Evolution of Phage Capsid and Genome Size

Viruses come in all shapes and sizes. From the very small, such as the picornaviruses or the parvoviruses, to the very large like mimivirus, or the herpesviruses, and poxviruses. These large viruses are not just large in physical size, but in the size of their genomes as well.

At the recent 2008 ASM General Meeting, Roger Hendrix of the University of Pittsburgh, laid forth a rather interesting hypothesis as to how large genomes, and the capsids that hold them came into existance and how they managed to be competitive in the gene pool.

Using phage P1 as an example, we know that larger capsids can be created "simply" by a single mutation allowing capsid subunits (capsomers) to come together in a quasi-equivilant matrix that is larger than the previous. This matrix follows the mathematical model set by Casper and Klug, and has discrete sizes (triangulation numbers, such as T=1,3,4,7,13). An increase in T number, as in our P1 example, causes a dramatic increase in capsid volume. Hendrix proposes that a mutation causing a such a shift acts as an evolutionary ratchet, and therefore smaller capsid sizers would no longer be available.

Now that we have a larger capsid, the phage now has the ability to package much more DNA. Not only does it have the ability, but in many cases, the phage MUST package DNA until its capsid is filled (headfull-packaging). With a larger capsid, phages who package via headful mechanisms now must package more DNA creating a greater amount of redundancy in its genome.

Hendrix explained that a greater amount of terminal redundancy leads to greater resistance from DNA damaging agents, specifically UV light. Although some in the session contended this, Hendrix described large amounts of genomic redundancy as an evolutionary advantageous trait for phages which live on the surface of the ocean and soil.

Furthermore, the extra space in the genome acts as a virtual genetic laboratory to aquire and mutate genes without disrupting the ability of the phage to survive. Gene aquisitions and subsequent mutations could create genes which provide some sort of marginal (or large) benefit to the phage or the host it infects.

With a simple click of the ratchet and a headfull of DNA, the role that large phages play in novel gene development are only now beginning to become clear.

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My posts on similar topics

Mosaicism: Life on a Small, Ever Changing Scale

The Definition of Life; and, Taxonomy as We Know It

Mosaicism: Life on a Small, Ever-Changing Scale (Part 2)

In the my last article, I briefly discussed the role of horizontal gene transfer in bacteria...specifically the development of mosaic pathogenicity islands in Escherichia coli. However, the formation of mosaics are not just limited to operons within bacterial genomes. In fact, we can see such events in phage and viral genomes. This article is part two in a brief series on genomic mosaicism.

Current research shows that illegitimate recombination events create mosaics of unique genes within the genome. Each gene then acts as an individual unit upon addition, whereby beneficial genes are selected for and remain in the genomes, and non-beneficial genes build up mutations or are dropped. Illegitimate recombination, by definition, occurs within gene boundaries, and as such, highly unique and variable regions within genomes are formed.

Looking within the sequenced genomes of the mycobacteriophages (an obvious interest of mine) we see a large amount of mosaicism. Small sets of genes, individual genes, and parts of genes are constantly being shuffled in, out, and around genomes. There are countless examples of gene insertions and deletions in the phage genomes, adding to the case that genomes are liquid and constantly changing. We see gene swaps not only between phages, but also between hosts.

These events show themselves by the presence of unique genes in genomes. For instance:
1) the presence of photosynthesis genes in many cyanophages
2) a portion of mycobacterial MetE in far right arm of the mycobacteriophage Giles genome (a conclusive example of illegitimate recombination between virus and host)
3) the protein Ro in mycobacteriophage Bxz1 (a eukaryotic protein implicated in the human disease lupus)
4) Bxz1 also contains a human peptide chain release factor
5) a portion of Bacillus VIP toxins in a mycobacteriophages PBI1 and PLot
6)Mycobacteriophage Che8 contains a human prion-like protien
7) motifs of resuscitation promoting factors in a wide variety of of mycobacteriophage tape measure proteins
8)not to mention the countless (30-50%) number of open reading frames with absolutely no homologs in the current databases or which only match other phage proteins.

