Monday, January 19, 2015

My thoughts on "The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer"

I was on a late Christmas break last week, when I caught wind of a newly published study (here) and associated write ups (best one by Ed Yong here) which suggested that natural transformation and type VI secretion (T6S) were linked in Vibrio cholerae. Given my research interests into microbe-microbe interactions, and my experience studying and writing about natural transformation and evolution, I was naturally intrigued. I was also wary, however, because these kinds of studies tend to oversell correlations and tend towards "just so" stories. Having now read the paper a couple of times, I actually think it's quite a good example of a microbial genetics story and much less so evolutionary biology story.

I won't go into the gory details too much, but the authors start out pointing out that little is known about regulation of the T6S system in Vibrio. The main take home result of this paper is that the T6S system operons are controlled by TfoX and also by quorum sensing through HapR and QstR. That's a solid story and worthy of publication in a pretty high tier journal. However, due to a happenstance of history moreso than anything else, TfoX is also said to be a master regulator of competence for natural transformation in Vibrio. This association arose because TfoX was originally identified as a regulator of competence in the presence of chitin. Looking back in hindsight, maybe TfoX should be referred to as a master regulator of pathways associated with chitin presence.

The authors decide to run with with regulatory association between T6S and competence, and test whether killing of cells by T6S facilitates horizontal gene transfer through natural transformation. As a way to suggest that there is a more evolutionary link between these processes, the authors set up experiments to demonstrate that genetic exchange dependent on T6S killing can occur. For this experiment, the authors test for the ability of their focal T6S wielding strain to be transformed by a kanamycin resistance gene integrated into the genome of another Vibrio cholerae strain. Surprise, surprise (/sarcasm) the experiment works and T6S facilitates genetic exchange. I say that snarkily because ANY process that releases DNA from cells can facilitate horizontal transfer by natural transformation...heat, lightning, whatever you can imagine. My problem here is that the authors place their finger on the scale to effectively rig an experiment whereby they will get the "sexy" result that would be undoubtedly overspun in press releases.  The problem, and this goes for a lot of papers (especially of the microbiome sort) is that just because something is possible under experimental conditions doesn't make that phenomenon evolutionarily relevant.  How did the authors bias this experiment and why am I annoyed enough to hastily craft a blog post?

1) Natural transformation frequency of genomic DNA is highly dependent on similarity of donor and recipient genomes. Transformation by plasmids is a bit different because these don't require recombination. The authors used two (relatively closely related) different Vibrio strains ensuring that recombination could occur. I doubt this experiment would have nearly the success rate (if at all) if different Vibrio species were used as prey. The chances of success fall with genomic divergence from the recipient strain. I have no clue of what the spectrum of other bacteria that live on planktonic crustaceans that would be killed by Vibrio, but the more diverse they are the less likely that T6S truly affects genetic exchange.

2) The type of selection matters. The authors set up the experiment with kanamycin resistance, because they can plate out strains onto antibiotics and strongly select for transformants. Not critiquing that part, and it's certainly how you'd do the experiment, but I'm not sure that such selective environments are representative life on crustaceans or in the ocean. For T6S to have evolved to significantly affect genetic exchange requires a constantly changing environment with strong selection pressures whereby prey strains can be more adapted than predator strains. To this point, laboratory experiments have begun to show that natural transformation can increase rates of adaptation, but generally only in "stressful" environments. It's possible that such conditions could consistently arise for Vibrio, but it's a hard sell.

3) Since T6S preferentially targets dissimilar strains, there is a much much much greater chance that transformation of DNA from prey cells would be detrimental than beneficial. Rosie Redfield has already made the case (here and here) that transformation of DNA from closely related strains is likely detrimental because transformable DNA will contain more deleterious alleles on average than living cells. Additionally, there is always the chance of incorporating alleles that lower the transformation rate and which can't easily be replaced once incorporated. Transformation of DNA from prey cells targeted by T6S systems introduces two related problems. Although transformable DNA won't inherently contain deleterious mutations (unlike Rosie's paper, cells are killed by other cells rather than by deleterious mutations) many of the genes within this pool will be diverged from those in the recipient genome. Therefore, it would be much (much much+++) more likely that predator cells would be transformed by alleles of housekeeping genes that wouldn't function efficiently when placed into a new genomic context than by beneficial genes (here although see here). Is it likely that Vibrio cells will grow equally well if you replace their copy of rpoD with that of Pseudomonas? Probably not. On average then, forgive the lack of a mathematical model but I could whip one up if you'd really like, it is probably much easier to lower fitness of Vibrio through transformation after killing by T6S than to increase fitness. Added to this, analogous to alleles that lower competence in Rosie's model, is that genes that render strains sensitive to killing by T6S will be overrepresented in the transformable DNA pool.

