BEACON Researchers at Work: An Adventure in Thailand

This week’s BEACON Researchers at Work blog post is by MSU postdoc Eben Gering.

I. The Land of the Leech

Figure 1. Golden Naga in Emerald Buddha temple Wat Pra Kaeo, Bangkok, Thailand

Figure 1. Golden Naga in Emerald Buddha temple Wat Pra Kaeo, Bangkok, Thailand

This spring I received a last-minute invitation to join a French film crew in Thailand, which left me a) totally stoked! b) with virtually no prep time. And so I found myself arriving in Khao Yai National Park with a mess of hastily borrowed equipment, abundant enthusiasm, and very little basic information about the region.

For example: did you know that Southeast Asian forests are home to a 150 million-year-old, ten-eyed beast whose name means blood thirsting guts? If you don’t perhaps you are picturing something along the lines of the fantastical Naga (Figure 1) – a ferocious serpent believed to protect the Buddha. Naga guard temple entrances throughout Thailand, but are nowhere near as ubiquitous as Haemodipsidae (Figure 2) which, FYI, are hermaphroditic land leeches.

Figure 2.  Haemodipsidae zylandica. One of ~90 species of terrestrial (“land”) leeches.

Figure 2. One of ~90 species of terrestrial (“land”) leeches.

When our guide first offered us prophylactic “leech sox” I didn’t want them. In my undergraduate days I had read a terrific book1 about tropical biology which eventually helped land me in this Thai jungle many years later. One of the book’s most memorable chapters (recapped in a recent radiolab episode) concerns an evolutionary biologist who lets a parasitic botfly pupate in his head for heuristic (learning) purposes. Botfly husbandry has since become a fashionable act among a small and hardcore subset of tropical biologists.

The idea of joining Club Botfly makes me a little queasy, and involves an apparently painful initiation too. But leeches – painless, virtually incapable of transmitting disease, a boon to human health and medicine for at least three millennia – provided an attractive alternative source of field “cred.” In fact, I’ve been quite curious about these animals since meeting Mark Siddall, a passionate and persuasive expert on leech biology who “fishes” for his study subjects by dangling his legs in murky water. In parallel manner, Dr. Siddall advocates for these greatly maligned creatures. He lures his listeners past the ‘ick’ reflex into a reluctant appreciation for their elegance and mystery. It is, after all, an animal which once fed on dinosaurs, and outlived them, and will probably outlast us.

An enlightened view of the leech is also apparent in Khao Yai’s local, Buddhist residents, who point out their relative harmlessness with the saying:

The hero of Khao Yai National Park…

Leeches…

They eat blood but not the trees! 2

Will I be able to get a leech if I don’t wear the sox? I asked Tony, our guide. But I needn’t have worried. In the coming days I would have many chances to observe sanguivory (bloodfeeding) first hand. Leeches would be so abundant at times that we would see and hear them moving towards us on the forest floor.

II. Why won’t the chickens cross the road?

Figure 3.  Red Junglefowl (Gallus gallus) eating ectoparasites from a sambar deer (Rusa unicolor). Photo courtesy of Tontantravel.

Figure 3. Red Junglefowl (Gallus gallus) eating ectoparasites from a sambar deer (Rusa unicolor). Photo courtesy of Tontantravel.

The chief purpose of our expedition was to visit the Red Junglefowl (Gallus gallus) within its native range. These extremely wild and elusive birds (figure 3) are the domestic chickens’ closest relatives. The French guys (Figure 4) were out to gather the world’s first 4k footage of wild Red Junglefowl. I, in turn, hoped to gather audio recordings and compare vocalizations to those from a non-native (Hawaiian) G. gallus population. Our recent research3 indicated that Pacific island G. gallus share both Red Junglefowl and “chicken” (i.e. domestic) ancestry. We are now investigating which wild, domestic, and/or hybrid traits have been favored in these feralized populations.

Figure 4. Wildlife filmmakers Benoit Demarle and Nicolas Cailleret

Figure 4. Wildlife filmmakers Benoit Demarle and Nicolas Cailleret

During our first days out, we occasionally heard Red Junglefowls calling from deep within the forest. That’s a somewhat surreal experience for western eyes and ears, because they look and sound just like backyard roosters, yet glide through primary tropical forests without effort, thriving among leopard cats and pythons.

From day one, I was able to collect audio recordings, but our efforts to film (with a menagerie of cumbersome gear) typically sent our targets fleeing dozens, or even hundreds of meters into impenetrable growth. A day’s work yielded only a few precious minutes of 4k video.