(Yes, I did say that phage Bxz1 of the mycobacteria contains a human antigen gene, with significant sequence similarity, see here.....I'll leave this discussion for another day, mainly because as far as I know, no one has looked at such a seemingly odd phenomenon outside of bioinformatic comparison. How did a human gene sneak its way into a bacteriophage?)

What I am trying to drive home here, hopefully with some success, is that life at the small scale is ever fluctuating and massively interconnected. Nothing in biology is genetically isolated. We are the current homes of many genes, where they end up next is up to our viral and phage couriers.

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PEDULLA, M. (2003). Origins of Highly Mosaic Mycobacteriophage Genomes. Cell, 113(2), 171-182. DOI: 10.1016/S0092-8674(03)00233-2

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My Similar Articles
Mosaicism: The World of Horizontal Gene Transfer (Part 1)
The Definition of Life, and; Taxonomy as We Know It
ReQ's Role in Illegitimate Recombination
I'll Have My Bacteria Extra-CRISPR

Also, visit The Phamerator to explore the sequenced mycobacteriophage genomes on your own.

Mosaicism: The World of Horizontal Gene Transfer (Part 1)

Commonly, gene transfer is thought of as a vertical line from parent to offspring, along which all evolutionary traits are passed. However, as we began delving into genomic sequences, we found that this may not be true and that the lines between "species," especially on the microbial level, are quite fuzzy.

Horizontal gene transfer is the transfer of genetic elements between species. The microbial world is filled with examples of this phenomenon. This article is the first in a 3 part series that will explore the ever fluctuating genetic world of our microbial majority.

Escherichia coli is perhaps the most well studied and utilized bacterial organism on the planet. This bug also has the capability for a wide range of illness in humans including: non-pathogenic, enterohemorrhagic (such as popular strain 0157:H7), enterotoxigenic, and uropathogenic. The question is obviously raised--how can the same organism have such pathotypes, and what allows it to be versatile enough to thrive in such different environments as the urinary tract vs. the intestinal tract.

An article coming out of the University of Wisconsin-Madison shows that E. coli consists of a general common backbone that is nearly 100% identical across strains, yet only takes up 75%of the genome. However, they show that within this backbone are regions (islands) that are highly divergent between strains (See figure at left). It is no surprise that within these divergent regions contain the genes which allow for survival in different environments (pathogenicity islands)

These islands are distinct from each other, the surrounding genome, and the corresponding areas in other strains. However, the striking similarity between islands of different strains is that they all seem to be located in nearly the same locations.

Furthermore, strains of the same pathotype tend to have the same/similar genes located in islands (see figure below)--but not necessarily in the exact location or orientation. These islands tend to occur in areas at tRNA regions--commonly utilized by bacteriophages for integration.

Phages are clearly agents of horizontal gene transfer, further examination of these island regions show the presence of cryptic (non-viable) prophages. This indicates that at least 5 phages integrated, but subsequently lost genes required for the creation of viral progeny. It is possible that the other islands are also prophages, but gene loss has occurred to the point where they are no longer recognizable.

What this means as a whole to the definition of species is to me, yet, unclear. Strains of E. coli all share a distinct backbone of metabolic genes, but differ drastically in their specialized regions. How do we decide which belongs in a species?...especially when we differentiate humans from chimpanzees (only ~6% genetic differences).

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Welch, R.A. (2002). Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proceedings of the National Academy of Sciences, 99(26), 17020-17024. DOI: 10.1073/pnas.252529799

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My Similar Articles

The Definition of Life; and, Taxonomy as We Know It

Reductive Evolution in Mycobacterium leprae

I'll Have My Bacteria Extra CRISPR

Phages With Horns?! What's Next?

In the world of phage, uniqueness rules. The total number of phages in the biosphere is dramatic, with estimates numbering the population at 10³¹! Despite such high numbers, since the discovery of phage no identical phage has been found in the environment twice. They have often been termed "Nature's Most Successful Experiment." Their genomes are in constant flux amoung themselves and their hosts. It therefore should no longer surprise us when we discover a phage that doesn't quite fit the mold.