4) Last but not least...I can understand why authors and press releases would be spun to suggest a tight evolutionary link between T6S, competence, and genetic exchange. As Rosie has pointed out, it's a much cleaner evolutionary story to think that predator cells are killing prey for nutrition. Also see her comment on Ed's blog post (here). The authors chose to play up the genetic exchange angle rather than test whether DNA from killed cells could be used as a nutrient. They don't even mention that DNA (and proteins, and a bunch of other things from lysed cells) could be used as a nutrient even though they use the terms predator and prey. Now to bring everything full circle, TfoX is actually the ortholog of Sxy, the gene in Haemophilus influenzae that Rosie's nutrient research is focused on. C'mon folks, at least acknowledge the literature.

So in conclusion, it's a nice genetics story.

Monday, November 10, 2014

We're Recruiting a Graduate Student to Study Microbiomes Inside Fungi

The Baltrus lab is looking to recruit a graduate student to start in the Fall of 2015. We have funds to support this student for at least 2 years, but am very willing to work with students to apply for outside fellowships to cover expenses past this point. Outside of this, the School of Plant Sciences at U of A does have opportunities for TA support on a competitive basis. Students are also encouraged to apply for the ABBS program (here), which provides support for rotations during the first year of graduate school. There's also the chance the grant gets renewed in a couple of years, fingers crossed. As such we're interested in recruiting either a Masters or PhD level student, given implicit funding caveats described above.

The student is being recruited to work on an emerging model system for multi-host symbioses. The Arnold lab (a close collaborator on this project) recently described a phylogenetically diverse group of facultative bacterial symbionts, found within a phylogenetically diverse group of fungal endophytes, found within a diverse group of host plants (here). Yes, even fungi harbor microbiomes! Betsy's lab has also demonstrated that these bacterial symbionts can mediate fungal metabolism (here). To this point, all of the bacterial symbionts are able to be grown and maintained under standard laboratory conditions. We have since established a protocol to cure and reinfect fungi with different bacterial symbionts and have demonstrated that effects on fungal metabolism are 1) specific to the bacteria isolate 2) specific to the fungal host. We have also been able to obtain complete genomes for 12 diverse bacterial symbionts. The new graduate student will work with me to tease apart the molecular basis for symbiotic phenotypes using basic microbial genetics approaches coupled with comparative genomics and transcriptomics.

There are many, many open questions at both evolutionary and ecological levels within this system. I am particularly interesting in setting up laboratory evolution experiments using these bacterial and fungal strains. In addition to the experiments described above, I'm open to helping this graduate student develop new research directions within the context of this system and encouraging of experimental independence. The deadline for admissions to UA is December 15th, and additional details can be found at the Plant Sciences admissions page (here).

If you have any questions, please feel free to contact me by email.

EDIT: even if you don't have questions feel free to email me. It's really important that you get along with your grad school advisor, so set up this connection early!

Saturday, October 4, 2014

CAREER grant post mortem

I received the grant rejection email last week...and waited until today to look at the reviews. For a couple of reviewers I hit everything just right, and for a couple of other reviewers it was the opposite.

I'm not going to go through these line by line, but I think overall I got a fair shot. I could have gone into a lot more detail about what reviewer 2 wanted to see, and I actually have in previous iterations of this grant, but decided to not go to heavy on lots of work on gene duplications. Suffice it to say I grew up as a scientist in the EvoDevo program at Oregon, and my first paper is actually on gene duplications. Will admit to being a bit stung by the "overgeneralization" part, because anyone that knows me knows I am well aware of every nuance...but  in science these days, you win some you lose a lot more than some. The reviewers made good points, I just tried to go heavy on the outreach part of the grant and had to sacrifice some science to make the page limit. I would have also liked to have had some more preliminary data under my belt (KOs of some of the genes and phenotyping), but right now I've got two very capable undergrads working on that for next year. C'est la vie.