On day two, the production director (Benoit) put his cameraman (Nicolas Cailleret, a professional falconer back in France) in a camouflaged bird blind. We left him there before sunrise in an area where we’d seen junglefowl eating figs. Later a hornbill polished off the figs, so we decided to bait the area with another of the Red Junglefowl’s preferred food sources: elephant dung (figure 5).

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Figure 5a,b. Elephant dung collected in an attempt to lure Red Junglefowl into the open

Figure 5a,b. Elephant dung collected in an attempt to lure Red Junglefowl into the open

As I grabbed my first handfuls of the dung, which was surprising lofty, fibrous, and not the least bit stinky, I thought how much I love my job. I was 11 time zones from home, and almost intoxicated by the nearly deafening whirs of the cicadas, the sweet and mournful ‘bwoooooops’ of gibbons, the bright flapping of birdwing butterflies gliding on hot, humid air.

While we picked the freshest dung we could find, the waste of our planet’s largest herbivore was already becoming a complex ecosystem. By coincidence, the book I mentioned earlier 1 (and highly recommend) also contained a whole chapter on poop – specifically, how scatophagic4 organisms colonize and compete for this rich resource within minutes of its “birth.”

III. In praise of Bill Nye

Figure 6. Stealth camerawork by Nicolas Cailleret

Figure 6. Stealth camerawork by Nicolas Cailleret

While our brief experiment with elephant dung brought on an existential rapture, it did not bring any Junglefowl in front of the camera. Soon, however, Benoit and Nicolas found other ways to catch clips of the birds in stealth (figure 6). They crouched underneath a camouflage tarp in the bed of Tony’s rolling truck, ready to start shooting when Junglefowl were spotted. They learned and patrolled a few territories’ boundaries. And then, when they had acquired sufficient footage of their non-human prey, they turned the camera on me.

I am curious to see myself on French television, but not worried a bit. I cannot possibly look worse than I felt. For two days I had been crowing for the camera, a one-eyed monster that emitted so much heat it required its own water breaks. Usually this work was done standing in direct sunlight, sometimes while peeling off leeches… whose novelty was waning (sorry, Mark!). The heat seemed to fog up my brain, making it a struggle to follow simple instructions or speak intelligibly. And I could see that this was beginning to try the (extraordinary) patience of the crew – two men who had just spent uncomplaining days in a sauna-like blind surrounded by elephant poop.

On our last afternoon, we all spent an hour crouched in that small, poorly vented vinyl blind collecting footage of me photographing imaginary junglefowl. Nicolas held the camera a few inches from my face while Benoit held the microphone over my head. We were as close together as the heads of the Naga (Figure 1), and it was so hot in there – so very hot and humid (and the whole shot seemed so unimportant) that I wondered if perhaps they were trying to kill me in order to increase their film’s marketability.

But apart from such moments of heat and performance-induced panic, it was a joy to watch Benoit and Nicolas work. There was boldness and creativity in every step of their process, from handling equipment failure, to avoiding extortion by corrupt officials, to framing a narrative arc across this wild landscape, its diverse wildlife, and their bumbling biologist that would eventually reach into the homes of French families.

I was eager to participate in Benoit and Nicolas’ film because nature documentaries helped cultivate my interest in biology. And as a scientist on the more right-brained end of the spectrum, I enjoy seeing artists at work. While I would jump at another such chance, our filming of just one segment (25%) of a 45 minute show was both physically and mentally exhausting. I couldn’t do this work everyday, and have redoubled my respect for the stamina of David Attenboroughs and Bill Nyes.

When I first read Tropical Nature1, my game plan was to become a writer for a documentary production company. But I have since become increasingly fascinated with basic research questions, which take sustained (and sometimes boring) effort to answer. Benoit, on the other hand, began his career as an acarologist (mite expert) before transitioning out of research and into film. The mite work, he said, was much too specialized to match his interests. I could understand this, I said, but also asked him to tell me more about the mites. I think we have chosen wisely.

1 Forsyth, A., & Miyata, K. (2011). Tropical Nature: Life and Death in the Rain Forests of Central and South America. Simon and Schuster.

2 Translation by Elizabeth Borda

3 Gering, E., Johnsson, M., Willis, P., Getty, T., & Wright, D. (2015). Mixed ancestry and admixture in Kauai’s feral chickens: invasion of domestic genes into ancient Red Junglefowl reservoirs. Molecular ecology.

