Evolution by gene loss

Sometimes it’s amazing just how little we know about the microbes around us. For precious few microbes, we know a good deal about virulence factors–genes and proteins that, when present, increase the severity of disease either in animal models or in humans (or both). However, much of this research has been done investigating acute infectious diseases, where one is infected, becomes ill, and gets better in the course of a few weeks to a month. Much less is known about factors that affect long-term (or chronic) infection. A recent study addressed one gap in this research, examining what happened in patients with cystic fibrosis who were chronically infected with the bacterium Pseudomonas aeruginosa.

Cystic fibrosis (CF) is a genetic disease that currently affects approximately 35,000 Americans, with about 1,000 new diagnoses every year. The average lifespan of CF patients is currently around 30 years; patients eventually succumb to the chronic lung infections that they suffer from during much of their lives. Common pathogens are Burkholderia cepacia and Pseudomonas aeruginosa. Both are gram negative soil bacteria. In humans, they’re opportunistic pathogens, infecting those with compromised immune systems. Both of these species are notoriously difficult to treat, having high levels of antibiotic resistance. In this study, they examined the changing population of P. aeruginosa in chronically infected cystic fibrosis patients.

The basic question they asked was this: how does the bacterial population evolve during the months and years of chronic infection?

Though P. aeruginosa is common in CF patients, studies have found that patients generally acquire the bacteria from environmental reservoirs, rather than other patients. Therefore, each patient will start out with a “wild type” isolate, adapted to an environment quite different from that found in the lung. In this study, Smith et al collected an isolate of P. aeruginosa early in a patient’s infection (6 months) and then a second 96 months into the infection–then they sequenced both of them and compared the two, detecting 68 mutations, including one that removed 188 kb of DNA (~139 genes). Approximately a quarter of the other mutations caused a stop codon, a frameshift mutation, a transposon insertion or a gene deletion. A number of genes previously reported to play a role in virulence, as well as antibiotic resistance genes, were also mutated. Interestingly, the mutated genes caused the 96-month isolate to have reduced biofilm formation. This was unexpected, as it’s been thought that biofilm formation was one reason that P. aeruginosa was such a successful pathogen in these types of infections.

Additionally, while some antibiotic resistance genes were mutated, the 96-month isolate still had increased resistance to several aminoglycosides. (This was as expected, as the patient had received this class of antibiotics). It was also found to have a mutation in the mutS gene, which encodes for a protein involved in DNA mismatch repair. Mutations in this gene, therefore, result in an increased mutation rate–again, this fits with their other results.

Interesting stuff by itself, and then they toss in a bit of extras. They also looked at an isolate collected from this patient at 30 and 60 months into the infection to see if the mutations in the 96-month isolate were present (and if so, how many). They found almost half of the mutations (26/68) in the 60-month isolate, and half again (13/26) in 30-month isolates. They also discovered several mutations that weren’t present in the 96 month isolate, allowing them to get an estimate of heterogeneity in the population at that point in time. They note:

Viewed together, the known mutations in patient 1 isolates indicate that parallel lineages existed during the early years of the infection and coexisted, in some cases, for several years. For example, mutations in mucA commonly occur during CF infections, and mucA mutations arose independently in three different lineages during the patient 1 infection. Isolates from a single time point are also heterogeneous, e.g., five of the six isolates collected at 36 months had detectably different genotypes.

Again, interesting, but are the mutations unique to this individual patient? Or are some of them common to other CF patients who acquire P. aeruginosa? That was their next question, where they examined other P. aeruginosa isolates from 29 additional CF patients that had matched P. aeruginosa isolates (from early infection to late infection). In these isolates, they sequenced 24 of the genes that were mutated in patient 1’s isolates, along with 10 genes that other studies had shown were frequently mutated. Most of them weren’t found to have a large amount of mutations, but they did find a few that were hotbeds of change. In these, mutations were selected for: 5 synonymous (silent) mutations were found in these genes, versus a whopping 103 non-synonymous mutations (that is, mutations that change the amino acid sequence of the encoded protein). They again saw a high number of loss-of-function mutations in these genes as well.

