Bacteria Can Talk! (and now we can silence them)

Most cellular biology has focused on eukaryotes, but just as interesting are the prokaryotic bacteria. As the most ancient life form, bacteria have primitive features and are characterized as single celled organisms. Presumed to be asocial and reclusive, bacteria were once thought to lack cell to cell communication systems. Evidence of cell-cell communication was discovered in the marine bacterium Vibrio fischeri (Nealson & Hasting 1979). In dilute concentrations, Vibrio did not bioluminesce, but when the bacteria density was sufficient, secreted chemical signals reached neighboring cells and triggered bioluminescence.

Quorum sensing was first detected in the bioluminescent Vibrio fischeri.

The mechanism involves the production, then release, and finally adherence to receptors of hormorne-like molecules called autoinducers (see figure 1). Termed quorum sensing, this intra and inter-species communication blurs the definition of multicellularity (Waters & Bassler 2005). Quorum sensing can occur through species-specific autoinducers or through general broadcast autoinducers shared by many species. The interplay between autoinducers and their receptors controls gene expression and ultimately the synchronized behavior in bacterial populations.

Figure 1: Autoinducers (red triangles) are produced by LuxI synthase. When concentration of autoinducer is sufficient, it binds to the LuxR receptor, initiating the light producing reaction. From Waters & Bassler 2005.

Quorum sensing circuits are responsible for virulence gene activation in many pathogens. Knowing this, a novel antibiotic concept has been developed which utilizes antagonists to jam the receptors and prevent the expression of virulence genes in a bacterial population. The autoinducers used by gram-negative bacteria are usually acyl-homoserine lactone molecules(AHLs) (Chen et al 2011). Numerous pathogenic bacteria are known to use the LuxR protein family quorum sensing mechanism shown above to detect the AHLs (Fig 1). Previous work has identified several quorum-sensing antagonists, but their mechanism had previously remained a mystery.

Chen et al. studied the antagonist mechanism used in blocking the AHL:LuxR complex in the human pathogen Chromobacterium violaceum. Using a variety of methods from genetics to x-ray crystallography, the researchers characterized the LuxR type protein CviR. Without an autoinducer the proteins are unstable, but a stable complex is formed when the autoinducer is present (see Figure 2). In the stable form, virulence genes are activated. Chen et al. show that it is possible to develop antagonists that function by stabilizing transient interdomain interactions, giving rise to an inactive configuration (Chen et al. 2011). Essentially, the antagonist blocks the autoinducers by binding in its place, rendering the site inactive. Such a system worked as long as the antagonist induced a closed conformation, which is unable to bind to the DNA domain. Future research is being conducted to identify molecules that preferentially bind in place of the autoinducer and that favor an inactivating interaction. These new molecules may lead to the application of anti-quorum sensing molecules to therapeutic antibiotics.

Figure 2: The two monomers are colored orange and blue. Each monomer of two domains, a ligand-binding domain (LBD) and a DNA-binding domain (DBD). The antagonist induced a closed conformation, so the CviR is unable to bind to the DNA domain.

In a period when the development of antibiotics is declining and the number of antibiotic resistant bacteria is on the rise, advancements in the field of quorum sensing have promising application. The autoinducers are both species specific and generic, so particular pathogenic bacteria can be targeted through this type of antibiotic. Intuitively one could also create a pro-quorum sensing molecule to increase the function of beneficial bacteria. Now that scientists have cracked the code, we are on our way to ending bad communication.

Works Cited:

G. Chen, LR Swem, DL Swem, DL Stauff, CT O’Loughlin, PD Jeffrey, BL Bassler, FM Hughson. (2011) A Strategy for Antagonizing Quorum Sensing. Molecular Cell 42, 199–209.

CM Waters & BL Bassler. (2005) Quorum Sensing: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 21:319–46.

KH Nealson & JW Hastings (1979). Bacterial Bioluminesence: Its Control and Ecological Significance. Microbiiological Reviews 43:496-518.

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Photosynthetic organisms survive in mammalian cells: Are Dryads no longer for fairy tales?

In “On the Difficulty of Conjuring Up a Dryad,” Sylvia Plath writes,

But no hocus-pocus of green angels
Damasks with dazzle the threadbare eye;
My trouble, doctor, is: I see a tree,
And that damn scrupulous tree won’t practice wiles
To beguile sight:
E.g., by cant of light
Concoct a Daphne;
My tree stays tree.

Perhaps if Plath had witnessed the extraordinarily leaf-like appearance of the sea slug Elysia chlorotica, or the supernatural glow of green algae within salamander eggs, it would have been easier for her to imagine a tree, anthropomorphized.

The intracellular relationship between photosynthetic algae (zooxanthellae) and coral animals was first reported in the late 1800s (Rosenberg et al. 2007).  Symbioses between photosynthetic algae living within salamander eggs (also discovered over 120 years ago) were long thought to be intercellular.  Recently, Kerney (2011) discovered that this relationship, where the algae are thought to confer valuable oxygen to the developing embryo, is actually intracellular.  The sea slug Elysia chlorotica harbors green algae after ingesting it as a food source.  The algal plastids continue to photosynthesize within the sea slug, providing it with energy and carbon.  Careful investigation of this fascinating relationship has revealed that genes from the algal plastid (psbO) are transferred horizontally to the sea slug nuclear genome where it is expressed.  These are just a few instances exemplifying the evolutionary impacts such intimate symbiotic relationships can have on organisms and biodiversity in general.  Indeed, ancient endosymbiotic events are thought to have resulted in all the eukaryotic diversity seen on our planet today (Keeling 2010).

Elysia chlorotica

In an effort to better understand the evolution of endosymbiosis, as well as to provide new tools for advancing synthetic biology, researchers at Harvard Medical School have successfully engineered photosynthetic cyanobacteria to invade and divide inside mammalian macrophages.  In the first of a series of experiments, Agapakis et al. discovered that Zebrafish embryos injected with ~ 10 million cyanobacteria at the single cell stage survived up to twelve days intracellularly with no apparent effects on the embryo (compared with E. coli, which killed Zebrafish cells within two hours).  A second investigation proved cyanobacteria genetically engineered with invasin and listeriolysin  (genes needed for mammalian host cell invasion into the cell surface and cytoplasm, respectively) could effectively enter mammalian cells. Incredibly, a third test showed that engineered cyanobacteria taken up by mouse macrophages were able to survive and begin dividing, rather than being digested (Agapakis et al. 2011).