4scat feeding

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BEACON Researchers at Work: The Social Lives of Bacteria

This week’s BEACON Researchers at Work blog post is by MSU faculty member Chris Waters.

Nature red in tooth and claw”-Lord Alfred Tennyson

Tennyson’s famous phrase eloquently describes the adversarial nature (pun intended) that arises from Darwin’s concepts of natural selection and survival of the fittest. From a human perspective, these concepts are easy to understand. One only needs to attend my son’s little league games to see how competition is ingrained in humans. Competition for limited resources leads to genetic winners and losers. This idea permeates throughout the tree of life; indeed, visit the Lenski lab at Michigan State to see the ongoing results of a 25 year Escherichia coli “cage match”.

But we all know that natural interactions are not always adversarial. Humans are also highly cooperative individuals, often helping one another at their own expense. Bees, wasps, and ants, live in highly cooperative communities in which individual members forgo their own reproduction for the net good of the group.

Bioluminescent Vibrio harveyi actively quorum sensing.

Bioluminescent Vibrio harveyi actively quorum sensing.

It has become abundantly clear that even unicellular lifeforms participate in highly complex social interactions. The first appreciation for social interactions in bacteria came with the discovery of chemical communication in bioluminescent Vibrios. It was found that these ocean growing bacteria prematurely induced bioluminescence when exposed to cell-free supernatant harvested from a dense culture. It was subsequently determined that bacteria produce and secrete small chemical signals, termed autoinducers, to coordinate behaviors in response to cell density. We now appreciate that this process, known as quorum sensing, is widespread in bacteria and it is generally hypothesized that all bacteria engage in some form of chemical communication. Autoinducers, and their sensing apparatuses, come in many flavors, suggesting that this is a highly beneficial trait that has coevolved multiple times.

Quorum sensing is often considered to be a mechanism for coordinating cooperative behavior in bacteria. While I think this aspect of quorum sensing is often overstated, it is quite true that many cooperative tasks are controlled by quorum sensing. For example, secreted “public goods” (i.e. shared benefits that all of the members of the community can utilize, not just the producers) are often clearly induced by quorum sensing. This cooperative situation leads to a strong selection for freeloading cheats which can gain the benefit of the public good without paying the production cost. Imagine five students working on a group project-at least one is typically a freeloader who gets the benefit of the grade without putting in the effort. This is an evolutionary smart strategy for the freeloading individual, but this strategy destabilizes cooperation within the group.

Eric Bruger pondering the evolutionary underpinnings of quorum sensing in bacteria.

Eric Bruger pondering the evolutionary underpinnings of quorum sensing in bacteria.

Eric Bruger, a graduate student in my laboratory, is studying this fundamental social evolution question-the evolution of cooperation-in the bioluminescent bacterium Vibrio harveyi. Studying social evolution using bacteria has a multitude of benefits including fast generation times, huge population sizes, easily measurable phenotypes, exquisite control of the environment, and, perhaps most importantly, the ability to genetically manipulate the cooperative state of test subjects (the ethics of genetic manipulation are much less stringent with microbes!).

Eric has found that quorum sensing in V. harveyi does indeed induce public good production, and he has identified environments where public good production is required for growth. When Eric genetically manipulated V. harveyi to unlink public good production from quorum sensing control, leading to constitutive public good secretion, he found that this mutant strain was rapidly invaded by cheating cells. This led to a population crash, typically referred to as a “tragedy of the commons”. However, having the public good linked to quorum sensing stabilized cooperation and the cheats could not invade. Thus, Eric experimentally demonstrated that the ability of cooperating cells to communicate stabilizes cooperation!

Eric then extended these experiments to study the natural emergence of cheats following experimental evolution of V. harveyi for over 2,000 generations. He observed that quorum sensing control of cooperation delayed cheater invasion, but eventually cheaters did emerge. However, communication prevented cheaters from sweeping the population, leading to a complex mixture of cooperators and cheats that is reminiscent of V. harveyi strains observed in the natural world.

Will Soto and Chris Waters glean wisdom from all sources including Bob Marley and Star Wars to study quorum sensing.

Will Soto and Chris Waters glean wisdom from all sources including Bob Marley and Star Wars to study quorum sensing.

