Procrastination Blogging: Eek My Science Honors Proposal is Due Edition

Ugh, proposals. I need a little space to think out loud, and to get my doubt and self-loathing out of my system so I can be productive. I know it's just an internal undergrad research grant and not an NIH R01, and I am making it a lot harder than it needs to be probably. I had a really hard time getting my (easier) STEP proposal out last quarter too. A big part of it is feeling like I don't completely understand the project, in large part because I haven't been working in Dr. P's lab (or any labs for that matter) unlike most of the other kids applying. So here goes trying to sketch out some thoughts about the background and experimental design--I think best out loud and process by talking, what can I say. Thanks in advance for listening--you guys are the best!

Soap Lake, Microbial Observatory Extraordinaire

The overall area is looking at how viruses drive microbial evolution and genetic diversity in extreme or stressed environments, in this case a super salty and alkali lake in central WA known as Soap Lake. Such lakes are common in areas where there is more evaporation than rainfall or inflow--the minerals and such become concentrated. Soap Lake is an interesting study area for microbes, because it is so salty and the pH is so high that not much else can live in it besides some bacteria, archaea, and their phage. There are also two distinct layers to the lake due to its geomorphology. The whole area of central and eastern WA was shaped by the massive Missoula floods.

The bottom layer, or monolimnion, was formed first in an earlier flood; the top layer, or mixolimnion, formed on top of it in a subsequent flood. The interesting thing is that due to the difference in densities and composition, the two layers don't mix. The trippiest part is that the zone dividing them, called the chemocline, is surprisingly thin--about 0.5 meters. Dr. P tells me that when they take water samples from the monolimnion, if they forget to weight the instrument it literally strikes the chemocline and floats there. The monolimnion is a constant 7-8ºC, and total dissolved solids of about 85 grams per liter, and there is pretty much no dissolved oxygen--it's pretty much all anaerobes down there. The mixolimnion varies by season in terms of temperature, oxygen levels, and composition (average about 14 g/L).

The mixolimion represents stressed environments because of this flux, particularly in the shallower coastal areas, or even the sediment left when lake levels lower. The takeaway is that since the two layers don't mix, and there are different communities of microbes in each layer, it is interesting indeed to consider how common genes get between them. One should be able to compare the numbers of phage and bacteria in stressed areas (coastal mixolimnion) vs. stable areas (monolimnion) to see whether there is support for they hypothesis the greater the stress on an organism, the more that viruses are movers and shakers of genetic diversity.

Bacteria and the Viruses that Love Them

There have been a number of studies showing that viruses represent one of the richest repositories of genetic diversity on earth, and that one of the key drivers of elevated mutation rates in bacteria is antagonistic coevolution with parasites, vis-a-vis viruses, or phage. Further, because of how virus "lifecycles" (I don't know what to officially call them, since phage aren't technically alive) work, stressed bacteria show evidence of greater horizontal gene transfer.

Horizontal gene transfer is simply any transfer of genetic material that isn't passed from parent to child, which would be vertical transfer. In bacteria and archaea, which are single-celled organisms that reproduce by basically simply dividing, there are only 3 ways this can happen. There's conjugation, in which one bacteria can transmit genetic material directly to another, and is sometimes called bacterial sex, even though it doesn't result in mixing gametes because bacteria don't produce gametes. In conjugation, the receiving bacterium just gets some genes from the donating bacterium. However, bacteria of different genera don't conjugate in natural environments, so that doesn't explain all of the genetic mixing one sees in microbial communities.

There is also transformation, in which bacteria can incorporate free DNA in their environment. However, in an environment that is so salty and alkaline, the free DNA is not particularly functional, and in shallow water areas it is damaged by UV radiation as well. Thus, transformation doesn't really represent a likely vehicle for microbial gene mixing.

That leaves transduction, which is mediated by viruses (which are essentially free DNA or RNA protected by a protein capsid. The reason viruses aren't considered to be alive is because they can't reproduce their genetic material on their own--that is why they infect a host cell and hijack the host's cellular machinery to reproduce themselves. Viruses can undergo lytic or lysogenic reproductive cycles, and some can do both. Lytic phage copy themselves inside a host cell without inserting themselves into host DNA, and then burst or lyse the cell open (killing it) right after replication. Sometimes, when the viral particles are packaging the newly minted DNA, they can incorporate bits of host DNA with them, which get carried to new hosts with subsequent infections. This is called generalized transduction, because any bacterial gene can be transferred this way.

Lysogenic, or temperate phage, have enzymes that allow them to insert themselves into a host's DNA. The host then continues to replicate its "enhanced" genome without incident, creating several copies of new bacterial cells with incorporated prophage, until some sort of stress to the host (lack of nutrients, unfavorable environmental change) signals the prophage to cut themselves back out of the host genome and kick over to the lytic cycle. Sometimes, when these activated phage are excising themselves out they don't cut in exactly the right place, and they end up taking bits of host DNA with them. When they go forth and infect new host cells, they transfer those genes, which can then be incorporated into the new host's genome and reproduced. This is called specialized transduction, because the enzymes of the phage can only insert themselves into specific places in the host genome. The takeaway from this part is that in Soap Lake, the only real avenue for significant genetic recombination is bacteriophage transduction.