Microscopy image of Zebrafish embryo. Red dots are cyanobacteria throughout the embryo.

A 2005 Nature review written by Drew Endy of Stanford University underscores the importance of foundational synthetic biology to progress in bioengineering.  Endy states, “for biologists, the ability to design and construct synthetic biological systems provides a direct and compelling method for testing our current understanding of natural biological systems.” The work of Agapakis et al. illustrates this idea.  In considering research that has shown photosynthetic bacteria surviving inside mammalian cells, popular media or science fiction fans might conjure images of people simply basking in the sunlight a few times a day in lieu of making that weekly trip to the grocery store. If you are anything like me, the thought of a future that does not involve ingesting cheese is just dreadful.  Rest easy, caseophiles.  The authors are not implying such engineered relationships could serve in place of biological systems (i.e. photosynthetic skin!), at least not yet.  However, they do a really nice job of putting their research into the context of creating a unique platform for bioengineering (that could potentially fill gaps in our knowledge of the complex evolutionary dynamics of endosymbiosis).

As Endy eloquently suggests, “it is possible that the designs of natural biological systems are not optimized by evolution for the purposes of human understanding and engineering… thankfully, these concerns are best evaluated by attempting to surmount them.” With that in mind, a number of questions that might be addressed by follow-up studies of this engineered system come to mind. What is the response of the host immune system?  How is it similar or different to that of other innocuous associations, such as in algae and sea slugs or salamander eggs?  Does gas exchange appear to be beneficial to Zebrafish embryos as it has been in salamander’s eggs?  Is it possible that the cyanobacteria could ever survive to adapt (assuming that photosynthesis would not be possible at some level of the hosts’ development due to sunlight not penetrating multicellular structures)?  And due to their benign nature in vertebrate cells, might the cyanobacteria make safe and effective gene delivery vehicles for therapeutic treatments or vaccines?

This post has been submitted for the NESCent (National Evolutionary Synthesis Center) research blogging competition.  Winners receive a travel award to attend the ScienceOnline 2012 science communication conference at North Carolina State University.

Agapakis CM, Niederholtmeyer H, Noche RR, Lieberman TD, Megason SG, Way JC, & Silver PA (2011). Towards a synthetic chloroplast. PloS one, 6 (4) PMID: 21533097

Endy, D. (2005). Foundations for engineering biology Nature, 438 (7067), 449-453 DOI: 10.1038/nature04342

Keeling PJ (2010). The endosymbiotic origin, diversification and fate of plastids. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 365 (1541), 729-48 PMID: 20124341

Kerney R., Kim, E., Hangarter, R.P., Heiss, A.A., Bishop, C.D., & Hall, B.K. (2011). Intracellular invasion of green algae in a salamander host. PNAS 108: 6497–6502.

Plath, Sylvia.  (1957).  On the Difficulty of Conjuring Up a Dryad.  Poetry 90: 235-236.

Rosenberg, E., Koren, O., Reshef, L., Efrony, R., & Zilber-Rosenberg, I. (2007). The role of microorganisms in coral health, disease and evolution Nature Reviews Microbiology, 5 (5), 355-362 DOI: 10.1038/nrmicro1635

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Dino-soars: Reptiles with feathers

ResearchBlogging.org
Dinosaurs have long captured our hearts and our curiosity.  From our first field trips to museums where their fossilized skeletons towered over us, it was difficult not imaging what they looked like and how they lived.  How many species were there?  What did they eat?  Did they have social groups?  How did they die?  Science and paleontology have slowly been able to answer these and many other questions about their biology, ecology, and environment.

Until recently one of the hottest debates about dinosaurs were the mechanics behind such a successful species’ extinction.  Schulte et al. (2010) were able to definitively show that the Chicxulub asteroid was the cause of the dinosaurs’ demise.  When the 7.5 mile wide asteroid blanked the sun for a decade with debris from an impact a billion times more powerful the the Hiroshima nuclear strike, the dinosaurs (and many other forms of life) stood no chance.

With this issue finally laid to rest a new debate has surfaced; did dinosaurs have feathers? We’ve all seen the renditions of dinosaurs as scaly, drab colored creatures with skin similar to modern reptiles.  The recent evidence from McKellar et al. (2011) and Wogelius et al. (2011) indicate that this too is incorrect. Dinosaurs had feathers, colorful ones even (see images).  These amber entombed feathers were discovered in Late Cretaceous amber from Grassy Lake, Alberta, Canada.  These findings fill a large gap in feather evolution and coincide geologically with Chinese compression fossils of feathered non-avian dinosaurs.  The combined evidence strongly support the theory that dinosaurs possessed protofeathers and that feathers from a variety of developmental stages were present in the Late Cretaceous on both dinosaurs and birds (McKellar et al. 2011).

This is fascinating on two separate levels.  First, dinosaurs had feathers!   I’ve studied biology my entire life, I am constantly amazed at what we learn as science and technology progress.  Second, and more scientific, the implications this discovery has on the evolution of feathers is priceless.  As these feathers have been preserved in amber, structures that would be lost in any compression fossil are literally frozen in time.  Complex features of the (proto)feathers including the calamus, rachis, and barbules (see images) are clearly identifiable.  These findings have been able to fill in the missing stages in feather evolution and provide a clear picture of the development of extremely complex features.   To paraphrase a bit of Hollywood “Fifteen hundred years ago we knew the earth was the center of the universe, 500 years ago we knew the earth was flat [and 3 months ago we knew dinosaurs were scaly and dull] imagine what [we’ll] know tomorrow.” (Men in Black) Amazing.