Public good production is but one of hundreds of traits regulated by quorum sensing in V. harveyi. How do the other regulated behaviors impact the stability of quorum sensing? Enter Dr. Will Soto, a BEACON funded postdoctoral fellow in my laboratory. Will is exploring the impact of quorum sensing on central metabolism in V. harveyi. Will examined the growth of three strains-the wild type strain with functional quorum sensing, a strain that cannot communicate, and the constitutive quorum sensing strain-in a hundred different laboratory growth media. He found dramatically different growth patterns of these strains, but, surprisingly, the wild type quorum sensing strain did as well or better than both quorum sensing mutants in virtually all environments examined! This result shows that communication in bacteria not only stabilizes cooperation, but it is also a mechanism to enhance colonization of many different ecological niches, suggesting that quorum sensing provides bacteria a large fitness benefit in the real world when faced with ever changing environments.

Quorum sensing is but one of many social traits in bacteria. Most bacteria can also form multicellular communities encased in a protective matrix called biofilms. Some photosynthetic cyanobacteria actually grow as multicellular filaments and exhibit striking differentiation and division of labor. Yet others, like Myxococcus, undergo complex cooperative development and form multicellular fruiting bodies upon starvation. All of these systems are excellent unicellular models to test concepts of social evolution.

Clearly, cooperation is not limited to us multicellular organisms; unicellular organisms have a robust social life. It is important therefore to consider not just nature’s “tooth and claw” but perhaps also “Nature gentle with helping hand”.

To learn more about research in the Waters lab follow us on twitter (@WatersLabMSU) or visit our website: https://www.msu.edu/~watersc3/.

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3rd Annual Big Data in Biology Symposium at UT Austin

Summary by UT Austin graduate student and symposium organizer Rayna Harris.

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The 3rd Annual Big Data in Biology Symposium on May 15, 2015 was hosted by the Center for Computational Biology at UT Austin and organized by some BEACON members. This annual symposium provided an ideal opportunity to interact with attendees from UT Austin and nearby institutions with an interest in computational biology, bioinformatics, and systems biology. Here, I summarize scientific content of the talks, breakout sessions, poster session, and industry dinner.

The Talks

The symposium talks covered research into epigenetics, genomics, transcriptomics, and immune repertoires in a wide range of organisms. We heard from graduate students, postdocs, and faculty members, providing a diversity of experience and perspective of the impact of big data on biology.

This year’s keynote lecture was delivered by Dr. Shelley Berger from The University of Pennsylvania. She discussed her research into the epigenetic mechanisms regulating of caste-specific phenotypes in eusocial ants. Amelia Hall, a graduate student in the Iyer lab, also focused on epigenetics, describing her analyses of histone pattering in glioblastoma tumors and the effects on gene expression.

BEACONite Dr. Jeffrey Barrick talked about technological advances for mapping beneficial mutations in the E. coli Long-term Experimental Evolution Experiment and the exciting implications for evolution and adaptation. Dr. Kasie Raymann, a postdoc in the Moran lab, talked about her thesis research using large scale genomic data to understand the evolutionary origins of eukaryotes.

Justine Murray, graduate student in the Whiteley lab, discussed her use of RNA-seq and Transposon (Tn) Seq to understand the dynamics of poly-bacterial infections. Dr. Mikhail Matz talked about recent computational tools his lab has developed for analyzing RNA-seq data. BEACONite Dr. Becca Young (Hofmann lab) also described new computational tools that can be used for identifying homologous genes groups, an important step for comparative transcriptomics. Jeff Hussmann, a graduate student in the Press and Sawyer labs, discussed how systematic biases in ribosome profiling experiments can lead to an incorrect understanding of translation speed.

Dr. Jenny Jiang talked about using high-throughput sequencing and single cell analysis to characterize immune repertoires, and Dr. Oana Lungu, a postdoc in the Georgiou and Ellington labs, shared her in silico approach for characterizing changes in protein structure of immune after antigen experience.

The Lunch Breakout Sessions

The lunch breakout sessions provided attendees the opportunity to have small-group discussions with various big data professionals over a catered lunch. These sessions were aimed at helping attendees network with other like-minded researchers and discover resources for different aspects of and opportunities in data science. The three breakout session topics included 1) Big Data in Medicine & Health, which highlighted the tremendous opportunities and technical challenges for evidence-based medicine arising from electronic health records; 2) Careers in Biotech/Industry, which provided insights into non-academic careers; and 3) Open Science, which discussed the importance of data sharing, public access to research, and the increasing role of social media in scientific communication.

