Bringing Together a World through Science

This post is written by UT undergraduate researchers Zachary Martinez and Andrew Ly

UT Austin undergraduates (L->R) Rachel Johnson, Zachary Martinez, Andrew Ly, Andrea Martinez, and Milki Negeri. Behind them is their poster, entitled “Yo GABA GABA”. The student researchers also presented their work orally.

The University of Texas at Austin is known for many things: from being a powerhouse in Division 1 sports, to leading the world in innovation and cutting-edge research. However, there is one historic fact that many Longhorns do not know, and that is the success of the UT Austin iGEM team. For the past six years at the International Genetically Engineered Machine (iGEM) conference, UT Austin has earned a gold medal each year, an honor bestowed only to teams fulfilling the highest and strictest research requirements. This annual synthetic biology conference takes place in Boston, where over 300 teams from universities around the world present their research. This year, the UT Austin team consisted of a wide range of students, from underclassmen that have just started doing research, to more seasoned upperclassmen that have participated in iGEM previously. The 2017 project focused on engineering an effective GABA-producing probiotic.

The indigenous gut flora of humans possesses the ability to synthesize neurotransmitters, such as GABA, that are hypothesized to influence behavioral, cognitive, and emotional processes of the body via the gut-brain axis. The microbiome-gut-brain axis is a bi-directional communication system in which the microbiome of the gut affects the central nervous system, and vice-versa. Using this information along with our background in microbiology, molecular biology, and synthetic biology, we set out to engineer this microbiome as a way to potentially treat mental illnesses.

Gamma-Aminobutyric acid, or GABA, is the chief inhibitory neurotransmitter in the body and is responsible for reducing neuronal signaling in the central nervous system. Medications, such as alprazolam and diazepam, that increase GABA signaling are typically used for treating anxiety disorders. However, such drugs can lead to a physical dependence, and if given to children, a “pill-popping” habit. Due to these reasons, we began researching potential probiotics that we could study and engineer in order to produce GABA. We ended up picking Lactobacillus plantarum, which is not only indigenous to the human gut, but also expresses GABA in small amounts by converting glutamate to GABA via a glutamate decarboxylase enzyme encoded by the gadB gene. Our goal was to engineer this microbe to produce high levels of GABA and implement it into fermentable foods (such as kombucha, kimchi, or yogurt), which could then be ingested as an alternative form of medicine for patients suffering from anxiety.

Bacterial plate of transformed L. plantarum.

In order to engineer our probiotic to produce high levels of GABA in the human gut, we first wanted to assemble a plasmid in which the gadB gene was overexpressed. To accomplish this, we employed a cloning technique called Golden Gate Assembly, which utilizes type IIS restriction enzymes that cut adjacent to the recognition sites. This allows for the scarless and simultaneous interchanging of different DNA parts, such as origins of replication, antibiotic resistance cassettes, coding sequences, and promoters, all while maintaining directionality in a single reaction. As such, we chose this assembly method due to its ability to rapidly create functional plasmid prototypes that would allow us to interchange parts quickly as we begin experimenting with L. plantarum. After successfully assembling our intended gadB overexpression plasmid using Golden Gate Assembly, we would then introduce it into our probiotic.

While trying to overexpress GABA, we observed various mutational inactivations of our gadB gene. Given that glutamate is an important substrate in biosynthesis and that GABA production requires the conversion of glutamate into GABA, we hypothesized that the functionally active form of gadB was ultimately toxic to cells. As a result, cells containing a mutated gadB gene were more evolutionarily fit and thus selected for. This explains why we were only able to obtain cells with the mutated gadB gene. We then constructed plasmids with either lower copy numbers and/or inducible promoters that would downregulate or control the expression of the gadB gene. However, we still found mutations within the gadB gene. Some possible solutions to address this issue are to utilize an inducible promoter with tighter regulation in our plasmid assembly, perform DNA transformations with a strain with a lower mutation rate, or even simply growing the bacteria in media supplemented with high levels of glutamate. Our future directions include developing a quorum sensing system in our engineered probiotic for controlled GABA production and potentially introducing our probiotic into the microbial ecosystem of the fermented beverage, kombucha, which was the main focus of our iGEM project last year. This year’s project, much like our 2015 iGEM project regarding evolutionary stability, has highlighted the importance of creating evolutionarily stable genetic circuits with low metabolic burdens: a problem synthetic biology has long had.