Schulte, P., Alegret, L., Arenillas, I., Arz, J., Barton, P., Bown, P., Bralower, T., Christeson, G., Claeys, P., Cockell, C., Collins, G., Deutsch, A., Goldin, T., Goto, K., Grajales-Nishimura, J., Grieve, R., Gulick, S., Johnson, K., Kiessling, W., Koeberl, C., Kring, D., MacLeod, K., Matsui, T., Melosh, J., Montanari, A., Morgan, J., Neal, C., Nichols, D., Norris, R., Pierazzo, E., Ravizza, G., Rebolledo-Vieyra, M., Reimold, W., Robin, E., Salge, T., Speijer, R., Sweet, A., Urrutia-Fucugauchi, J., Vajda, V., Whalen, M., & Willumsen, P. (2010). The Chicxulub Asteroid Impact and Mass Extinction at the Cretaceous-Paleogene Boundary Science, 327 (5970), 1214-1218 DOI: 10.1126/science.1177265

McKellar RC, Chatterton BD, Wolfe AP, & Currie PJ (2011). A diverse assemblage of Late Cretaceous dinosaur and bird feathers from Canadian amber. Science (New York, N.Y.), 333 (6049), 1619-22 PMID: 21921196

Wogelius RA, Manning PL, Barden HE, Edwards NP, Webb SM, Sellers WI, Taylor KG, Larson PL, Dodson P, You H, Da-qing L, & Bergmann U (2011). Trace metals as biomarkers for eumelanin pigment in the fossil record. Science (New York, N.Y.), 333 (6049), 1622-6 PMID: 21719643

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To TA or not to TA

As a graduate student and a TA, I have often looked at my non-TA peers and thought about how fantastic it would be to be in their shoes.  No more grading papers and no more long hours sounded fantastic.  A recent study showed that there may be more to being a TA than the fanfare and 7 digit salary* that come with the job.

The study, conducted by Felton et al (2011) focused on 2 groups: those who only conduct research, and those who conduct research and teach.  The 95 participants in the study submitted research proposals in the early fall and resubmitted them in late spring.  The fall and spring proposals were scored based on a rubric and compared.

The study showed that there was a significant difference between the two groups in 2 areas: improvements in writing testable hypotheses and improvements in experimental design.  For both, the group of students that taught and conducted research showed a greater amount of improvement.

The authors claim that these findings should substantially effect the way programs are designed, and that the merit of learning-by-teaching is very much at work in this type of environment.  While I do not disagree that learning does happen while teaching I would like to see more work done in this area before I change my dream of no longer teaching while I do research.  Does a second year of teaching yield the same amount of improvement as the first?  Did the students who were not teaching do a significantly better job the first time around?  And finally, if the main goal of a student’s graduate work is to contribute to the scientific community as a researcher, does teaching help, or hinder, the quality and quantity of work?  Hopefully follow-up studies will answer these questions, and we can take a second look at the nature of graduate research programs.

Feldon, David; James Peugh; Briana E. Timmerman; Michelle A. Maher; Melissa Hurst; Denise Strickland; Joanna A. Gilmore; Cindy Stiegelmeyer. Science 19 August 2011.  Vol. 333 no. 6045 pp. 1037-1039

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The Spawn of Medusa or Evolutionary Tinkering?

Living at the end of the world, Medusa, the creature with twisting serpents for hair, was such a vile looking creature that one look would turn a man to stone. Greek mythology purports that drops of blood from her severed head fell on the desert and gave rise to venomous snakes. While this account is compelling, venomous snakes predated the Greeks and until recently the exact origin of venom was just the source of speculation.

Picture 1: Medusa executed by Perseus painted by Peter Paul Rubens. Medusa was once a beautiful maiden who dared to compare her beauty to that of the god Minerva. Angered by this, Minerva turned her into a gorgon, and changed her hair into writhing snakes. Medusa was a terror until Perseus was able to sneak up while she was asleep, and by using his shield as a mirror to avoid a direct look at her, cut off her head.

Snakes are reptiles that have no legs and are strict carnivores which swallow their prey whole. Primitive snakes do not produce venom, but the advanced snakes of the superfamily Colubroidea contain all venomous snake species Venom molecules come in many forms that attach to proteins and receptors; blocking neurons, loosening the walls of blood vessels, and confusing the immune system. These toxins evolved from duplication events, which through the accumulation of mutations were recruited to the snakes’ venom weaponry. Although the venoms undergo variation in sequence and structure, the molecular scaffold of the ancestral protein is usually retained (Fry and Wuster 2004). By constructing the phylogenies of the toxin sequences, the resulting pattern of recruitment and diversification of the venoms can resolve the evolutionary history of snake venoms.

Figure 1: Phylogeny of the superfamily Colubroidea. “Colubridae” refers to the groups of snakes that lack front fangs. The 3FTx gene was believed to be exclusive to the Elapidae but it was recently discovered that it was recruited prior to the split between elapid and colubrine lineages. Viperidae is the most basal and elapidae is the most derived group.

The vipers and elapids are the most distantly related of all the Colubroidea lineages, so their recruitment of toxins can be informative to the clade as a whole (see Figure 1). Phylogenies were constructed by Fry and Wuster to resolve whether a single recruitment or independent recruitment events led to the presence of venom families in the both vipers and elapids. Of the eight toxin families analyzed, five were monophyletic to the exclusion of non-venom proteins, but were non-monophyletic among the vipers and elapid lineages. These genes, BPTI-Kunitz, CRISP, M12B, NGF, and GBL (see Figure 2) are indicative of a single early recruitment. The elapid and viperid PLA2 and natriurec peptides (see Figure 2) arose from independent recruitment events but are homologous in function. The cystatins remain unresolved as the phylogeny is consistent with both a single recruitment with losses and also multiple recruitment. The distance values indicate a single recruitment, placing the cystatins at the base of the clade (see Figure 2). The last common ancestor of the colubroid snakes had an arsenal of at least five and possibly six or more venom families existing in the vipers and elapids today.

Figure 2: The pattern of recruitment of toxin families present in both the viperids and elapids. The Cystatins remain unresolved. Recruitment events are represented by perpendicular gray bars.