In this paper by Pope, et al. they describe a phage (named Syn5) of the cyanobacteria that comes equipped with a "horn." Described as a "slender elongated fibrous protrusion," the horn clocks in at 50nm in length. To put perspective on this, the horn is almost as long as the capsid is wide (60nm) and is longer than the tail (25nm). It is however, quite thin 10nm wide at its thickest point and 2nm at its thinnest.

The figure below is a cryoEM taken of Syn5, showing the fibrous horn structure directly opposite the tail.
Every Syn5 capsid has this protrusion, and it only occurs once in its specific location.

The purpose is yet unknown, however there is a running hypothesis. These phages live out in the open ocean where there hosts are not around in very high numbers. Therefore, it would be highly beneficial for the phage to increase its chances of finding and attaching to a host. The phage would not want to be oriented in the wrong direction as a host cell passed overhead. If this horn functions as a host recognition site, clearly it would increase the phage's chances to find a host cell.

To further amaze you, the vast majority of cyanophage carry their own photosynthesis genes!

I can only imagine what we will find next as we continue to explore this small world. Truly, All the World's a Phage.

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Suggested Reading

The Bacteriophages

The Cyanobacteria: Molecular Biology, Genomics and Evolution

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POPE, W., WEIGELE, P., CHANG, J., PEDULLA, M., FORD, M., HOUTZ, J., JIANG, W., CHIU, W., HATFULL, G., HENDRIX, R. (2007). Genome Sequence, Structural Proteins, and Capsid Organization of the Cyanophage Syn5: A “Horned” Bacteriophage of Marine Synechococcus. Journal of Molecular Biology, 368(4), 966-981. DOI: 10.1016/j.jmb.2007.02.046

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I Got You Phage

Back in August of 2006, I wrote and performed this song for a lab meeting to summarize my work I had done that summer and the previous year (All regarding a bacteriophage called BPs).

Some of the lyrics are hard to catch, you can find them below the embed. Enjoy!


Lyrics
Will I find you? Well, I don't know.
I won't find out until my smeg will grow.
I scraped the dirt right off my shoe
You were there and phage, I found you.

(Chorus)
Phage, I got you phage. I got you phage. I got you phage.

They say you won't kill TB, you'll only infect Bovis BCG
I can tell from your lack of spots
You won't infect all the bacteria we got

(Chorus)

You have recE in your genes
You have Halo on your team
And when your cruising round the town
Bateria die when you're around.

So let them say your tail's too long
I don't care, I wrote the song
So put your little head by mine
It has the smallest genome we can find

(Chorus)

I found you from dirty sludge
I found you while eating fudge
I got micrographs of you
I still got lysogen work to do
I want you to integrate, I'll watch it when you replicate
I found you, I won't let go
I found you, I love you so

Phage
I got you phage
I got you phage I got you phage I got you phage
~End Lyrics~

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There will hopefully be some more microbiology songs coming soon (as soon as I get a camera up and running). With titles such as:
1. It's Gonna Be There (The E. coli song)
2. Brillant Dance of the Starvation Response
3. Phage in the Soil

Just For Phun!

I found this in my photo archives, I thought some of you may enjoy it! (2005)

I'll Have My Bacteria Extra-CRISPR

The microbial cold war between species has been widely studied and is, in my opinion, one of the most interesting topics in current biology.

Fungi make compounds to destroy bacteria, bacteria make compounds to destroy fungi. But it doesn't stop there. Phages mutate constantly to evade bacterial defenses. We've known about a handful of bacterial defenses against phage-- repressor systems, restriction enzymes, receptor modification, DNA modification, etc.

We have utlized these systems to our advantage--our first effective antibiotic (Penicillin G) came from the fungus Penicllium, a vital antifungal (cyclohexamide) comes from the bacteria Streptomyces, our ability to clone and subclone is only useful because we can specifically cut DNA with restriction enzymes, etc.....