The Poster Session

A poster session followed the symposium and allowed trainees to explain their work and facilitate fruitful exchanges. There were twenty posters on various topics, including genomics, transcriptomics, epigenetics, and proteomics. BEACONite Dr. Daniel Deatherage (Barrick lab) and Claire McWhite (Marcotte lab), respectively, won the best postdoc and student poster awards.

Figure 2. The Poster Session. Left: The poster session took place in a lovely ballroom. Right: The symposium ended with a postdoc and graduate student poster award presentation. L-R: Dr. Scott Hunicke-Smith, BEACONite Dr. Daniel Deatherage (postdoc winner), BEACONite Dr. Hans Hofmann, Claire McWhite (grad student winner), and BEACONite Rayna Harris.

The Poster Session. Left: The poster session took place in a lovely ballroom. Right: The symposium ended with a postdoc and graduate student poster award presentation. L-R: Dr. Scott Hunicke-Smith, BEACONite Dr. Daniel Deatherage (postdoc winner), BEACONite Dr. Hans Hofmann, Claire McWhite (grad student winner), and BEACONite Rayna Harris.

The Industry Partners Dinner

This year, we hosted an Industry Partners Dinner for representatives of the local biotech and high-tech industry corporations to meet a diverse set of graduate students in the College of Natural Sciences who are interested in careers in industry. Graduate students from multiple graduate programs and a handful of faculty members networked with associates from Asuragen, Bioo Scientific, Dell (who generously sponsored this event), IBM, Lab7, Macromoltek, and Sonic Healthcare USA.

We invited 12 industry partners, 12 faculty/staff members, and 24 students. We have found that 8-person tables work well for promoting discussion, so seats were assigned such that every industry partner and faculty member was flanked by two students. Everyone agreed that this seating arrangement works very well for facilitating conversation between people at different career stages and with diverse backgrounds, many of whom had never met.

Based on the many positive and encouraging comments we received most of the attendees, our first formal event with our Industry Partners was a huge success, as it opened many doors for new graduate students interested in different career options and for industry-academia partnerships.

The Industry Partners Dinner. This cocktail hour and dinner provide more than 20 graduate students the oportunity to chat with people from the thriving biotech and bioinformatic industry in Austin Texas. Seating was arranged so that Industry partners were flanked by students, providing ample oportunity to learn about graduate research at UT Austin.

The Industry Partners Dinner. This cocktail hour and dinner provide more than 20 graduate students the oportunity to chat with people from the thriving biotech and bioinformatic industry in Austin Texas. Seating was arranged so that Industry partners were flanked by students, providing ample oportunity to learn about graduate research at UT Austin.

Acknowledgments

The Organizers

The symposium and the dinner would not have happened without the efforts of Hans Hofmann (Director of the CCBB) and Scott Hunicke-Smith (Director of the GSAF). BEACONite Laurie Alvarez (CCBB) is a crucial team member who handles all the finances and so much more. Nicole Elmer (CCBB) is an excellent graphic designer and helped design and distribute all communication materials. Thanks to Becca Tarvin and Sean Leonard for helping organize the Industry Partner Dinner. 

The Sponsors

We are grateful to BEACON for providing travel support to BEACON Managing Director Dr. Danielle Whitaker and to The Graduate School Academic Enrichment Fund for providing travel support for Dr. Shelley Berger. Graduate Student Assembly Appropriations were used for printed materials and speaker gifts. We want to extend a special thanks to Dell for generously sponsoring the Big Data in Biology Industry Partners Dinner.

For more information about the Big Data in Biology Symposium, visit the website at http://www.ccbb.utexas.edu/dataconference.html

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BEACON Researchers at Work: What ice cream and biofuels have in common: vanillin and the microbes that eat it

This week’s BEACON Researchers at Work blog post is by University of Idaho postdoc Jessica Audrey Lee.

Greetings, BEACON fans. I’m writing from beautiful Moscow, ID, where I work as a postdoctoral researcher in the Marx Lab at the University of Idaho, and where our incubator smells like a cookie factory. It smells that way because the model compound we’re working with at the moment is vanillin, which is not only an important substance in the global food and fragrance industries, but is also a key intermediate in the microbial degradation of lignin.