With the evolutionary location of the recruitments resolved, the origin of the recruited genes was still unknown. Many scientists retain the idea that the venoms are modified saliva proteins.  Fry (2005) constructed phylogenies of amino acid sequences of the venoms and related non-venom proteins to discover the origin of the venoms. He discovered the toxin recruitment events occurred at least 24 times. After constructing the evolutionary trees of these 24 genes, Fry discovered that in only two cases did venom genes arise from salivary glands. The 22 other genes arose from genes originating in a variety of organs including the brain, trachea, heart, lung, spleen, and large intestine. Following gene duplication in one of these organs, a copy retains its function allowing the other to accumulate mutations. The mutated gene can then be recruited to producing proteins in the venom gland. Mutations that made venom more deadly not only increased the survival of the snakes but also ensured their strange status in myths and legends.

References:

Fry, B.G. and Wüster, W. (2004) Assembling an arsenal: Origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol. Biol. Evol. 21: 870-883.

Fry, B. G. (2005) From genome to ‘venome’: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res. 15: 403– 420.

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Novel Defenses: Gene Duplication Events Shape the Evolution and Fitness of Daphnia pulex

Daphnia are keystone species in their natural ecosystems, and model organisms for scientists.  Because they are speciose, ubiquitous in freshwater habitats, and can develop a wide range of phenotypes in response to environmental factors, these crustacean arthropods are often used to assess a variety of ecological impacts. Because their ecology is well understood, the research team of Colbourne et al. considered it logical and useful to sequence and begin to analyze it’s genome; the results appear in a 2011 article in Science.  The researchers combined genome sequencing, assembly, and analysis with microarray methods that enabled them to look at gene expression under various environmental conditions such as salinity or food availability, as well as the presence of infectious bacteria or toxicants.

Colbourne et al. determined that D. pulex has a ~200 Mb genome comprising 12 chromosomes and containing many duplicated genes.  A total of 30,907 protein-encoding genes were predicted with almost all highly conserved eukaryotic genes present in the assembly.  In D. pulexthere is a substantial percentage of genes with no homology to other crustacean species, and a net increase in the number of gene paralogs in the Daphnia lineage. In the phylogeny shown below (which Colbourne et al. included to illustrate the history of gene gains and losses in pancrustacean and deuterostome genomes) Daphnia are, remarkably, more similar to the outgroup (Nematostella vectensis) than more closely related organisms with respect to gene family expansion.

Scale bar corresponds to 1000 genes gained. Gene gains along each branch of the tree are scaled by the maximum value along the branch leading to D. pulex (blue); gene losses scaled by max loss along branch leading to C. elegans (yellow).

In sum, this lineage has been very good at gaining and retaining duplicated genes.  Gene families that exhibit this trend include the opsins, for light-sensing, and the hemoglobin genes, which respond to oxygen availability.  Both of these are important to fitness in Daphnia’s aquatic environment.

To examine the nature of duplicated gene families further, Colbourne et al. sequenced the hemoglobin gene cluster in D. magna, another species of Daphnia, for comparison.  The two species showed nearly identical arrangement of genes in this cluster, except D. pulex possessed an additional gene (Hb6).  Because of this, they hypothesized that the duplication and divergence of the hemoglobin genes occurred before the two species diverged.  However, an initial phylogenetic analysis of the protein-coding sequences in these two organisms suggested the hemoglobin genes duplicated independently within the two species, as seen in this figure:

A second phylogenetic analysis, this time of the non-coding sequences, revealed a different story, supporting the original hypothesis of duplication before speciation.  This tree is shown below:

In analyzing both sets of evidence, the researchers concluded that indeed, evolutionary tinkering prior to divergence of D. magna and D. pulex led to the homologous hemoglobin gene clusters.  However, their ordered arrangement within the genome subsequently begot gene conversions that, when considered alone, confuse the evolutionary history of these organisms.

Because gene duplication ultimately can lead to new functions, Colbourne et al. conducted microarray experiments to test whether patterns of transcription differed under various environmental conditions.  They found that the older the duplicated gene, the more likely it was to exhibit divergence in expression.  By looking at interacting genes with known function, they were able to investigate the functional role of gene paralogs and determine that duplication history is not necessarily coupled with functional association.  The authors hypothesize there are three possible evolutionary outcomes for duplicated gene paralogs.  If gene duplication leads to increased gene product, this increases the probability that this new gene will be retained if the product has adaptive value.  If expression patterns do not lead to proper transcription or regulation, the gene will be lost, and if new combinations of genes create products that are beneficial in the face of a certain environmental condition, they are likely to be preserved.  This study showed that genes unique to Daphnia and genes that reside in these tandemly duplicated gene clusters were significantly over represented under varying environmental conditions, and likely play a very important adaptive role in these organisms.  Because many of the genes responsive to environmental change are of unknown function, continued research will be needed to annotate the genome, and to sequence a diverse number of additional genomes for comparative purposes.

Colbourne et al. state than Daphnia possess a “compact” genome with reduced intron size compared with insects, mice, and nematodes, but although the mean size of the intron fragments are shorter (possibly due to smaller repeat elements), D. pulex have over twice as many introns per gene as Drosophila as well as an estimated intron gain that is much higher than intron loss, resulting in 78% of intron gains being unique to the Daphnia lineage.  So, Daphnia genomes are becoming more compact in the size of their introns, but not in the number of introns acquired.  This is noteworthy, since generally a relationship is seen between increased intron number and increasing intron size (Lynch & Conery, 2003).

The markedly large percentage of intron gains by Daphnia compared with others within its clade was striking, so I looked to references that would help me better understand the significance of introns in eukaryotic evolution.  A subject of debate that is addressed by Scott Roy in the 2006 Trends in Genetics review “Intron-Rich Ancestors” is whether there has been a steady loss of ancestral eukaryotic genome complexity as is shown in his studies, or rather, if intron number has repeatedly increased and decreased among different lineages, as other analyses have suggested.  Roy explains that in lineages experiencing significant intron loss, intron gains are likely to be subsequently lost, which could confuse the actual frequency of intron gain in such organisms, and may be one of the reasons why intron gain in Daphnia appears so unlike that of its nearest relatives.  A potentially steady state of gains and losses might confuse their true evolutionary history— another example of the challenges inherent in studying such phylogenies.

Roy also notes that intron loss tends to be correlated with stronger selective pressures, but on the contrary, Daphnia (possessing significant intron gain) are remarkably sensitive to environmental change. There is, however, some argument that natural selection may eventually exploit introns for adaptive purposes (Lynch & Conery, 2003). In any case, the complexity of the Daphnia genome likely plays a major role in the high level of phenotypic diversity displayed among this genus.