But now, there is another form of bacterial defense. We have evidence for, what I will call, the "special forces" of bacterial defense systems. Heck, I'd liken this to a form of adaptive immunity.

CRISPR (clustered, regularly interspaced, short, palindromic repeats) sequences have been found in 40% of all sequenced Bacteria and 90% or sequenced Archaea. Essentially, you have an array of nearly identical repeats which form RNA stem-loops. Between the repeats, you find unique DNA that has sequence similarity to phages. There are also a handful of unique genes that are closely associated with the arrays.

The idea is that the bacteria are using an RNA interference mechanism. The CRISPR array gets transcribed and then spliced at the loop structures--forming small RNAs. These sRNAs associate with phage nucleic acids, recruit degradation genes, and subsequently denature the phage nucleic acid. See the figure at right taken from the review article.

You find a distinct correlation between the number of CRISPR sequences and resistance. You also can add specific phage sequence to the array and grant immunity. If you then take that sequence away, immunity is lost.

What is interesting (but not necessarily surprising)is that a small population of phage remained resistant to the system. It was found that these phage had a slightly different nucleic acid sequence at the site. BUT, given time, the bacteria can acquire CRISPR sequence against the phage.

It's an evolutionary arms race!

That we should be exploiting!There are a variety of ramifications that this system can and will have on the future of molecular biology.

For one, a system of spoligotyping could be developed based on the unique spacer regions in the CRISPR array.

Apparently a large problem in dairy industry (specifically yogurt and cheese) is that bacterial cultures get lost to phage. This system could help prevent that by allowing the development of ultra-resistant strains.

A more unique (and as far as I know, untested) use would be the development of a system to knock out genes in vivo. You could imagine setting up a CRISPR system on a plasmid with specificity for a specific gene (or multiple genes) you want to knock out. The CRISPR system would do what it does naturally and degrade the gene's transcript. Since these regions are only 26-72bp, it would certainly be an awesome tool to have--especially for organisms notoriously recalcitrant to genetic manipulation (like my favorite bugs: the Mycobacteria). Genes could be effectively silenced using only a plasmid, without the need for messy recombination systems.

Sorek, Kunin, and Hugenholtz. "CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews Microbiology. 6, 181-186 (March 2008)

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Sorek, R., Kunin, V., Hugenholtz, P. (2008). CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology, 6(3), 181-186. DOI: 10.1038/nrmicro1793

Phage Therapy's Positive Attributes

Let's discuss now what makes phages ideal for human therapy and food treatment.

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1. Phages are very specific with what they infect.

They only infect bacterial cells, never your cells! They also only have the ability to usually infect one type of bacteria as well. This means that a phage used to kill off harmful gut bacteria, isn't likely to kill off beneficial bacteria. We can also easily make cocktails of different phages to ensure that the harmful bacteria are completely destroyed.

• Current antibiotics do not differentiate between harmful and beneficial bacteria. Phages do.

2. Phages don't harmfully interact with your cells.

Unlike some antibiotics, you won't experience any negative affects directly from exposure to a bacteriophage. For one, they are already ubiquitous to the environment. Studies have also shown that phages do not illicit an immune response, meaning that allergies are not likely. Also, they are able to traverse to nearly every part of the body quickly and easily.

• Current antibiotics can cause allergic reactions and other negative responses Phages likely won't.

3.After doing their job, phages disappear.

They don't linger around in your body. Since they require their specific host bacteria to replicate, once that type of bacteria is gone, they can no longer replicate. Studies show that phages leave the system in a little over 24 hours.

4.Phages are "nearly" living organisms and will evolve to surpass bacterial resistances.

As bacteria grow to be resistant to a phage, that phage will evovle around the resistance. A classical predator-prey relationship. Cocktails of phage will likely be used, so this is not a huge concern.

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So let's imagine some ideal ways that phages can be used in combating bacterial illness.

1. Topical application of a bacteriophage salve onto skin lesions.

Everything from pimples and acne, to diabetic foot ulcers and Staph infections could be treated this way. This is perhaps the best use of phages for therapy.

