I spent a good part of my time as a postdoc figuring out how to sequence microbial genomes cheaply, and learning how to deal with the flood of data that ensued. After my first post on this project, I was asked a very good and relevant question by Olin Silander...specifically, why not just spend a couple of hundred dollars and sequence the interesting brown mutant rather than go through transposon mutagenesis? I'm guessing that if you're reading this and are somewhat up to date on modern microbiology, you probably have the same question. Here's a couple of reasons that I came up with with varying levels of importance:
1) I'm a sucker for old school genetic screens. To be honest, it's fun coming into the lab and checking whether cultures are brown or yellow. There's so much about being a PI that can stress you out, and that feeling of "I got one!" is one little simple pleasure that I love about research. Agar plates, media, and bacterial conjugations are cheap...and I've got a very talented recently post-grad summer student that's helping me out with this experiment to ease the time investment so it's really not a big waste of anything.
2) It's possible that sequencing and transposon mutagenesis will yield different results. Whereas sequencing will likely tell me exactly what the mutation is, it's possible (maybe likely given that my gut is telling me that the brown color is a product of overproduction or disruption of negative regulation) that the exact mutation yielding the brown phenotype is in some regulatory region/protein. However, I'll admit that it is equally as likely at this moment that the brown phenotype is due to disruption of an enzymatic pathway and that what I'm seeing is the buildup of an intermediate product that is getting oxidized. IF the mutation is regulatory though, the transposon screen will likely yield disruptions in the pathways producing the brown product and that act downstream of the initial mutation. With only the sequence of a regulatory mutation I could be left guessing what pathway was being affected and what the next step is.
3) Transposon disruptions give me knockout mutants, and I can directly verify cause and effect of the transposon disruption on knocking out the brown coloration by transforming these loci back into the original brown mutant. If this ever is publishable, I need to be able to demonstrate genetic causation of a phenotype and natural transformation of the brown mutant with an antibiotic resistance locus present within a transposon is slightly easier than the molecular biology gymnastics necessary to recreate the original brown mutant in a wild type background. Additionally, more phenotypes (in this case disruption of the brown color) are never bad things.
4) This one is a little inside baseball...but I'm teaching upper level microbial genetics next spring with a lab (about 80 students in the lab). As part of this lab, I want to have the students perform transposon mutagenesis and go over basic ideas like screens, selections, phenotypes. This little project gives me the opportunity to fine tune this procedure in P. stutzeri using an easily scoreable visual readout and provides a good base for figuring out what experiments I can have the students perform next April.
5) I'm going to sequence it:) However, given how much of an overkill it is to sequence bacterial genomes using Illumina's HiSeq technology, I'm waiting to collect 23 other bacterial genomes to sequence along this one (I've got about 17 right now) in a single lane. I'll be blogging about those results too, so don't worry.
Tuesday, July 10, 2012
Friday, July 6, 2012
Being a PI can be stressful for a variety of reasons, but I love my job as a researcher. There is no greater thrill for a curious mind than asking questions, designing experiments, and figuring out how nature works. Partly inspired by Rosie Redfield, one of my motivations for writing this blog is to relay the fun/not so fun moments of everyday science. In the spirit of openness I'm periodically going to be writing about a little side project that I've got going on in my lab. I actually have no clue where this will go, and at the moment there is absolutely no hypothesis, but I'm curious to see how this turns out because we've found something phenotypically interesting (to us at least). Science is about asking questions after all, and following the biology (currently reading an ASM book dedicated to John Roth, and "Follow the biology" is advice John liked to give to folks in his lab).