I recently read Sean B. Carroll’s Making of the Fittest, in which he details the role of gene duplication in allowing the evolution of new opsin proteins that produce a range of color vision in, for example, diurnal vs. nocturnal mammals, aquatic vision in cetaceans or deep-water fish, and ultra violet light detection in birds. Carroll extends his explanation to gene duplication events in ruminating colobus monkeys, which possess three copies of the pancreatic ribonuclease gene (instead of one), allowing them to digest large quantities of plant material.  Analysis of DNA and protein sequences of the duplicated ribonucleases indicate that there is a much higher ratio of nonsynonymous to synonymous changes in the new genes, providing evidence for selection in the monkeys.  If we broaden the gene family argument to include the whole genome (or at least many gene families) it is certainly conceivable that the high rate of gene duplication events in Daphnia may have lead to their characteristic phenotypic adaptability as inhabitants of highly changeable environments.

References:

Carroll, S. B.  (2006) The Making of the Fittest.  New York.  W. W. Norton & Co.

Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S, Aerts A, Arnold GJ, Basu MK, Bauer DJ, Cáceres CE, Carmel L, Casola C, Choi JH, Detter JC, Dong Q, Dusheyko S, Eads BD, Fröhlich T, Geiler-Samerotte KA, Gerlach D, Hatcher P, Jogdeo S, Krijgsveld J, Kriventseva EV, Kültz D, Laforsch C, Lindquist E, Lopez J, Manak JR, Muller J, Pangilinan J, Patwardhan RP, Pitluck S, Pritham EJ, Rechtsteiner A, Rho M, Rogozin IB, Sakarya O, Salamov A, Schaack S, Shapiro H, Shiga Y, Skalitzky C, Smith Z, Souvorov A, Sung W, Tang Z, Tsuchiya D, Tu H, Vos H, Wang M, Wolf YI, Yamagata H, Yamada T, Ye Y, Shaw JR, Andrews J, Crease TJ, Tang H, Lucas SM, Robertson HM, Bork P, Koonin EV, Zdobnov EM, Grigoriev IV, Lynch M, & Boore JL (2011). The ecoresponsive genome of Daphnia pulex. Science (New York, N.Y.), 331 (6017), 555-61 PMID: 21292972

Lynch, M. & Conery, J.S. (2003)  The Origins of Genome Complexity.  Science 302: 1401-1404.

Roy, S.W. (2006) Intron-rich ancestors. Trends in Genetics 22: 468-471.

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QOD: An alternative approach to multiple genome comparison

Mancheron et al. are attempting to re-work our current methods of genome comparison with their new program, QOD. The problems addressed by comparative genomics are annotating genes, inferring homology or orthology, and to reveal syntenic regions and rearrangement events; basically, comparative genomics are used to identify the genomic regions that are either shared or specific to individuals, strains, or species. There have been two main methods that been used to meet the needs of comparative genomics in the past. The first is the highly time consuming BLAST –hit approach, which is the method currently being used by members of the Lane Lab. This method is widely accepted but highly computationally demanding. In the lab many of our jobs take days to run, and even using the powerful Oscar ccv cluster at Brown University they can take hours to days to run. The second method commonly used is whole genome alignment which is, according to Mancheron et al, is a “highly computationally difficult optimization problem” which requires trained users and even when the complex method is done correctly may not lead to clear results. Mancheron et al set out to create a new method that was able to compute genome comparisons, and I believe they may have delivered it.

The GUI for the QOD Program

Mancheron et al use a novel concept called ‘maximum common intervals’, which they define as a genome region that cannot be extended and is shared across all genomes. This can be solved with a fast algorithm which yields a unique solution. First, they prepare the input for their algorithm. They are given a target genome and any number of reference genomes. For each reference genome, all local pairwise similarities whose statistical significance lies above a user defined threshold are returned as a set of short genomic intervals, paired up so that one sequence from the reference is paired up with one sequence from the target. An interval is considered to be ‘common’ if every reference genome has a copy of that interval. A common interval is considered to be the maximum common interval if there do not exist any larger common intervals on that region.

Mancheron et al. demonstrated the capabilities of their new technique by comparing the three available strains of E. ruminantium.  They were able to identify what percent of each strain had common interval in the other strains and returned the genes that were not common.

Because of the ease of its GUI and the speed at which it can operate, I believe that this program could overtake BLAST and whole genome alignments in the near future. Regardless, it is worth looking into if you are doing genome comparisons.

Reference:

Mancheron A, Uricaru R, Rivals E. An alternative approach to multiple genome comparison. LIRMM. Nucleic Acids Res. 2011 Jun 6, 2011.

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Limited or Lazy: Materials and Methods are Lacking Methods.

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Imagine asking a friend for a recipe to their homemade chocolate cake and getting one of the following two responses: First: Collect the material, mix with a KitchenAid Stand Mixer (Model KSM150PSOB) for 65 seconds with a whisk attachment, and bake using a GE wall oven (Model PK916SRSS) using the default settings. That is not going to be an easy recipe to follow.  What are the ingredients? Can I get the same results with a different mixer or oven? Default settings, these two words are the bane of my existence, they are meaningless and should be taboo from scientific writing.  The second response: It’s my grandmothers recipe (see Grandmas Cookbook, 1954).

What do these analogies have to do with science?  The more I read my peers’ papers the more I see trending towards incomplete Material and Methods sections. Typically, I find that there is a fairly even mix of the above mentioned strategies to explaining what was done in a paper.  Personally, I don’t find either explanation useful.

Below I will highlight some guidelines for writing Methods sections, some examples of useful and less useful descriptions, and some potential solutions for people writing their own papers.

The Goal: Before we start we need to know what we are aiming to achieve.  This may be the reason this problem has been perpetuating.  I will use Day’s How to Write and Publish a Scientific Paper as our benchmark.

…careful writing of the [Materials & Methods] is critically important because the cornerstone of the scientific method requires that your results, to be of scientific merit, must be reproducible; and, for the results to be adjudged reproducible, you must provide the basis for repetition of the experiments by others.