2. Inhalation of a phage cocktail to fight lung infections.

Pneumonia and tuberculosis could be treated despite increasing antibiotic resistance.

3. Injection of phages into the bloodstream to combat systemic bacterial infections.

4.Ingestion of phages into the GI tract to combat things like cholera, E. coli, etc.

5. Phage treated food supplies to protect us from E. coli laden spinach, or Listeria infected hotdogs.

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Next time, we'll discuss the downside to the uses of phages in therapy.

Phage Therapy

Phage therapy is the use of bacteriophages (usually purely lytic) to treat pathogenic bacterial infections. Phages are viruses that invade only bacterial cells and, in the case of lytic phages, cause the bacterium to burst and die, thus releasing more phages. Phage therapy is a potential alternative to antibiotics, being developed for clinical use in the 21st century by many research groups in Europe and the US. After having been extensively used and developed mainly in former Soviet Union countries for about 90 years, phage therapy is now becoming more available in other countries such as USA for a variety of bacterial infections.Phage therapy has many applications in human medicine as well as dentistry, veterinary science and agriculture.

An important benefit of phage therapy is that bacteriophages are usually more specific than common drugs, so one can be chosen to be harmless to not only the host organism (YOU), but also other beneficial bacteria, such as gut flora, reducing chance for opportunistic infections. They also have no known side effects as opposed to drugs, and do not appear to stress the liver or immune system. Because they replicate inside the pathogen, a single, small dose is usually sufficient.

Phages are currently being used therapeutically to treat bacterial infections that do not respond well to conventional antibiotics. They tend to be more successful where there is a biofilm covered by a polysaccharide layer, that antibiotics typically cannot penetrate.

The origins of phage therapy can be traced to the origins of the discovery of phages themselves. Felix d'Herelle discovered and implemented phages as therapeutic agents back in the 1920s. His story is fantastic and should be known by all microbiologists. Read more about him here Felix d`Herelle and the Origins of Molecular Biology

Intro to Bacteriophages

So, Tim studies bacteriophages...well, what the heck are they anyway. Here's a very brief introduction as to what they are and do. There may be some links around the site that will tell you more.

A bacteriophage (from 'bacteria' and the Greek "phagein", meaning 'to eat') is any one of a number of virus-like agents that infect only bacteria. The term is commonly used in its shortened form, phage.

Typically, bacteriophages consist of an outer protein hull enclosing genetic material. The genetic material can be dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and 200 nm in size.

Phages are estimated to be the most widely distributed and diverse entities in the biosphere, numbering 10^31 virions. Phages are ubiquitous and can be found in all reservoirs populated by bacterial hosts, such as soil or the intestine of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 10^9 virions per milliliter have been found at the surface, and up to 70% of marine bacteria may be infected by phages. They are also found in drinking water and in some foods, including fermented vegetables and meats e.g. pickles, salami, where they serve the function of controlling any growth of bacteria.

Phages are very specific to bacteria, and thus are more accurate and potent than antibiotics. They have been used for over 60 years as an alternative to antibiotics in the former Soviet Union and Eastern Europe. They also have no known side effects, as opposed to drug therapy. They are now seen as a hope against multi drug resistant strains of many bacteria.

Research Synopsis

So my grad school application process continues. This is part of my personal statement....it is a synopsis of my research. Let me know what you think, any changes I should make, etc. Thanks in advance!

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For the past two and a half years, I have had the privilege to work as an undergraduate researcher in the lab of Dr. Graham Hatfull at the University of Pittsburgh. Dr. Hatfull is one of a unique group of researchers adamantly recruiting young students into biological research. Students as young as 6th and 7th grade are able to work in his lab through his Phagehunting program. Although I did not begin my work in the program at that young of an age, many would consider my start as a college freshman early compared to most.

I began my research in January of my freshman year. The first project was to use bioinformatic methods to analyze and annotate the genome of a Bacillus phage, MP15. Utilizing programs such as DNA Master, Glimmer, and GeneMark, I called the most likely protein coding regions in the genome. These putative genes were then compared to GenBank databases to assign potential functions and evaluate the novelty of the phage’s genome. Not surprisingly, this phage proved to be highly novel; its genome had a large amount of genes with no significant sequence similarity to those genes in the databases.

