Thursday, February 4, 2016

We tend to be harshest those we love...

Last year a paper was published investigating links between natural transformation and type VI secretion mediated killing, and I cobbled together a blog post with some select thoughts about this paper (here). It's recently come to my attention (should have written this earlier, it's my fault that I dropped the ball) that that particular post could be viewed in a couple of lights depending on your the context. I want to make it completely 100% crystal clear, that it is never my intention in writing in this space to include ad hominems in posts (I'm not Dan Grauer...)*. I write quickly and don't ever really sit on posts, but really I'm writing here because I love what I do and I care for certain topics in science. I'd like to see these topics understood as clearly as possible. For those that "know me", I really enjoy critical discussions about science and sometimes my words get ahead of my inner filter. Suffice it to say that I've made a conscious decision to try and not use this space to trash people and papers personally, but to be critical about science involving topics that are near and dear to my heart. Sometimes when I do this my inner New Yorker comes out, but know that my intention is not to critique the person but to focus on the science. If I do this, let me know and I'll directly tackle/ammend/try to fix the issue in public.

Figure 1. An example of me responding a little quickly in public to a recently published paper

This being said, I'm writing this post because another paper was published recently that linked together natural transformation and microbe-microbe killing. Honestly, I haven't had the chance to read the paper yet because I was sitting in airplanes all day yesterday. I'm guessing that I would probably have similar thoughts as I did about the type VI secretion paper last year. These thoughts come out in public sometimes:

Figure 2. An example of me responding to a recently published paper after sitting on airplanes all day and without reading the paper

All right, so what's my beef with these studies? Let me be completely clear, I respect the authors and inevitably I have no problem with the experimental design or the actual reported science. In the type VI secretion case, and probably with this new paper, I have absolutely no problem with the experimental design or with the genetics. I don't think the papers are wrong science-wise. I'm writing about these papers because I spent the better part of 5 years in graduate school huddled in the fetal position thinking about the evolutionary effects of natural transformation in bacterial populations. My problems with a lot of these papers are usually directed solely at the evolutionary interpretations and spin within discussion sections and press releases.

There's a historical legacy that surrounds researchers of natural transformation in bacteria, where there are a couple of entrenched camps that tend to argue past each other. These fights usually flare up around disagreements that conflate questions about original evolutionary benefits of natural transformation and benefits of natural transformation that are measurable today (after these systems originally evolved). After many years of thinking about this, I'm actually agnostic when it comes to the original evolutionary scenarios for natural transformation. Rosie Redfield is one hell of a thinker and I defer to her about such things (so I guess that firmly places me within one camp). I tend to be more interested in wondering about how strong the selective forces on natural transformation are within present day bacterial populations and on trying to figure out realistic parameters that could affect our evolutionary interpretation of gene exchange in bacteria.

Like I said above, I think the genetics and molecular biology within these papers are tip top and have absolutely no quarrel with those. I get caught up when the discussions start to extrapolate from  the controlled conditions of the lab environment into natural populations. Natural transformation certainly leads to gene exchange in natural populations of bacteria. However, suggesting that pathways are linked in regulation because of evolutionary benefits of natural transformation is a leap of faith that no paper out there has been able to tackle as of yet. What leads to the disconnect? There is a long standing tradition across many, many, many papers that describe evolutionary "just so" stories whereby we witness results in the lab or under certain conditions and think/assume that natural selection must act that way across many different conditions or environments. My comments about "hand waving" are usually directed at such extrapolations.

This is getting long, so I'll save the nitty gritty for another time. However, long story short, these extrapolations hinge on critical parameters of these experiments being similar in the lab and under natural conditions. There are no natural populations of bacteria for which we have realistic estimates of things like A) the DNA pool available for natural transformation B) natural selection pressures over space and time within and between bacterial populations that are exchanging DNA C) the repeatability and direction of these selection pressures D) how often cells encounter other cells that they can kill in nature E) having killed these cells in nature, how often these cells take up DNA F) evolutionary costs of natural transformation in nature G) I'm missing something because it's early in the morning but there are other parameters. When one sits down to write mathematical models that account for all of these above parameters, it ends up being REALLY difficult to find parameter space whereby natural transformation is GENERALLY beneficial. That's not to say that gene exchange doesn't matter within natural populations (as it certainly does) but it's hard to find situations where there are clear results where natural transformation is beneficial even a majority of the time. Under laboratory experiments like the ones in the type VI secretion paper (and probably the bacteriocin paper, again, haven't read yet) all of these parameters are actually controlled for pretty cleanly:

A) the DNA pool is controlled by the experimenters so that there is no contaminating DNA from other strains/species that could compete with genes of interest for uptake

B) Natural selection is really strong because these experiments are typically selecting for antibiotic resistance where cells pick up the relevant DNA survive and those that don't die. The same would be true if we experiments were set up to investigate phage predation, etc...