That is a perfect reflection of what someone should expect when reading about methods. Let’s look at two examples from published research and see how our peers have done.

Example 1: A method describing how oomycete cultures were prepared for experimental manipulation (Volz & Beneke, 1966)

Hyphal tips were removed from stock cultures and placed on agar medium containing 0.4 ~o maltose and 0.1 ~o peptone. After approximately one day, 5 mm discs of hyphae were cut out of the medium from the perimeter of the colony with a cork borer. Nine of these discs were then placed equidistantly in Petri plates. Distilled water was added to slightly submerge the plugs. Hemp seed halves were then placed on top of each agar disc.

Example 2: A method describing how gene predictions were generated (Denoeud et al., 2011).

GeneID [96] and SNAP [97] ab initio gene prediction software were trained on 300 genes from the training sets.

These examples were chosen to highlight the growing problem with our ability to report in the digital age.  As a group we are, generally, very good at putting our benchwork into text with enough detail that others could follow our experiments (Ex. 1).  As technology has taken a front seat with current research I feel that we haven’t developed a successful strategy for documenting our computer based work.

Added to the lack of effective communication we have journal page limitations, thus, further forcing us into brevity.  However, I don’t think that we can continue to use that as an excuse. With more and more journals going to online formats and the ability to create supplemental documents we must still adhere to the Scientific Method and provide detailed explanations.  According to Day, but not to the reviewers, Example 2 should not have been “adjudged reproducible.” I cannot take their gene set and return the same results using the information they supplied.  I have used SNAP in my own research and here are the steps I did to create one file:

What Example two MAY look like from my use of SNAP

  1. Moved to SNAP folder on computer for next steps
  2. Used inputs as described in MAKER readme; entered on Terminal command line
  3. ./fathom “filename”.ann “filename”.dna -gene-stats
  4. ./fathom “filename”.ann “filename”.dna -validate
  5. ./fathom “filename”.ann “filename”.dna -categorize 1000
  6. ./fathom uni.ann uni.dna -export 1000 -plus
  7. mkdir params
  8. cd params
  9. move ‘forge’ into params
  10. ./forge ../export.ann ../export.dna
  11. move ‘forge’ out of params
  12. cd..
  13. perl hmm-assembler.pl “filename” params > “filename”.hmm

I don’t think that ‘I used SNAP’ qualifies as “basis for repetition.” That is 11 different commands send to SNAP.  The example above shows only one program and only one file manipulation. Most modern genome papers use and average of 6 different pieces of software; and of those 6 most require other programs to function, compounding the problem further.  How do we report on these computational processes in a concise AND useful manner?

Solution. My first suggestion: error on the side of too much information.  If you’ve used a program you need to describe your settings.  For the commands found in my SNAP example above, I did exactly what was described in the MAKER readme file. In that case there would be no issue with stating “Custom HMM’s were created using SNAP as described in the MAKER readme.” However, if I’ve deviated in anyway from that procedure I must discuss those changes.

My second suggestion: if page limitations are an issue submit supplemental documents, or indicate where someone can go to get the detailed information required.  Dequard-Chablat et al. (2011) provide an excellent example of this.  Most journals don’t even print hard copies any more and all have SI online, do your part to make useful information available without worrying about destroying the rainforest.

My third suggestion: if you are going to cite previous work(s) make ensure the reference has done a proper job of detailing procedures.  Perpetuating a terrible description is lazy and a cop-out; do the right thing and provide a decent protocol (your paper could become the new “see” paper and up your citations). Do not daisy chain methods citations! If you say ‘procedures followed as in Calvin & Hobbes (2001)’ and I go to that paper and it links me to another reference it becomes very annoying and time consuming.

Conclusions: Ninety percent of the people that read your publication will not even look at the methods, so I know that putting in the proper amount of information seem futile. However, for the 10% that need to know what you did give half-hearted details hinders the progression of their work, and ultimately slows down our progress as scientists. We’ve all built off of previous efforts and know how difficult it can be to recreate our own experiments lets make things easier for our peers and future graduate students by doing what we are supposed to, and be as transparent as possible.

Day, RA. How to Write & Publish a Scientific Paper. 4th Edition. Phoenix: The Oryx Press, 1994.

Volz, P., & Beneke, E. (1966). An aquatic fungal bio-assay method for detection of carcinostatic agents Mycopathologia et Mycologia Applicata, 30 (2), 97-114 DOI: 10.1007/BF02130356

Denoeud F, Roussel M, Noel B, Wawrzyniak I, Da Silva C, Diogon M, Viscogliosi E, Brochier-Armanet C, Couloux A, Poulain J, Segurens B, Anthouard V, Texier C, Blot N, Poirier P, Ng GC, Tan KS, Artiguenave F, Jaillon O, Aury JM, Delbac F, Wincker P, Vivarès CP, & El Alaoui H (2011). Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome biology, 12 (3) PMID: 21439036

Déquard-Chablat M, Sellem CH, Golik P, Bidard F, Martos A, Bietenhader M, di Rago JP, Sainsard-Chanet A, Hermann-Le Denmat S, & Contamine V (2011). Two nuclear life-cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina. Molecular biology and evolution PMID: 21273631

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It Survived a Freeze, but can Ascophyllum nodosum take the Heat?

Ascophyllum nodosum is a fucoid intertidal seaweed found along North Atlantic coastlines. Its northerly range extends from Arctic Canada, Norway, Greenland, and Iceland down to Portugal and Long Island where it has an abrupt southern limit at the 42° N latitude. A. nodosum is a dominant rocky intertidal species and provides habitat and canopy for other intertidal organisms. Because of its role as a foundational species, change in the range and abundance of A. nodosum has substantial effects on entire intertidal communities (Olsen et al. 2010).

The distribution of present day intertidal species was affected by the last glacial maximum (LGM) that occurred 20,000 years ago.  The advancing ice pushed many northern algae and invertebrate species to southern refugia. Refugia do not always exist at the southern most edge, but most expansion after the glacial retreat has been from the southern edge to the north. As a result, post-glacial populations have been genetically characterized by “southern richness and northern purity” (Hewitt 1999). At the front of the dispersal, the northern populations typically exhibit low genetic diversity. Southern populations, however, usually have increased diversity due to the fusion of once isolated subpopulations. Olsen et al.  (2010) tested this hypothesis that links genetic variation and population structure to climactic periods on A. nodosum, using six microsatellites and an mtDNA intergenic spacer as markers.