Having enjoyed my first research experience, I then continued work in the lab, moving from bioinformatics into wet lab projects. I became part of a larger project to isolate and characterize novel mycobacteriophages on Mycobacterium smegmatis. I isolated, amplified, and concentrated a novel mycobacteriophage named BPs. Using Sanger sequencing procedures, the phage’s genome was sequenced. The final sequence was analyzed and annotated. Lysogeny experiments showed BPs to form stable lysogens in M. smegmatis, likely integrating at a tRNA^Arg gene in the host. Furthermore, host range studies showed that BPs was able to infect not only its initial host, but also M. chelonae, M. bovis BCG, and notably, M. tuberculosis (M. tb.).

Following up this host range study of BPs, I began to characterize the ability of over two hundred mycobacteriophages (published and unpublished) to infect M. chelonae, M. bovis BCG, and M. tb. Dr. Hatfull approached me with a few ideas for projects that could stem from the large amount of host range work I was performing. One of these projects involved the discovery of a phage capable of performing generalized transduction in M. tb.





Generalized transduction occurs when a phage capsid accidentally packages host DNA, resulting in a viral particle capable of inserting that DNA into a new cell. This mechanism has been exploited in a variety of bacterial systems. Currently, there are only two published phages with the ability to transduce Mycobacteria; Bxz1 and I3. However, neither of these is able to infect or transduce M. tb. My goal was to find which of our published phages could infect M. tb. and which could function as transducing phages. I developed and performed transduction assays with two different genetic markers. At the same time, I finalized the host range of our published phages.

The results showed that 11 of our 30 published phages were able to transduce M. smegmatis and that 7 of these 30 were able to infect M. tb. However, no phages appear in both groups. Bioinformatic analysis of putative genes showed that there was no particular gene present only in the group of M. tb. infecting phages. Rather, the only genetic element common among M. tb. infectors is the presence of cohesive genetic termini. Those phages capable of mediating generalized transduction all have terminally redundant genetic termini. Currently, I am asking whether or not the presence of terminally redundant ends prevents phage from infecting M. tb.

The nature of a phage’s genomic ends determines how the DNA circularizes upon infection. Phages with cohesive termini circularize via complementation; while phages with terminal redundancy circularize by homologous recombination. M. tb. is notorious for their large amount of illegitimate recombination, which potentially may inhibit phages with terminal redundancy from circularizing and subsequently replicating. Previous studies have shown that recombination proteins are necessary for certain phages with terminal redundant ends, such as Salmonella phage P22, to circularize and replicate. Previous work with mycobacteriophages has discovered functionally analogous proteins for recombination in Mycobacteria. These proteins, encoded by phage Che12, have been successfully utilized to promote homologous recombination in both M. smegmatis and M. tb. My current work is establishing whether the presence of these proteins can overcome a recombination deficiency in M. tb. and allow terminally redundant phages cause infection. If this is true, generalized transduction in M. tb. may be a possibility.

Aside from learning laboratory techniques and critical thinking, this research experience has provided me with the opportunity to present my work at a wide variety of scientific meetings. These meetings include: the 2005 Molecular Genetics of Bacteria and Phages Meeting, the 106th and 107th Annual American Society for Microbiology General Meetings, along with meetings hosted by the University of Pittsburgh.

My work with phage has opened my mind to the world of microbial interactions. The constant flux between host and pathogen, on any scale, is vast and decidedly interesting. Studying these interactions can provide the focus for a wide range of projects. Microbial interactions have components in vaccine development, industrial fermentations, novel antimicrobial development, genetic tool creation, and evolutionary studies. It is my hope that graduate school will allow me to delve deeper into the interactions between microbes and humans, themselves, or their environment.

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This is the first 2/3 of a ~1000 word essay. The remaining will be geared towards which ever school I am applying too. Thanks for your opinions.