One limitation of this part of the analysis, however, is that they were starting from the genes that had already been identified in patient 1’s bacteria. It’s likely there are other genes mutated in these additional patient isolates that weren’t found in the isolates from Patient 1, so they’re missing some measure of the diversity (and, therefore, missing some genes that may play a role in this transition from acute to chronic infection). What would have been ideal would be to have the complete genetic sequences of all isolates–which, for now, remains cost-prohibitive.

A fascinating portion of this study showed that, in these chronic P. aeruginosa infections, many virulence factors were selected against. Now, virulence factors are generally called as such because they’ve been shown to be critical for a pathogen to establish an infection, or because when they’re lost, the lethality of the pathogen decreases substantially, as I mentioned above. But these analyses are generally based on acute infections, where infection is established (often by an artifical means, such as injection) and then either resolves, or the animal dies. As I mentioned in the first paragraph, much less is known about factors which are important for chronic infections. So-called “antivirulence factors” have also been identified: genes that, when knocked out or mutated, make the organism more virulent instead of less virulent. (Another example I’ve described previously is a mutation in a regulatory gene in Streptococcus pyogenes, where a frameshift mutation increased the virulence of isolates which possessed it). These are nice examples of how an organism can evolve due to loss-of-function mutations. Creationists like to crow about how “information can’t be added” and suggest that evolution is uni-directional, as if proceeding up some ladder. Of course, that’s simply not the case. In addition to this study, there are many examples of a population of organisms evolving–and losing unnecessary genes along the way. Carl Zimmer (in his new digs at Scienceblogs) gives two more examples, describing how a plant microbe and an animal pathogen (another species of Streptococcus, as a matter of fact) lost genes for functions that were no longer necessary for their new life in yogurt–genes encoding proteins responsible for metabolizing some sugars, or genes associated with virulence. Examples like this are so common that a new ASM book, Evolution of Microbial Pathogens, devotes an entire chapter to the phenomenon. How sad that all of this is dismissed as “mere microevolution” and waved away by creationists–they’re missing all the good stuff.


Smith et al. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. PNAS. 103:8487-8492. Link.

Image from http://www.ikp-stuttgart.de/ps-aerug.gif

13 Replies to “Evolution by gene loss”

  1. I would love to know what would happen if you put the mutated or long-term infection version of P. aeruginosa back in the dirt. Would any survive? Would the genetic changes reverse or find brand new pathways?

  2. “Examples like this are so common that a new ASM book, Evolution of Microbial Pathogens, devotes an entire chapter to the phenomenon. How sad that all of this is dismissed as “mere microevolution” and waved away by creationists–they’re missing all the good stuff.”

    I think you’re missing the point. Creationists (myself in particular) do enjoy this stuff and agree that this _is_ “the good stuff”. What Creationists (myself in particular) don’t agree with is that this has anything to do whatsoever with large-scale evolutionary theory. When Creationists are against “evolution”, it’s not that they don’t think that variation is a hugely interesting subject worthy of great amounts of attention. Instead, it’s that there is a fundamental difference between what is being observed in evolution and what it is being claimed of being capable of doing.

    In fact, Creationists believe in a much faster evolution than do evolutionists, precisely because it is a constrained change rather than a haphazard drift.

    I think perhaps you grew up on a view of Creationism that was focused on being “anti-evolution”, rather than persuing positive research programs. Surely there have been and are now many people in the Creation movement like that. However, I don’t think you should judge the Creation movement based on its expression to and by lay people any more than Creationists should judge evolution based on its expression in high-school textbooks and by high-schoolers. I think you’ll find a different story when talking to biological researchers in Creationism.

    You should check out the proceedings of the latest BSG conference and see if perhaps Creationism from a research perspective is different than what you had assumed.

  3. “What Creationists (myself in particular) don’t agree with is that this has anything to do whatsoever with large-scale evolutionary theory.”

    This is exactly what Tara means – the creationists preconcieved notions blocks them from seeing the information and beauty that the scientists see.

    “In fact, Creationists believe in a much faster evolution than do evolutionists, precisely because it is a constrained change rather than a haphazard drift.”