Oenophiles (of which I am one) may already know this: vanilla-like flavors in wine are almost always a sign that the wine has aged in oak barrels, because oak wood, with its high lignin content, flavors the wine by releasing aromatic bits and pieces of that lignin into it. Foodies (of which I also am one) may also already know this: more than 99% of the vanilla flavoring used today doesn’t actually come from Vanilla planifolium (the vanilla orchid, the source of “real vanilla extract”), but instead is produced industrially either from fossil fuels or from chemical processing of lignin (Walton et al., 2003). And biofuels researchers also know that vanillin (and its derivative, vanillic acid) can be used as a decent stand-in for the monomers that make up the very complex polymer that is lignin.

vanillin_vanillate_MethylobacteriumMy current research is part of a larger project, with collaborators at BU and LBNL, to create a microbial consortium for degrading lignocellulosic biomass (that is, assorted plant parts, some of them woody) into fatty acids that can be used as biofuels. Our consortium will include some of the usual suspects—bacteria from the well-known soil-dwelling genus Streptomyces, and the fatty-acid-accumulating yeast Yarrowia lipolytica—as well as a more unusual player, Methylobacterium extorquens. This Methylobacterium species is typically found on plant leaves—not in the soil, degrading dead plant matter—but we’re introducing it to our consortium because it has a special talent: handling formaldehyde.

Lignin is a difficult compound to degrade, and one of the reasons it’s difficult is that it contains a great number of methoxy (-OCH3) groups. The typical microbial approach for dealing with these methoxy groups is to remove them and turn them into formaldehyde, but the subsequent process of detoxifying the accumulating (very toxic!) formaldehyde can severely slow down lignin degradation (Mitsui et al., 2003). Happily, Methylobacterium, because it typically eats methanol for a living, is very efficient at processing formaldehyde as a metabolic intermediate, so if we could just get it to pull the methoxy groups off of the lignin monomers we could potentially make significant gains in biofuel production from lignocellulose.

M. extorquens (and most Methylobacterium species) carries pink sunscreen-like pigments. M. nodulans, adapted to living in the dark, is white.

M. extorquens (and most Methylobacterium species) carries pink sunscreen-like pigments. M. nodulans, adapted to living in the dark, is white.

This is where the evolution and ecology get interesting. Methylobacterium is in the order Rhizobiales, and therefore not too distantly related to some other plant-associated bacteria that have been shown to degrade lignin, for instance Bradyrhizobium japonicum (Sudtachat et al., 2009) (which can often be found in plant root nodules). In fact, if you compare the two genomes at the locus of the B. japonicum vanillate-demethylating genes (vanAB), you’ll find that all Methylobacterium species also have a gene that looks similar enough to be part of the same pathway (I call it vanA-like). Sadly, M. extorquens can’t demethylate vanillin (hey, it was worth a try—it’s possible we’re the first lab that ever bothered to ask). However, we’ve found that it has a close relative, M. nodulans, that can! We’ve located the M. nodulans gene cluster that we think is responsible, and it seems to have nothing to do with the vanA-like gene that I mentioned earlier; it’s located far away in the genome and doesn’t resemble anything that any of the other Methylobacterium species has (as far as I can tell). What’s even cooler is that M. nodulans is the one species of Methylobacterium that lives not on leaves but in nodules on plant roots. So I suspect that lignin degradation might be a talent it retains specifically because it’s useful in the soil environment.

So, in M. nodulans, we’ve found a great source of lignin-degradation genes for engineering into M. extorquens—a close family member willing to donate an organ, if you will. What I find even more interesting, though, is that at the same time we’re working toward a tangible, potentially industrially important, goal, we also get to peek into the ecology and evolution of some really cool plant-associated bacteria. When several species of a genus diversify to fill radically different niches (leaf versus root), how and when do they pick up the genes they need, or lose the genes they no longer need? I like to think that when we clone a new set of lignin-degrading genes into M. extorquens, we’ll be either restoring a function it once lost, or repeating an acquisition that an ancestor of M. nodulans once experienced. (Which one? I don’t know, but I’m definitely inspired to delve deeper into the phylogeny of these genes across the diverse species that have them.)

We’re likely to follow up cloning with evolution in the lab to help M. extorquens get used to its new genes… and when we do, what kind of changes can we expect to see? I’m new both to experimental evolution and to the metabolism of aromatic compounds, so I have a great deal still to learn. I’m looking forward to finding out more about the evolution has happened in the environment to separate M. nodulans and M. extorquens on the plant, and the evolution that will happen soon in our lab, on the way to creating a microbial consortium for biofuel production.

References

Mitsui R, Kusano Y, Yurimoto H, Sakai Y, Kato N, Tanaka M. (2003). Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid. Appl Environ Microbiol 69:6128–6132.