So, as a resource for everyone out there:

There were a total of 36 grants in my panel, MCB Genetic Mechanisms. 1 was ranked High priority, 21 (including this one) were ranked Medium priority, 10 were Low priority, and 4 were Non-competitive.

My grant can be found here
Reviews of the grant can be found here

Tuesday, September 16, 2014

My daughter's first birthday

One year ago I was sitting in a hospital room holding my daughter for the first time. That feeling was incredible and she has made the last year of my life indescribably happy. The last year of my life has also coincided with my fourth year as a PI. This blog is a product of me wanting to sit down and reflect on how those two parts have intersected. Everyone will obviously have different experiences, and the experiences will change from place to place, but I wanted to provide a bit of an optimistic view on an academic family.

1) I am my own greatest enemy

Through grad school and my postdoc, I always wanted to work as much as possible. Research felt a bit like a competition and I figured the more time I put in the more I would get out. I wasn't necessarily wrong, but I did get a bit burned out at times. The most difficult feeling I've encountered since having a munchkin is that it's inevitable to feel like you aren't accomplishing as much at work as before. Truth is you probably aren't. I quickly started to view this feeling like I view my own impostor syndrome though...that little voice is always going to be there no matter how much I work, so I might as well be satisfied with what I do get done. Each institution has its own bar for "good enough". My guess is that this bar is lower than the one in my own head. This has by far been the best year I've ever had for publications and grants, and that has to be OK. If it's not, then c'est la vie.

2) Efficiency and saying no are key

I had to be more efficient to survive if I was only on campus for X hours a day. I figured out what I could get done at home, and what I could only get done at the lab. I learned how to work from home and became quite good friends with Arizona's VPN system. While I highly valued walks around campus for getting my thoughts straight, I don't really have time for this anymore and found other ways. I became a much better planner than I was before. Although I spent less time preparing for lectures, I still managed to relay all the information I wanted to (I think). In class, I tried to limit the emotional energy spent on students to a realistic level and set up firm boundaries (no responding to emails after 6PM or before 9AM). I gave more online quizzes to limit my grading by hand. I actually said no to some reviews. There is only so much time in a day and I learned how to cut unnecessary expenditures, because that's what had to happen. You have to make sacrifices, the key is learning and cutting out superfluous actions.

3) Your community is key

I won't go too much into it, but both my wife and I worked full time during this last year and somehow managed to avoid daycare. She is, quite frankly, a superwoman. 

I also wouldn't have been able to survive the last year without the support of my department. They value my contributions, and understand my need for work/life balance (as far as I know, check back in two years when I'm up for tenure). Arizona even started granting paternity leave last year. I don't know how it is other places, but I can only hope that the tide is turning like this. 

I've also been able to survive because I've lucked out and had very good people in the lab. I've tried to embrace an environment where the only metric for success is having a set of goals and hitting certain checkpoints rather than working a specific number of hours. I don't question vacations or time spent with families, and magically enough we still come up with really good data. I fight for them whenever I need to. They know what I expect from them, and I know what they expect of me. I give everyone a lot of rope and foster independence. This certainly doesn't work for everyone, but it's the only way I can survive as a PI. 

4) Sleep is not overrated

When there is an infant in the house, your sleep patterns go out the window. This translates to a lot more spelling mistakes in documents (and lectures!) and a much lower tolerance for the everyday BS you deal with in academia. My advice is to proofread documents first thing after sleeping or a nap (or get others to proofread). My other advice is to try the best you can to avoid saying things you shouldn't while sleep deprived, even if you're teaching a class of 100. You'd be surprised how some of your comments are actually interpreted by the students, so it's better to just shut up. 

5) The fringe benefits of a munchkin are awesome

Research is hard. Our lives are filled with rejection day in and day out. There is no better cure for rejection emails than coming home and playing with my daughter. Absolutely nothing. Her little smile puts everything else into perspective.