    The rate of change is measurable, not a matter of belief. (Though I gather there are difficulties.) Since the local selection in an RM+NS model is constrained to find incerasing fitness, your description is in error. Isn’t even neutral drift selected for? Likewise I think evolution rate is selective, which your haphazard drift can’t explain.

    Yes, creationism is different.

  4. BTW, Jonathan, since Tara is doing research which concerns biology, don’t you think it is rather besides the point to refer to ideas that can’t pass peer review in biology?

    One current relevant discussion here is http://www.pandasthumb.org/archives/2006/06/laudan_demarcat.html which is looking at whether creationism is bad science in the sense of Laudan or pseudoscience in the common sense. The basis for the discussion is its vacuity, as demonstrated by the lack of results for example.

  5. “You should check out the proceedings of the latest BSG conference and see if perhaps Creationism from a research perspective is different than what you had assumed.”

    Uh, no. The entire proceedings are a succession of ad hoc arguments, based on compatibility with the Bible. No original experiments or observations. Sounds like creationism to me.

  6. Larsson —

    “The rate of change is measurable, not a matter of belief.”

    To some degree this is true, but not entirely. First of all, there are different kinds of change. Erwin and Davidson pointed out that different kinds of changes are of a fundamentally different kind, and some of the ones required for evolutionary theory are completely absent from modern observation. Therefore, to extrapolate large-scale changes requires belief in a _kind_ of change that is currently unobserved.

    Gene regulatory networks and the evolution of animal body plans

    Likewise, in light of a decaying genome and a relatively stable set of current environments, we have a decreased amount of change because (1) the change mechanisms have degraded and (2) the environment is no longer as extreme as it has been historically [as well as potentially other factors].

    “Since the local selection in an RM+NS model is constrained to find incerasing fitness, your description is in error.”

    But this is begging the question. You are assuming that localized RM+NS can actually find ever-increasing targets. Modern conceptions of mutation are going away very fast from random mutations as a substrate of evolution. The problem is that this is a giant road sign for teleology. Two good papers to read are:

    Chance Favors the Prepared Genome
    A 21st Century View of evolution

    frank —

    “Uh, no. The entire proceedings are a succession of ad hoc arguments, based on compatibility with the Bible. No original experiments or observations.”

    Several points:

    (1) There were many original observations.

    Art Chadwick, one of the plenary speakers, gave a talk about the dinosaur bone digging he is personally in charge of. He is digging out a dino bed, and using high-precision GPS locating techniques to create a high-resolution map of every bone excavated.

    The talk on adaptive radiations was based the speaker’s personal first-hand research of island floral species.

    The talk on the Green River Formation was based both on the authors’ personal visits and experimentations as well as on secondary research results. Some of the author’s previous work on the area has been presented at the GSA.

    There were several taxonomic studies as well, which, while using published datasets, used statistic analysis methods which were developed by a Creationist and have been put to use by an evolutionist and published secularly.

    I also think it is naive in the highest degree to think that global studies of stratigraphic tendencies such as presented in Kurt Wise’s talk could be obtained by primary research alone.

    (2) Compatibility with the Bible

    Personally, I have trouble seeing what the big deal about this one is. Why is it controversial to use a historical primary source in hypotheses about history? A fuller answer along these lines can be found here.

  7. “In fact, Creationists believe in a much faster evolution than do evolutionists, precisely because it is a constrained change rather than a haphazard drift.”

    “Constrained change”?

    What precisely are these “constraints”?

    What precisely are the forces which ensure mutations do not break through these “constraints”?

    Where precisely in nature is the evidence which would demonstrate mutation conforming to these “constraints”?

    If evolution is a “constrained change”, then surely you should be able to tell us exactly what those constraints are and where they came from, right?

    Now, a hypothetical question: Let us say that a researcher (for example, someone such as the disease researchers analyzing P. aeruginosa in this article; or the persons who analyze the effect of gene deletions on an organism in the ASM book) observes the development of a mutation which in all measurable ways appears to be an example of a mutation violating the “constraints” you refer to above. What would be the proper way for the researcher to respond to this evidence?