The predicted haplotype diversity is not exhibited by A. nodosum across the 28 locations (see Fig 1 below). The coasts have retained a genetic signature that indicates isolation that predated the LGM. Population divergence between the North American and European halpotypes is estimated to be between 1.8 million years ago and 0.08 million years ago, indicating separate populations existed on both sides of the Atlantic long before the LGM. Private alleles also confirm its existence on both Atlantic coasts prior to the LGM.

Figure 1. A. nodosum haplotype distribution and haplotype network (inset) for the IGS-trnW locus (mtDNA marker). Haplotypes are widely distributed, there is no distinct southern richness and northern purity pattern. The 20 thousand years since the LGM does not account for the amount of sequence divergence and for the distinct alleles found in northern populations.

A large effective population size (Ne), estimated in A. nodosum to be between 12,000 and 486,000, was sufficient to maintain rare haplotypes.  This ancestral Ne is approximately the same in the present population. The migration rate from Europe to North America is 5 to 60 fold greater than migration in the reverse direction.  However, there is minimal contemporary population differentiation across the Atlantic. Like the findings of other algae and invertebrate studies, Olsen et al. found Brittany was clearly a refugium and hotspot for diversity. Evidence supports an additional mid-range refugium in southern Maine, which preserved private haplotypes and North American genetic diversity (see Figure 2 below).

Figure 2. Neighbor joining tree for the six microsatellite loci. Grey shaded areas indicate refugia locations, upper circle is the Southern Maine (USA) refugium and lower circle is the Brittany (France) refugium. 

Like a “marine tree”, Olsen et al. characterize A. nodosum by its long lifespan, large size, high fecundity, high within population diversity and weak large scale differentiation. Like an old growth forest, Brittany is an important site for genetic diversity and is important in species conservation. A. nodosum is an old species and has survived many glacial and interglacial cycles.

Understanding how a species reacted to climate change in the past can provide a substantial basis for future projections. It has been reported that intertidal species in the North Atlantic have experienced ranges shifts of 50m per decade in response to current climate change (Helmuth et al. 2006). Externally fertilized eggs and sperm produce non-buoyant zygotes, which typically disperse less than 7 meters but to a maximum of 30m (Olsen et al. 2010). A rapid shift in summer sea temperatures could wipe out the southern populations, if unable to disperse to a more northerly range. As the northern locations increasingly become ice-free year-round, the northern population of A. nodosum will likely experience expansion.

The indirect influence climate change has on biotic interactions is particularly interesting to me since I study Vertebrata lanosa, a red algal epiphyte on A. nodosum.   Helmuth (2006) points out that organisms exposed to the same conditions can exhibit very different core temperatures and therefore different levels of physiological stress. A. nosodum is the host to 3 algal epiphytes, 2 brown and the red algae V. lanosa.  Longtin et al. (2009) found that V. lanosa may be less tolerant to high irradiance and dessiation stress compared to both the brown epiphytes. However, V. lanosa is still able to exclude the other epiphytes from the middle frond segments. Some cold-water species, including A. nodosum, have not yet experienced a range shift. With the advance of climate change, V. lanosa may be exposed to different conditions, lose its dominance, and retreat into refugia or experience localized extinctions.

Figure 3. Epiphytic Vertebrata lanosa tufts on the brown algae, Ascophyllum nodosum. The red algae, V. lanosa is found in greater abundance at mid-intertidal elevation and within the canopy of A. nodosum rather than on the periphery (Longtin et al. 2009).

It would be interesting to see if the post-glacial V. lanosa population exhibits the same genetic framework as A. nodosum. The more resilient A. nodosum is better able to survive at the extremes of its limits, and as climate change progresses how will the warming seas affect the distribution of V. lanosa compared to A. nodosum?

Literature Cited

Olsen JL, Zechman FW, Hoarau G, Coyer JS, Stam WT, Valero M, Åberg P (2010) The phylogeographic architecture of the fucoid seaweed Ascophyllum nodosum: an intertidal ‘marine tree’ and survivor of more than one glacial-interglacial cycle. J Biogeogr. 37: 842-856.

Hewitt, GM (1999) Post-glacial re-colonization of European biota. Biological Journal of the Linnean Society, 68, 87-112.

Helmuth, B, Mieszkowska N, Moore P, Hawkins SJ (2006) Living on the edge of two changing worlds: forecasting the responses of rocky intertidal ecosystems to climate change. Annual Review of Ecology, Evolution, and Systematics, 37, 373-404.

Longtin, CM, Scrosati RA, Whalen GB, Garbary DJ (2009) Distribution of algal epiphytes across environmental gradients at different scales: intertidal elevation, host canopies, and host fronds. J Phycol, 45: 820-827.

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Gut microbiome studies lend significance to the idiom “you are what you eat”

The human intestine is an ecosystem that supports up to 100 trillion microbes—a cell number that is roughly ten times greater than the human cells that comprise our bodies. In addition to the vast number of cells comprising the microbial community of the gut, there may be over 100 times the number of bacterial genes present compared with the number of genes in our own DNA (Bäckhed et al., 2005). These beneficial microbes are instrumental in our ability to extract nutrients from food, and also play an important role in the development of our immune systems.

In a study conducted by Eckburg et al. (2005), 16S rRNA genes were obtained from DNA extracted from gut mucous or feces via PCR primers targeted to broad phylogenetic groups of bacteria.  The data indicated seven divisions (phyla) of bacteria, but by far the most dominant groups were the Bacteroidetes and Firmicutes.  Figure 1 from a review by Ley et al. (2006) highlights the Eckburg et al. finding by comparing the percent representation of divisions in a variety of environments.

Figure 1.  Comparison of Microbial Diversity in the Human Colon, Mouse Cecum, Fish Gut, Ocean, Stromatolite and Soil

Eckburg’s research team discovered that there were high levels of strain variation, but far fewer intermediate and deep lineages, supporting the idea that gut bacteria evolution is a classic case of adaptive radiation, where a few successful colonists gave rise to a variety of descendants, thus there was diversification of an initial population bottleneck into various strains that likely correspond to ecotypes.