Wednesday, August 27, 2014

Bacterial Genome Size and Ecology

 I often find myself wondering about general evolutionary pressures that shape bacterial genome sizes, and I'm going to use this space to try and crystalize some thoughts. Part of this is motivated by my interest in understanding how horizontal gene transfer affects adaptive trajectories (see here), and part is motivated by trying understand how to define (and what actually structures) bacterial populations in the context of ecology and selection (see here). In the latter case, Monod's famous quote doesn't necessarily hold true...if you wanted to define an elephant population you could go out and count them. This is sadly getting easier and easier every day. For bacteria, you can't go out and count total number of cells because micro-environments matter and all cells don't experience the same selection pressures. Chemical, geological, and biological gradients are much coarser for elephants than E. coli and this can be reflected in population subdivision. These kinds of questions don't really matter if you care solely about presence/absence of organisms...but if you want to try and predict evolutionary dynamics (strength of genetic drift, etc...), you have to understand what defines population size. This whole introduction is just a long winded way of introducing an interesting idea that has popped up across a couple of papers and lately in discussions I had with Steven Nayfach (from Katie Pollard's lab) over sushi. Can we use differences in average bacterial genome size across environments to say something about microbial ecology?

Bacterial genome size vs. number of annotated genes, from Wikipedia

Small Population Size + Host Association = Small Genome

Obligate microbial symbionts often have tiny genomes compared to free-living ancestors. This is due to the absence of purifying selection on genes no longer necessary within this symbiont lifestyle, an increase in effects of genetic drift due to small population sizes, and a slight deletion bias in mutations throughout the genome. Basically, when genes are no longer necessary in small populations they can accumulate and fix more mutations randomly, and these mutations tend to biased towards deletions. When vertical transmission is assured, all genes necessary for survival outside of this transmission cycle become superfluous. We see parallel increased rates of gene loss and inactivation (and overall smaller genome sizes) in some free-living bacterial pathogens as well with similar population size / relaxed selection explanations. For these cases, genetic drift is a key factor.

In terms of defining ecology, if you find a particularly small genome in your sequences with lots of pseudogenes, you might be able to a priori guess that this bacterium has particularly low effective population sizes and may be a parasite.

More DNA is Costly = Selection for Small Genome

Although patterns of genome evolution in symbiotic bacteria are likely driven by genetic drift, there are cases where selection appears to directly drive genome minimization. The best known example of this is referred to as "genome streamlining", and is seen in a wide variety of oceanic bacteria including the notorious SAR11 clade. These genomes are typified by a reduced but highly conserved core gene repertoire, a reduction in paralogs, and a reduction in intergenic spacer regions. Non-mutually exclusive explanations for such selective pressures include low Nitrogen and Phosphorous levels within the ocean (making extra DNA energetically costly) as well as optimization of cell surface to volume ratios. The cell surface / volume ratio theory is particularly interesting because it parallels discussions of genome size evolution all life (termed the C-value paradox). How are cell size and DNA content related...well, DNA takes up space and the more DNA in a genome the larger the cell size. An aside: there's scarce evidence that DNA replication is costly for bacteria across a variety of other environments where transcription and translation are thought to be the most costly processes.

So if you find a particularly small genome in your sequences (regardless of environment) with little evidence of genetic drift (low number of pseudogenes and low mutation fixation rate amongst core genes) it might be evidence of selection acting on genome size. This could in turn indicate competition for a scarce nutrient that makes up DNA or necessity of transport across cell membranes.

Evolutionary Correlates of Larger Genomes

It's possible that increased genome size can be selected as a correlate of cell size. I don't know of any cases where such selective pressures have been directly demonstrated in bacteria, but the correlation between DNA content and cell size certainly appears to hold true. That's not to say that there are other emergent ecological properties that could also select for larger genome sizes. As long as DNA isn't too costly (an important caveat), in variable environments where cells must be capable of metabolizing a wide range of compounds, genome size can increase as additional metabolic pathways are acquired through horizontal gene transfer (here and example here). These extra pathways can keep accumulating as long as they aren't selected against too strongly (which, you guessed it, is going to be dependent on population size). Just a correlation at this point as far as I know, but many "soil" bacteria have relatively large genomes: pseudomonads, Burkholderia, assorted Rhizobia, etc...*