    In what way would it improve the reliability of the scientific process if the researcher who encountered this evidence “believed” in the constraints on evolution which you refer to?

  8. Extremely clearly written summary of a very interesting article. Thanks for this.

    Especially nice that you point out the lack of understanding we have of chronic infections. There are some models out there — for example, chronic infection of tuberculosis in mice, a great system in which one can do genetic screens in the parasite and also have genetic access to the host. (For example see Glickman et al., Mol. Cell 2000.)

    But it is fascinating that the presumption that acute virulence factors will be the same as chronic virulence factors is turning out to be wrong.

  9. Tara,

    I heard one of the authors (Green) give this talk at ASM. He raised the idea that virulence factors should be divided into two classes:
    1) colonization/establishment loci
    2) persistence loci

    I think if you think about it that way, the loss of genes isn’t so surprising. We can think of this as a genetic basis for niche differentiation (although it remains to be seen if the evolved forms can recolonize another host).

  10. Greetings, Tara, thanks for sharing this!

    Is it possible that the genetic systems of P. aeruginosa (and other bacteria) have the ability to generate solutions to environmental pressures by triggering mutations at optimal points rather than relying on strictly random mutations at undirected points? Could a challenge from the environment trigger an optimized, focused “search” rather than a blind, global scan for a useful metabolic solution? Organisms that evolved such an ability in primordial times would clearly have been rewarded by natural selection with a veritable “thumbs-up”. Call it a primitive form of “immunity”-if you will. If larger organisms’ systems can mount “directed” responses to challenges (immune ones, for example), why not bacteria?

    Research should be mounted to determine differential rates of mutation in various parts of a subject genome (if it has not been started already). If distinct patterns of change are found, then chances are there is a mechanistic reason-that there are intrinsic genetic algorithms at work in these genomes that bias change towards more (or enough) productive mutations to keep the host bacteria “ahead of the curve” in an evolutionary sense.

    Perhaps microevolution as a whole is directed in part by still-poorly understood genetic forces latent in the DNA of organisms.

    Best regards,

  11. apollo230, a controversial phenomenon called “directed mutation” has been described that is what you’re thinking of. The phenomenon observed was that under stressful conditions (starvation for a particular nutrient), mutations arose in bacteria MORE OFTEN in a gene that would help them to survive that starvation than in a CONTROL, unrelated gene. The interpretation was that the bacteria somehow were “directing” mutation to the genes that needed it, rather than experiencing traditional (Darwinian) random mutation.

    This interpretation was later refuted by an additional observation: that the gene that was getting mutated was also being amplified many times. Thus, the new interpretation was a two-step model: (1) starvation selects for random mutation including gene amplifications where the relevant gene is duplicated many times, (2) random mutation occurs equally throughout the genome, but because the relevant gene is present in very high copy number, more mutations “hit” some copy of the amplified gene than hit a control gene (which is still present in single copy).

    If you think of it as a card game where you’re trying to pull out an Ace, first there’s a selection for stacked decks that have extra Aces, then a card is pulled at random — and of course often, it’s an Ace.

    This two-step model has been expanded on in a study that showed that mutation can also occur BEFORE amplification — that being starved induces “hypermutation” in which the overall mutation rate increases, and this hypermutation state can PRECEDE the amplification (or follow it).

    So the current thinking is that in a way you are right — there is evidence that stress conditions can trigger a program in a cell that increases the mutation rate. But I’m not aware of any evidence that says that the cell “knows” which genes to mutate or that the mutations are directed to the “right” gene — rather, the elevated rate of mutation is spread evenly around the genome, and only appears biased towards a single gene if that gene is present in many copies.

    hope that helps!

  12. How about evolution happens when an organism is plastic, doofae? Like in utero (eggsack), like flies exposed to ether, or high heat? How about taking that genome and its magical deletions out of the equation as the engine of change, and rather place it as the recorder of alteration?
    Environmental change=species change.


    (Send Nobel Prize to “flicky@the Dunderbunders, Whiley-on-the-Frem, Dunnyhill Manor 67458”)

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