A logical question then arises— How do the microbes withstand attack, by a bacteriophage for example, with such low levels of diversity?  It is suggested that genomic and/or transcriptomic diversity at the subspecies level results in a broad collection of responses to various environmental factors.  In other words, the microbes are functionally diverse in their adaptive responses despite relatively low levels of phylogenetic diversity (Bäckhed et al., 2005).  A fascinating example of microbial genetic differentiation via LGT was recently recovered by Jan-Hendrik Hehemann et al. (2010).  Working with Zobellia galactanivorans, a member of the marine Bacteroidetes, investigators discovered an enzyme (porphyranase) responsible for breaking down porphyran, an abundant polysaccharide in the red algal species Porphyra, on which Z. galactanivorans is often found.  While searching gene-sequence databases, investigators came across predicted porphyranase sequences in metagenomes derived from human feces and in the genome of the resident human gut bacterium Bacteroides plebeius, suggesting B. plebeius acquired the genes laterally from marine bacteria.  Because it turned out these sequences were present in Japanese individuals but not in residents of the United States, Hehemann et al. concluded that Z. galactanivorans were introduced via Porphyra (“nori”), the traditional seaweed used to wrap sushi and a common component of the Japanese diet.  The researchers hypothesize that by constantly consuming seaweeds, Japanese communities produced a selective force that led to retaining the beneficial porphyranase genes in their gut microbiomes.

But, if lateral gene transfer is considered to be a homogenizing evolutionary force, and we assume Bacteroidetes and Firmicutes have continually swapped genes over their evolutionary history, wouldn’t we expect to see little variability in the makeup and function of their genomes?  To the contrary, these two groups have a known difference in the GC content of their DNA, evidence that despite selection for functional redundancy, they are distinct enough genetically to occupy different niches in the gut environment.  It is likely they have complementary roles as well, where the product of one microbe becomes the substrate for another, and a complex yet efficient chemical food web results. Experiments that aim to quantify rates of LGT among key members of the gut microbiota will help to elucidate the degree to which microbes differ between these gut niches.

In general, the ability to aid the host in extracting energy and nutrients from food is considered an important benefit that has resulted in a long history of coevolution between humans and gut microbes.  However, recent research has demonstrated that resident microbial communities can become pathogenic within the context of other factors, such as host genotype, diet and behavior.  For example, groups that are highly efficient at extracting calories from food may confer a health advantage to human individuals or communities with limited access to food resources.  Conversely, these same groups could have negative health consequences (e.g. obesity) for those who have ready access to many types of food.  Ley and Turnbaugh et al. (2006) found that the proportion of genetic material from Firmicutes was higher in obese individuals than in lean individuals, and further, when obese individuals lost weight, the proportion of Firmicutes decreased to a level more like that of lean individuals.

The research team of Carlotta De Filippo et al. (2010) took the work of Ley et al. a step further by honing in on the coevolution of microbiota associated with different diets in human populations.  The study compared the gut microbiota of children aged 1-6 living in a village in rural Africa with those from European children of the same age group living in a typical modern western environment. The diets of the African children were characterized as low in fat and animal protein and high in starch, fiber, and plant polysaccharides, whereas the European children’s diets were high in animal protein, sugar, starch and fat and low in fiber.  Both groups of children were breast-fed, for up to two years in the African group and up to 1 year in the European group.  Researchers sequenced 16S rRNA from the fecal matter of several children in each population, and thought they found very similar bacterial phyla across groups, the African children had significantly more abundant Bacteroidetes and lower levels of Firmicutes compared with European children.


Figure 2.  Dendogram clustering of samples from African and European populations based on their genera.  Note that the subcluster at the middle of the tree are samples taken from the three youngest African children (aged 1-2 yrs) and two 1-year old European children, all of which represent children that are breast-fed rather than receiving the respective African or European solid food diets. 

Additionally, the African children contained bacterial genes for hydrolysis of cellulose and xylan (plant fibers), which the European children completely lacked.  De Filippo et al. suggest that the gut microbiota of the African children coevolved with their polysaccharide-rich diet.  The investigators propose that the European children may be predisposed to obesity due to the increased Firmicutes/Bacteroides ratio in their gut, likely resulting from their diet.  Most intriguingly, they found that short-chain fatty acids produced in the gut from the bacterial fermentation of whole grains were abundant in only the African children’s feces, noting the connection between these molecules and their protective role against gut inflammation.

Many hypotheses regarding the evolution of the human gut microbiome still remain to be tested.  Among these, what I find particularly interesting are potential connections between the makeup of the gut microbiome and cultural differences between developing and industrialized countries.  What are the impacts of improved sanitation on the gut microbiomes in developed nations, assuming we are reducing exposure to hosts of microbes that may have been able to set up shop in our gut?  What impact does this have on the development of the host immune system?  What about frequent use of antibiotics during postnatal development in western cultures? How might use of antibiotic treatments affect the establishment of long-term microbe communities in the gut?  Furthermore, might it be possible to establish particular microbial assemblages within the gut as a therapeutic strategy to treat health conditions such as inflammatory gut disease or autoimmune disorders?

Literature Cited:

 
Backhed, F. (2005). Host-Bacterial Mutualism in the Human Intestine Science, 307 (5717), 1915-1920 DOI: 10.1126/science.1104816

De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., & Lionetti, P.  (2010).  Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa.  PNAS, 107 (33): 14691-14696.

Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., & Relman, D.A. (2005).  Diversity of the human intestinal microbial flora.  Science, 308: 1635-1638.

Hehemann, J.H., Correc, G., Barbeyron, T., Helbert, W., Czjezek, M., & Michel, G.  (2010).  Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota.  Nature, 464:  908-912.

Ley, R.E., Peterson, D.A., & Gordon, J.I.  (2006).  Ecological and Evolutionary Forces Shaping Microbial Diversity in the Human Intestine.  Cell, 124: 837-848.

Ley, R.E. Peter J. Turnbaugh, P.J., Klein, S., & Gordon, J.I. (2006).  Microbial ecology: Human gut microbes associated with obesity. Nature, 444: 1022-1023.

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