It's also possible that emergent evolutionary properties will arise as genome size passes a specific threshold. Since the success of long-distance horizontal gene transfer increases with genome size (that's a bit circular, but them's the data....could also be confounded by observation bias and correlated to environmental proximity), but it's possible that free-living bacteria with larger genomes undergo fundamentally different evolutionary dynamics than free-living cells with smaller genomes. Likewise, cells with larger genomes appear to grow more rapidly than those with smaller genomes. This might be due to number of ribosomal operons but also to the presence of multiple large secondary replicons in bacteria with larger genomes (the more replication forks there are, the faster total genomic content is replicated). Bacteria with larger genomes might also be able to better tolerate secondary replicons like megaplasmids, which may again fundamentally and qualitatively shift phenotypic and genotypic evolution (here and here). We like to think that everything that is true for E. coli is true for Pseudomonas, I'm not so sure given possible evolutionary feedback loops that are correlated with genome size. For instance, you see a lot more megaplasmids in Pseudomonas.*

Conclusions

I'm definitely missing some citations and angles on this, so please feel free to point me in any relevant research direction. It's an interesting idea to imaging extrapolating ecological data and evolutionary trends from differences in average genome size across microbial populations. There are a couple of papers I've stumbled into that try and to just that. There are probably a lot more out there...

Wednesday, July 9, 2014

Flexibility

"Everyone has a plan, until they get punched in the mouth" - Mike Tyson

The best thing you can do when preparing to transition from postdoc to PI is plan out 5-year research goals. Talk with your postdoc advisor about what projects are yours, think about what questions you are interested in, design experiments to test these hypotheses, and make lists of every construct you need to create or reagent you need. Even though everything around you will be moving quite quickly, there will actually also be some time as you are setting up the lab to just sit and think. We often get asked, if there were unlimited funds, what kinds of experiments would you perform? Starting a lab with a pool of undesignated money (startup) will likely be the closest you will come to this "do any experiment you want" utopian world. I didn't realize the full weight of this until my startup ran out, but having a pool of money for which you don't have to specifically justify each experiment a uniquely powerful situation.

It's great to hit the ground running with a definitive plan in hand, but always keep in mind that the real world can intervene. Many people I've sought advice from over the years have suggested the importance of having multiple lines of research within the lab at any given time. Don't be wed to a single question or system, especially in times like now when funding is tight. Keep reading and don't be afraid to try new assays or experiment with different systems. The early years of your lab, startup money in hand, may be the best/easiest time to branch out and ask completely new questions. However, the flip side of having multiple irons in the fire is that juggling experiments requires a skill not easily learned. While it can be quite easy to dream up the "next" experiment, it's often difficult to know when it's time to pull the plug on failed projects. Sometimes it's just a gut call. At least IMHO, knowing when to stop a particular line of research is one of the most intrinsically important skills for being a PI.

I started my lab with ideas for "easy" projects that would be straightforward extensions of postdoc experiments. After moving from North Carolina to Arizona, I realized that my bacteria and plants didn't behave the same in the dry air of Tucson as they did in soupy Chapel Hill. It was frustrating to say the least, and I was stuck with the decision to slog through and figure out a way to carry out these experiments or to cut bait and try a new direction. I moved the plant based experiments somewhat to the backburner, which is a bit tricky because I'm housed in the School of Plant Sciences, and decided to focus on investigating interactions between microbes. We just started reading papers and trying stuff, building off of research interests shared across all lab members. Looking back (over the last four years), there have been a lot of starts and stops, but I'm quite happy at how things are turning out. There are still experiments that I know I could get to work given more money and time, and strains sitting in my freezer for experiments I haven't come close to trying yet. These side experiments fail much more frequently than they work, but you have teach yourself to do the cost/benefit analyses to know the difference between when to stick it out and when to move on.

All of this in mind...a brief sidenote. As I mentioned above, you will never be as free to experiment as when you have startup funds. The tendency can be to bring in people (technicians/postdocs) to carry out exact experiments written down in your 5-year plan. Although this may work in many situations, I made a conscious decision to do the opposite. I tried to hire independent postdocs whose interests overlapped with mine, but who wanted to branch out into completely new research directions within the context of my lab's interests. At the outset I had no clue how this would go, and there were certainly some nervous moments. Looking back, I can honestly say that this plan worked out about as well as possible. We have developed numerous new research directions and both postdocs have also contributed greatly to more "basic" projects. For the young PIs, don't be afraid to leap in directions that are uncomfortable because you might just find yourself in interesting new places. For the postdocs, given the lack of funding opportunities, don't be afraid to find your way into the labs of young PIs. They will often have freedom to spend money whatever way they chose (unlike if you are brought in on a grant), and you might have a greater chance of developing your own independent research programs.

Disqus for http://mychrobialromance.blogspot.com/