So, to begin, one of the bacteria that my lab works on is Pseudomonas stutzeri. The strain that we've been focusing much of our attention on usually makes vibrant yellow colonies during growth on agar plates, and this yellow tinge also comes through during growth in liquid culture (see test tube on left below). From my other work on Pseudomonads, I'm guessing that these colors might be due to iron scavenging molecules called siderophores, but I haven't had time to read a lot about the genetics of color in P. stutzeri, or even find out if tere are things to read. One day my technician picked a single colony from this workhorse strain and grew up an overnight culture. There was nothing particularly special about this colony or the overnight culture, but the next day this overnight culture was placed in the fridge to save for later. My technician later took the culture out of the fridge and, very surprisingly, noticed that the culture was now dark brown instead of the yellow tinge. We isolated single colonies from this brown culture and, sure enough, they continue to turn liquid overnight cultures brown. When colonies from this line are grown on plates, you can tell that the brown color is due to something extracellular to the colonies and which diffuses throughout the agar. Somehow, we were lucky (?) enough to randomly pick the very colony that possessed a mutation leading to an interesting phenotype (the odds are hugely against that). It's also not simply a contaminant...ruled that out already.
What's the first step of figuring out what this phentoype is (well, the first step is actually blogging about it and seeing if anyone has ever witnessed this before...if you have please let me know). If I were a biochemist I might try to isolate the chemical that's turning the culture brown, do some fancy analysis, and figure out what the composition is. I'm not a biochemist, I'm a geneticist. We like to figure out the underlying genes involved by breaking things and cleaning up the mess. To do this I am going to be using a Tn5 transposon. Transposons are pieces of DNA found widely in nature and can be thought about as chromosomal parasites. Tn5 is a naturally occurring transposon that has been "domesticated" for use in genetics labs. Once in a bacterial cell, it will randomly "hop" or "transpose" into the chromosome of the host. A good metaphor for this is that it cuts itself out of whatever previous piece of DNA it is present in, and pastes itself into the bacterial chromosome. The Tn5 transposon will hop only once into a (for the most part) random section of the new chromosome. Incorporation of the Tn5 will usually disrupt the function of whatever gene it incorporates into. We are going to hop a Tn5 transposon into the brown P. stutzeri isolates chromosome, with the hopes that this Tn5 will disrupt whatever genes are making the culture brown.
Overview of our Tn5 strategy
The particular strain of P. stutzeri that we are working with is competent for natural transformation in the lab, which basically means that it has the ability to suck up pieces of DNA from the extracellular environment and recombine them into it's own genome so long as there is some sequence similarity. Once we isolate a strain containing a transposon that eliminates the brown phenotype, we want to be sure that the transposon is causative for this phenotype rather than some other unknown mutation. To confirm this, we will isolate genomic DNA from the transposon mutant and transform the original brown strain with genomic DNA containing the transposon. If this Tn5 disruption is causative, we will be able to select for transformants that are no longer brown. Then, all thats left is to figure out what genes the transposon disrupted. We can do this using a few genetic tricks that I'll talk about later, but currently we are still trying to isolate non-brown isolates.
Wednesday, July 4, 2012
Bacterial genomes differ dramatically in size: from 140Kb to 13Mb (those numbers might be off now...please let me know if something has broken the record. Yes, I know the lower estimate can change based on semantics, but there are a bunch in that range). Although we have some clues as to how selection acts (or fails to act) on genome size, outside of intracellular parasites it's a bit of a mystery how selection shapes total genomic content. Perhaps the most interesting case out there involves genome streamlining in marine bacteria, which has been attributed to selection but which remains a just so story to this point.
One easy explanation is that "extra" DNA is costly in and of itself because it takes things like carbon, phosphorous, and nitrogen to physically make DNA. While probably true in the strictest sense, as far as I know there has not been a very clear test of the actual selective forces that act at this level. I would guess, especially given what's come out of the arsenic life debacle, that bacterial cells can survive just fine the way they are with low levels of phosphorous et al. I'm not sure how many environments are limiting enough for these elements to have direct selective effects on genome size (although see situations like this, this, and this). Other recent research points to the "cost" of extra DNA residing in the production of RNA and proteins. In this experiment, proteins and ribonucleotides are not inherently costly (*under the environments tested), but production of unnecessary proteins likely takes away cellular machinery that could otherwise be put to better use. There are only so many ribosomes in a cell to carry out translation. If these are occupied by unnecessary transcripts, they can't be used to produce more essential proteins. Interestingly, these costs may change based on previous environment. There are also some additional other hypotheses for genome size evolution that I may touch on in the future, but for now I would like to give a brief overview and thoughts about a paper relevant to this question that came out last week in PLoS Genetics from Dan Andersson's group.