C) Typically in these lab experiments, there is only one direction for selection to act and the environment doesn't change over time (i.e. there is only one antibiotic that the cells need to become resistant to, and therefore one locus that they need to pick up through natural transformation)

D) lab experiments are usually biased so that cells are encountering cells that they can kill at pretty much optimal frequencies (50/50).

E) lab experiments are done under conditions whereby cells are highly competent for natural transformation

F) there are few costs for natural transformation systems in these lab experiments because the experiments themselves usually occur in relatively cush situations for bacteria (media containing abundant nutrients, controlled temperatures, etc...) and only take place under limited amounts of time. In fact, for most bacteria, if you passage them under lab conditions for extended periods of time they usually lower competence levels (which suggests an evolutionary cost). Like I said though, the lab experiments are only performed over limited amounts of passage time.

So to sum this all together, I apologize for any perceived slights. That's not my intention (yeah, I know get out the bingo cards). If you feel I've been too personal, please let me know and I'll try and fix anyway I can. There are many great groups focused on understanding natural transformation in bacteria and I respect much of their work. I've just spent way too much time worrying about evolutionary scenarios that usually pop up in discussion sections of these papers without (what I perceive) is firm grounding within evolutionary biology. These papers usually aren't usually set up to be direct tests of evolutionary theory, but it's very easy to write about how we think evolution should work. These papers usually end up being very good at describing the if natural transformation works for gene exchange under certain scenarios rather than how it's actually happening in nature. That's completely OK, just be careful about extrapolating.

*c'mon, that one was way too easy


  1. I understand the fast comments part (I do tend to tweet sometimes before thinking), though it is not an excuse to the fact that blog commentaries are not PubMed listed, thus not read by all. No idea how to change this point other than contacting directly the authors...

    1. Just need to start posting links to Pubmed Commons. I don't do the paper critique thing much, but was lazy about posting link to this one.

  2. Thanks for your thoughts.

    1) I certainly agree with a lot of what you wrote and especially that lab experiments do not fully recapitulate what's happening in nature. But that’s not only the case for our paper (T6SS/transformation/V. cholerae) but true for almost every single paper out there including those that address biofilms, quorum sensing, T6SS, in vitro evolution, microbiota, CDI, host-pathogen interactions in cell cultures, animal models of disease, and you name it. But if you want to understand the whole world you have to start small.

    2) There are 10^4 Vibrios/ml ocean water (and the competence regulators / network of V. cholerae is conserved in most if not all Vibrios). Thus, if we measure a transfer frequency for a gene between two strains of 1 in 10^4, this would mean roughly one transfer for a given gene per ml. Now, even if these lab conditions are artificial and transfer "in the wild" might not occur in 1ml, it certainly will happen if we take the whole volume of the oceans into consideration. There are a lot of Vibrios out there!

    3) What is transformation good for? I did not subscribe to any of the 3 camps (DNA for HGT, DNA for repair, DNA for food) and I don't think the options are mutually exclusive. And transformation might very well be selected for because DNA is used for food as Rosie has been suggesting for many years and I very much respect her (not citing her in this one specific paper is based on the strict journal rules concerning numbers of references and the details + references on T6SS that were asked for by the reviewers and editor while revising the paper => remember, it's not a review on competence). But even if HGT is "only" a side-effect it will happen (and protecting ssDNA inside the cell from degradation + active methylation of the incoming DNA in some species, does hint towards integrating the DNA versus just chopping it up for breakfast).

    4) Concerning: "Natural selection is really strong because these experiments are typically selecting for antibiotic resistance where cells pick up the relevant DNA survive and those that don't die." => the experiment itself was done without ANY selection pressure (e.g., just growing strains together on a chitin surfaces in seawater medium). So there is no benefit of having the resistance gene throughout the experiment and the bacteria without the gene grow as well as the ones with the resistance gene (or even better). Only the enumeration afterwards is based on counting the total number of bacteria versus those that have incorporated the new resistance gene. So no enrichment of transformants in the actual experiment.

    5) For T6SS-mediated killing followed by gene transfer there might actually be an immediate selection advantage, namely T6SS toxicity and immunity. Indeed, the genes encoding the T6SS toxins and immunity proteins move horizontally in Vibrios (Unterweger et al., 2014 and Salomon et al., 2015). Thus, acquiring new toxin genes can provide a direct / immediate fitness advantage (probably at the cost of losing the old toxin+immunity genes). But I am sure that several labs are working on this right now and we will hear more about it in the future.

    Anyway, thanks for sharing your evolutionary thoughts and for stating "I think the genetics and molecular biology within these papers are tip top". Friendly open discussions are always welcome and I am happy to learn new aspects.
    However, comments like "How did the authors bias this experiment and why am I annoyed enough to hastily craft a blog post?" / "Surprise, surprise (/sarcasm) the experiment works and T6S facilitates genetic exchange." are personal and inappropriate.

    1. Thanks very much for commenting here. I wasn't sure how to change the original post given the last comment (I don't just want to delete), but I've included an apology and acknowledgement that I was wrong in tone.

  3. Thanks. I very much appreciate it.


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