Overall, the iGEM conference was an invaluable experience where we were able to meet and network with numerous people from around the world, ranging from China to Ghana. We spoke to researchers who were looking into creating more robust genetic systems in a wide array of bacteria, something we have had an interest in for several years. Additionally, students from Vilnius, Lithuania discussed how they were able to use multiple plasmids within a single bacterium while controlling the copy number and maintaining this entire set of plasmids (five in total!) over multiple generations. As we prepare for next year’s iGEM competition, we hope to take what we have learned from this year’s experience and apply it to our 2018 research project.

 

 

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Lessons from your parents: “Fool me once, shame on you. Fool me twice, shame on me” – Randall Terry.

This post is by UW faculty Cynthia Chang and Thelma Madzima, research tech Colin Feng, and undergraduate researcher Jackelyn Garcia

“I told you so” – All parents?

Can the lessons from your parent’s experiences be passed on to you for your benefit, and if so, how?

In many organisms, the memory of our experiences often influences our behavior and how we respond to similar situations in the future. In humans, these ‘learned’ lessons are often passed on verbally from one generation to the next. (Sometimes, no matter what our parents tell us, we have to experience things for ourselves). However, in some organisms like plants, life lessons and behavior are not ‘verbally communicated’, but rely on other methods so that clues of past experiences are passed on to offspring.

In living organisms, memory information can be passed on from one generation to the next at the molecular level; through signals added on top of DNA, referred to as ‘epigenetic modifications’ (Greek for epi = on top or above. Therefore, on top of genetic information). Epigenetic modifications (such as DNA methylation) can be inherited, are reversible and can influence phenotype or how an organism develops. Epigenetic modifications can also be induced by the environment, therefore, by studying the inheritance of epigenetic signals, we can understand how the environment experienced in one generation (parents) can impact developmental responses in the offspring. Thus, epigenetic modification may prove to be an important signal to understanding how species remember and respond to a rapidly changing climate (Donelson et al. 2017).

Climate change: “It’s getting hot in her(r)e” – Nelly

Current climate change models predict greater periods of drought as well as a more variable environment, both of which will drive the evolution of how plants respond to changing environmental conditions (Jump and Penuelas 2005). Our project focuses on determining if plants that experience high environmental stress (drought) pass on molecular signals (epigenetic modifications) to their offspring which allows the offspring to learn from their parents, and better adapt to a variable environment.

To do this, we are using the model plant Arabidopsis thaliana, a fast-growing, primarily selfing plant. As part of our experimental design, we will collect physiological and epigenetic data from Arabidopsis plants exposed to different stresses over multiple generations. In the first 3 generations, we will expose half of our plants to high-drought conditions, and the other half to normal conditions (low-stress; non-drought). Offspring seeds will be collected from each plant and planted in the same treatment their parent experienced. In the fourth generation, we will determine if these life experiences are inherited. We will compare historically stressed plants to non-stressed plants, when grown in either a low, high, or variable water stress environment. We hypothesize that historically stressed plants will grow better than non-stressed plants when grown in a high or variable water stress environment. However, it is also possible that plants have to ‘learn for themselves’ each time.

What’s in it for me?”

This research will provide insight into how a plant population’s past experiences can help or hinder its ability to adapt to a rapidly changing environment. Understanding how plants will respond to climate change is a major motivation for our whole research team.

“The molecular mechanisms of epigenetic inheritance are particularly relevant to all plants, especially in agriculturally important plants. But first, it’s important and more feasible to study these mechanisms in a model plant like Arabidopsis thaliana”. – Thelma Madzima

“I am excited to see how the epigenetic modifications will affect Arabidopsis in the future generations of this project. More specifically, I want to see what genetic differences do stressed plants have compared to unstressed (if any) and how that impacts their ability to respond to stresses.” – Colin Feng

With an interdisciplinary team, we are able to tackle this research question with our different areas of expertise.

Jackelyn Garcia, 2nd year undergraduate researcher at the University of Washington-Bothell, watering the first generation of experimental plants.

“Understanding the evolutionary implications of epigenetics is an exciting way to bridge the gap between molecular biology and ecology.” – Cynthia Chang

Finally, this Beacon research is providing first-hand research experience to young undergraduate researchers. Jackelyn Garcia, a 2nd year UW-Bothell aspiring Biology major has dedicated her time to understanding the phenotypic (trait) patterns of the plants. She hopes to use this research to connect her coursework to real research, and learn more about evolution, ecology, and genetics.