Schematic of how they isolated deletion mutants
This manuscript is basically laid out in three related, but independent parts. The first consists of measuring the rate of deletions throughout the Salmonella enterica var typhimurium LT2 chromosome (see figure above). They first hopped a transposon containing three phenotypic markers into random areas of the chromosome. The markers contribute 1) resistance to chloramphenicol 2) cleavage of B-galactosidase leading to blue colonies during growth on X-gal 3) sensitivity to chlorate. They can measure rates of deletion using this transposon because of a cool genetic trick: the moaA marker renders the cells sensitive to chlorate, so they can select on resistance to chlorate in order to identify when the transposon might have been deleted from the chromosome. Once they get these chlorate resistance mutants, they look for white colonies and those that are sensitive to chloramphenicol in order to eliminate cells that have only deleted small portions of the transposon or have inactivated moaA through mutation.
The "deletometer" transposon
They use this "deletometer" to measure rates of mutation in 11 chromosomal regions and find that this rate varies by 2 orders of magnitude. These deletions range up to 10's of thousands of bp in size. Kind of a sidenotes to the total story here, but they do find evidence for the existence of a RecA independent deletion mechanism in S. enterica by studying genomic context of the deletions (RecA needs 25bp of sequence similarity to recombine pieces of DNA, but they find evidence that there is much less similarity bordering many of their sampled deletions, and in some cases none). They also find that, as shown for other bacteria like Bordetalla, that the replication terminus seems to be a hotspot for deletions (higher rates at terminus). The one question in my mind that remains from this portion of the paper is how they control for genomic context. It seems like regions of the chromosome that have more redundant sequence should have higher deletion rates, but maybe I haven't thought through this enough.
Fitness effects of deletions across enviroments and assays
Next they use a subset isolates to test for fitness effects of the deletions (and therefore address the question of cost of extra DNA). By measuring growth of strains in two environments (rich and minimal media) they show that some of the deletions actually increase growth rate in 15 of 55 cases. They further reinforce that deletions can be beneficial using growth assays where two strains are directly competed against one another. Importantly, they find no relationship between the size of deletion and the fitness effects (strike for the DNA is costly in and of itself camp).
The third part of the manuscript consists of a 1000 generation passage experiment in rich media. Over the course of laboratory passage and adaptation, there are a suite of deletions that reach high frequencies and are therefore likely adaptive during lab passage and in the right genomic context. They recreate these deletions in an unadapted chromosome and are able to show that 2 out of 6 do increase bacterial fitness in the lab (the absence of effects for the remaining four they chalk up to epistasis...they are beneficial only in the presence of other mutations that are not present in the unadapted ancestral strain).
The overall, memorable, take home message from this paper is that random deletions can be beneficial under some circumstances. Although this has been previously seen, this paper extends the result. They don't test the mechanistic underpinnings of these selective effects, which is what I hoped might be in the paper given the title. Although the authors don't talk about this too much, there a lot of deletions that are detrimental under each condition. The data seems to indicate that specific regions of the chromosome are more costly than others (beneficial fitness effects are only found in a subset of chromosomal positions), and I'm curious whether there is some unifying theme to these regions. Maybe they contain highly expressed but unnecessary, and therefore wasteful, genes. I'm curious why there are differential fitness effects for mutations that affect the same region...seems like it would be straightforward to figure out what differs amongst these different deletions within the same region as a way to get at the cost of DNA question. There is some additional novelty in showing that deletion rate varies over the chromosome, that there seems to be a RecA independent deletion mechanism in S. enterica, and that there is a deletion hotspot in the terminus.
Here's the citation:
Koskiniemi S, Sun S, Berg OS, Andersson DI. 2012 "Selection-Driven Gene Loss in Bacteria". PLoS Genetics.
Here's the citation:
Koskiniemi S, Sun S, Berg OS, Andersson DI. 2012 "Selection-Driven Gene Loss in Bacteria". PLoS Genetics.