Sudtachat N, Ito N, Itakura M, Masuda S, Eda S, Mitsui H, et al. (2009). Aerobic Vanillate Degradation and C1 Compound Metabolism in Bradyrhizobium japonicum. Appl Environ Microbiol 75:5012–5017.

Walton NJ, Mayer MJ, Narbad A. (2003). Vanillin. Phytochemistry 63:505–515.

For more information about Jessica’s research, you can contact her at jessicalee at uidaho dot edu.

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BEACON Researchers at Work: The Many “Arms-and-Eyes” of Retinoblastoma Family Proteins

This week’s BEACON Researchers at Work blog post is by MSU graduate student Yiliang Wei.

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Hello BEACON members, I’m Yiliang Wei, a graduate student in the Arnosti Lab at Michigan State University (https://arnostilab.natsci.msu.edu). For my first blog at BEACON, I’m going to talk about my study of the functional diversity of the Drosophila Rbf2 protein, a recently evolved Retinoblastoma family member in Drosophila, and its possible role in regulation of ribosome biosynthesis. This study has been recently published on G3 (Genes|Genomes|Genetics) (doi:10.1534/g3.115.019166).

Our lab has been studying Retinoblastoma (Rb) tumor suppressor proteins in Drosophila. Rb proteins function as transcription co-repressors that bind to E2F/DP to control transcription of a diverse set of genes involved in cell cycle regulation. The Rb-E2F pathway plays central roles in cell cycle and development in most eukaryotes. The Drosophila Retinoblastoma family members Rbf1 and Rbf2 are structurally similar to their vertebrate counterparts and possess functionally conserved activities.

A canonical model for Rb-E2F pathway. Rb proteins interact with E2F and DP, and repress target gene expression, such as cell cycle related genes.

A canonical model for Rb-E2F pathway. Rb proteins interact with E2F and DP, and repress target gene expression, such as cell cycle related genes.

Many studies so far have been focused on Rbf1, since it has a dominant role in regulating the cell cycle control and mutants exhibit a lethal phenotype. A few years ago, our lab conducted a genome-wide analysis of the Rbf1 binding profile, and identified a number of non-canonical Rb targets, suggesting diverse functions of Rbf1 besides cell-cycle regulation, such as regulating signaling pathways (Acharya et al. 2012). This aspect of function of Rb appears to be conserved in mammals as well.

On the other hand, the study of Rbf2 has been less intense, mostly due to the mild phenotype and weak transcriptional activity. However, as we turned our eyes to Rbf2, we found some interesting features of this protein. First, the genus Drosophila is different from most arthropods in that it possesses this additional retinoblastoma family member – most arthropod genomes encode a single Rb gene. Second, the Rbf2 protein contains a distinct, derived C-terminus when compared to Rbf1. It is known that the C-terminus of Rb proteins influence regulation and promoter targeting in mammalian systems. These observations led me to consider why the rbf2 gene is strictly evolutionarily retained throughout the Drosophila lineage, despite the modest observed phenotypes; has the Rbf2 protein gained new functions during evolution, and might the unique C-terminus of Rbf2 contribute to differential targeting?

To answer these questions, I carried out parallel ChIP-seq analysis of Rbf1 and Rbf2 in Drosophila embryos. At first, we expected that Rbf2 genome-wide targets might be a subset of Rbf1, based on the canonical Rb-E2F interaction model that Rbf1 binds to both dE2F1 and dE2F2, while Rbf2 binds only to dE2F2. To our surprise, we found that Rbf2 has much more genome-wide targets than Rbf1, and in fact, Rbf1 targets are just a subset of Rbf2. As we noted in our G3 manuscript “this pattern either represents the neofunctionalization of Rbf2 with acquisition of novel gene targets, or alternatively, many of these genes may be bound by the Rbf1 homolog in sister species, with a Rbf2 acquiring some of these interactions through subfunctionalization of Rbf1.” This binding profile of Rbf2 runs contrary to the canonical Rb-E2F model, that Rbf proeins bind to their targets through E2F/DP. Our bioinformatic analysis indicates that Rbf2-only promoters may tend not to have strong E2F-like sites, suggesting that other transcription factors may recruit Rbf2- a property shared with mammalian Rb. In fact, over half of the Rbf2 targets were not associated with E2F binding when compared to another study (Korenjak et al. 2012). It has been noticed that the C-terminal regulatory domains of mammalian Rb proteins partially drive the specificity of these protein interacting with other cofactors. Likewise, the unique C-terminus of Rbf2 may allow it to interact with different types of regulators, other than E2F/DP cofactors.