And Now, the Experiment!

Now it follows to design an experiment that will not only show evidence of transduction, but whether there are elevated rates of transduction in areas where there is greater stress, since we know that 1) phage protects the integrity of DNA and RNA; and 2) stress induces both lysis of cells and conversion, or excision of prophage, which in turn propagates transduction. So, how to measure transduction? What evidence will point to transduction taking place? For me, this is easiest to conceptualize in steps.

1. # of Transducing Particles (ie Phage)

This step will show how many transducing phage are present in each sample area--if transduction is taking place, then transducing viruses must be present. It is also reasonable that the more viruses, or transducing particles, are present, the more transduction is taking place.

Stressed areas are represented by the coastal waters of the mixolimnion and by the steep chemical gradient of the chemocline. Stable areas would then be the monolimnion and perhaps deeper levels of the mixolimnion. Water samples can be collected for each. The numbers of temperate phage can be figured out by filtering out everything but viruses (which are super tiny compared to bacteria, archaea and zooplankton!) and then reacting the the isolated virus DNA or RNA with probes that will "tag" the temperate, transducing phage that I am interested in. The tagged phage DNA or RNA can then be quantitated to tell me how much of it is present in each sample. If my hypothesis is correct, then more transducing particles will be present in the stressed areas than in the stable areas.

I am in the process of developing those DNA probes right now, as a product of my STEP research grant, using the myriad wonderful gene databases to find genomes of phage and prokaryotes that we know are present in Soap Lake, and then using the equally wonderful free software to compare those genomes and find areas of common, or "conserved" DNA sequences. A probe is basically a short piece of DNA (or RNA, or even antibodies for that matter). DNA is double stranded, and the two strands bind to each other in a very specific way: of the 4 nucleotide bases you have to choose from, only adenine ("A") binds to thymine ("T") and vice versa, and only cytosine ("C") binds to guanine ("G"), unless something goes wrong. Thus, for a sequence reading C-A-T, the complementary strand would be G-T-A. This makes for a stunning and elegant mechanism for coding and replication.

Further, a gene is just a segment of DNA that codes for a product: it can be a protein, or a ribosomal RNA, for instance. So, if you are looking for presence of a specific gene, you can make a probe out of DNA that is a complementary sequence to the DNA of the gene you want. Then you can mix your sample DNA and your probe DNA together in specific temperature and pH conditions, and if your gene of interest is present, it will bind to your probe. Oh, and you can attach markers to your probe to make it easy to find: say, a glowing fluorescent signal. You can then separate the tagged DNA from non-tagged by running it out on a gel and transferring it to what is called a blot membrane, and the strength of your fluorescent signal will tell you how much tagged DNA, or gene of interest, is present in your sample. Pretty neat, yes?

2. Evidence for Actual Transduction

So, hopefully from the experiment above I will know whether or not there are more transducing particles in stressed areas than stable areas. But, that still doesn't tell me that those transducing phage are infecting bacteria and mixing up genomes. I need another arm to my experiment to show that.

For this part, I can collect water samples bacteria and archaea from the same stressed and stable areas as I took for my phage quantitation samples, and catch them on a 0.2 µm filter--the tinier viruses will go right through leaving just the prokaryotes to play with. Once the bacteria and archaea are collected and their DNA is isolated, I can use a DNA probe with a more narrow target, say specifically for viral integrase and excisionase genes (the enzymes temperate phage need to glue themselves in and cut themselves back out of host DNA). If the probe binds to the bacterial DNA, it means that those viral genes are in there--which means that those bacteria and archaea are infected with lysogenic phage! These can also be quantitated in similar manner as in Experiment 1. And, if my hypothesis is supported, then stressed areas will also have greater numbers of infected bacteria.

Finally...wow. That's about it, actually. Other than some snappy statements proving that Sarah Palin and her ilk are anti-science, anti-Greater-Good ingrates if they make fun of this research because they can't see the larger picture, that is. As stated above, viruses are major drivers of bacterial evolution in different ways. Specifically, this research is important because knowledge of viral interaction with bacteria in stressful environments has direct application to how viruses and bacteria will interact in other stressful environments, e.g. clinical medicine when combatting pathogens with antibiotics or antiviral compounds. Further, in an age when antibiotic abuse is epidemic and multiple drug resistant "super bugs" are on the rise, understanding the process(es) and conditions of how bacteria acquire resistance is critical.

So what do you think? Would you give me $3500 plus another grand or so for supplies to carry this out and present it at a conference or research symposium? What parts are good? What parts are missing or need work (other than some of the specifics of the methods, which Dr. P is going to help me fill in, and obviously some of the more breezy, conversational tone that will be formalized up for the actual review committee audience).