4 infects by adsorbing to the lipopolysaccharide (LPS) core of the E. coli cell envelope; (ii) the mutations that confer T4 resistance change the structure of the LPS core; (iii) novobiocin is a hydrophobic compound; (iv) the altered LPS core impacts the hydrophobicity of the cell envelope; and (v) that change allows novobiocin to enter T4-resistant cells at a much higher rate.
Given these points, Paul and I reasoned that we could propagate lines in a regime that alternated each day between exposure to T4 and novobiocin. Each round would impose lethal selection, and so we expected most lines to go extinct. But if a lineage happened to become resistant to one or the other killer by a mutation that also happened to increase the mutation rate in a gene encoding the relevant step in LPS synthesis, it would be more likely to survive the future back-and-forth challenges. Makes sense, right?
Given the lethality of the selection against sensitive cells, and the resulting high likelihood of extinction, Paul reasoned he would need a very large experiment. I forget the numbers, but he set up many tens or even hundreds of replicate lineages to start out.
After a few days, though, most or all of the lineages had survived. But how? Paul tested the evolved cells for their susceptibilities to T4 and novobiocin, and he got an unexpected result — the lines had become simultaneously resistant to both T4 and novobiocin!
We then realized that the reasoning behind our experimental design had been faulty. While mutations that disrupt the LPS core affect sensitivity to novobiocin, the cellular target of that antibiotic is a different macromolecule, namely the DNA gyrase that is required for genome replication. What had evidently happened, therefore, was sequential selection for double mutants that first became resistant to T4 by mutations in genes impacting the LPS core and then resistant to novobiocin by mutations in the DNA gyrase. The bacteria did not need localized hypermutability to solve the alternating environments that we imposed.
Edited to add: Paul Sniegowski and I were hoping to find a quick and easy route to building a contingency gene. Something like this, perhaps—that among, say, 1000 mutants that became T4 resistant, maybe a few would have, say, a new 4-bp sequence like AAGA. And then one of those that became novobiocin resistant might have an AAAA sequence, at which point some slippage and frameshifts would start happening. That was our thinking. The results of Barnett et al. were more subtle and complex than what we imagined, and required a lot more persistence, ingenuity, and insight to understand. Bravo!
Two lessons: Some experiments just don’t work out. And you get what you select for — in other words, evolution usually finds the simplest solution that is available to the organism, even if you were hoping for something else.
So perhaps a third lesson is in order: Persistence often pays off, as exemplified by both the LTEE and the elegant and sustained work on evolvability by Barnett, Meister, and Rainey.
Note: I know that many scientists, and especially early-career scientists, are concerned by other issues at this time. However, for many of us, one of the joys of science is to immerse ourselves in thinking about research and education. I offer this post in that spirit.
Telliamed Revisited 