As we further explored the nature of the bound genes, we found Rbf2 targets have diverse functional groups, similar to what we saw for Rbf1 targets. However, what we didn’t observe in Rbf1 is that the majority of ribosomal protein gene promoters were bound by Rbf2. Unlike the potent Rb activity on cell cycle genes, which are shut down by a small amount of Rb overexpression, the Rbf proteins’ repression activity on ribosomal genes is weaker, but widely spread to the majority of the ribosomal genes. We think that ribosomal protein genes are a different sort of target than cell cycle genes – they typically have less variation in expression levels than developmentally-regulated genes, which can be completely off in many settings. It is possible that the transcriptional regulation of ribosomal protein genes by Rb proteins may be subtle, but we think that it would have pleiotropic consequences. Interestingly, none or very few studies have shown any negative regulatory mechanism for ribosomal protein gene expression, it is rather surprising that this group of genes would be controlled only by positive input. Thus, our study offered an aspect of negative regulatory mechanism of ribosomal protein gene expression.

Intriguingly, one of the few reported phenotypes for rbf2 mutants may tie into the negative regulation ribosomal protein gene expression: rbf2 null flies lay eggs at a considerably higher rate than wild-type ones. This mild phenotype has been neglected, however, the egg laying is in fact tightly coupled to nutritional signals. Though it may sound good, laying too many eggs under certain conditions may have long-range fitness effects, perhaps reducing lifetime fertility. Therefore, Rbf2 may regulate Drosophila reproduction by fine-tuning the biosynthetic components, such as ribosome protein gene family.

An overlooked aspect of previous ChIP sequence data relating to human Rb and related p130 and p107 proteins is their similar enrichment on ribosomal protein promoters (Chicas et al. 2010). Interestingly, the mammalian Rb proteins are known to directly regulate RNA polymerase I and III activity. Together with the ribosomal protein gene regulation, this multi-layer gene regulatory network cover all aspects of ribosome production – what we usually consider as a potent break switch on cell cycle genes may also be regulating by numerous, subtle contributions the biosynthetic capacity of a cell. Clearly an activity relevant to cancer cells! Our findings provide a new view of the impact of Rb proteins in cancer.

Just like the Kuan-yin of a Thousand Arms, with each hand holding a unique power, the Rb family proteins have reached their “arms” to many aspects of the gene regulatory network. (Picture from The Palace Museum, http://www.dpm.org.cn)

Just like the Kuan-yin of a Thousand Arms, with each hand holding a unique power, the Rb family proteins have reached their “arms” to many aspects of the gene regulatory network. (Picture from The Palace Museum, http://www.dpm.org.cn)

As it turns out, 5000 years of Chinese culture provides an apt analogy for the action of the Drosophila Rbf proteins. Like Kuan-yin of a Thousand Arms and Eyes from Chinese Buddhism culture, where each hand holds a unique power, the Rb family proteins have reached their “arms” to many aspects of the gene regulatory network. Our studies of Rbf proteins indicate that there are many more functions of Rb, as well as non-canonical mechanisms that recruit Rb proteins to their targets. The appearance of the novel Rbf2 protein on the genome landscape, just as the more recently derived human Rb protein, indicates that gene duplication and diversification contributes to these central processes.

References:

Acharya, P., N. Negre, J. Johnston, Y. Wei, K. P. White et al., 2012 Evidence for autoregulation and cell signaling pathway regulation from genome-wide binding of the Drosophila retinoblastoma protein. G3 (Bethesda). 2: 1459–1472.

Chicas, A., X. Wang, C. Zhang, M. McCurrach, Z. Zhao et al., 2010 Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence. Cancer Cell 17: 376–87.

Korenjak, M., E. Anderssen, S. Ramaswamy, J. R. Whetstine, and N. J. Dyson, 2012 RBF binding to both canonical E2F targets and noncanonical targets depends on functional dE2F/dDP complexes. Mol. Cell. Biol. 32: 4375–87.

Wei, Y., S. S. Mondal, R. Mouawad, B. Wilczynski, R. W. Henry et al., 2015 Genome-Wide Analysis of Drosophila Rbf2 Protein Highlights Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis. G3 (Bethesda). In press.

For more information about Yiliang’s work, you can contact him at wyliang1987 at gmail dot com.

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