Scientific experience and inheritance

This research is being conducted in collaboration with the undergraduates phenotyping Arabidopsis knockouts (unPAK) network (http://arabidopsisunpak.org/). unPAK is a network of undergraduate research institutions working towards to goal of understanding the relationship between genotype and phenotype, using Arabidopsis. In addition to answering our own research questions, the plants grown in our experiment will provide data for this growing database of unPAK genotype-phenotype data. Both Assistant Professors are particularly excited to incorporate this research in their undergraduate Investigative Biology courses with the hope of adding to our growing understand of how plants can adapt to climate change and the molecular signals that are transmitted, and inspiring new researchers to tackle this complex problem.

Literature Cited

Donelson, J. M., S. Salinas, P. L. Munday, and L. N. S. Shama. 2017. Transgenerational plasticity and climate change experiments: Where do we go from here? Global Change Biology.

Jump, A. S., and J. Penuelas. 2005. Running to stand still: adaptation and the response of plants to rapid climate change. Ecology Letters 8:1010-1020.

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BEACON’s China Collaborations Expanding

BEACON Director Erik Goodman just returned from a 2-week whirlwind trip to four cities in China. He was seeking new collaborations and following up on existing ones, including two of long standing. His first stop was Shanghai’s Tongji University, visiting the laboratory of Prof. Lihong Xu, a longtime collaborator on the evolutionary design of greenhouse controllers for the new generation of greenhouses being built across China. MSU Ph.D. student José Llera-Ortiz is preparing to defend his Ph.D. thesis on controller evolution, and Goodman presented their latest results, and also heard from about a dozen Chinese researchers on a variety of topics concerning modern agricultural automation. Prof. Xu’s lab will provide some of the data to be used to validate Llera-Ortiz’s evolved controllers and greenhouse model.

Goodman, Prof. Lihong Xu (to his left) and some of Xu’s students (including Dr. Leilei Cao, a previous BEACON visitor) at Tongji University’s Jiading Campus

The second stop was Shantou University, hosted by Prof. Zhun Fan. He directs the International Joint Research Center “Evolutionary Intelligence and Engineering Applications” which was established between BEACON and the Guangdong Provincial Key Laboratory of Digital Signal and Image Processing. Goodman presented seminar for students and affiliated faculty members, and discussed their individual research projects with many of them. He also opened discussions on possible collaboration with the dean of the College of Science.

Stop three was the IDEAL Conference, this year hosted at Southern University of Science and Technology (SUSTech), in Shenzhen, very close to Hong Kong. This relatively new and gigantic city (population 20 million) is China’s high-tech capital, and SUSTech is hiring faculty from among the best in the world! Two of BEACON’s collaborators were among the three winners of Best Paper Awards. Goodman gave a keynote lecture at the founding of SUSTech’s new Key Laboratory for Computational Intelligence, directed by Xin Yao, one of the most distinguished researchers and leaders in the field.

Final stop was at the Chinese Academy of Science’s Beijing labs, where Goodman gave an invited talk introducing BEACON’s research on land use planning and on multi-level optimization of logistics. Researchers from the Academy and from Beijing University of Technology (BJUT) are interested in establishing future collaboration on these topics, and Goodman met with the BJUT president and with the director of the Beijing Advanced Innovation Center for Future Internet Technology.

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Fall 2017 elementary school science nights

We are excited to share some pictures from three local elementary school science nights that we had the pleasure of attending this semester. We ran booths at Marble, Whitehills, and Glencairn elementary schools here in Lansing introducing kids to evolution using a couple of highly interactive activities. Thanks everyone for volunteering and thank you Matthew Moreno for taking photos!

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Two BEACONites receive awards from Web of Science for their publication records

We are very excited to congratulate two amazing researchers, Amir Gandomi and Kalyanmoy Deb for their recent awards from Clarivate Analytics, formerly the Intellectual Property and Science business of Thomson Reuters, and owner of Web of Science.

Amir Gandomi was named a 2017 Clarivate Analytics Highly Cited Researcher based on his publication record, publishing Highly Cited Papers defined as those that rank in the top 1% by citations for field and publication year in the Web of Science. This list of researchers represent some of the world’s most influential minds as determined by a citation analysis of Web of Science data.

Kalyanmoy Deb was awarded the Lifetime Achievement Award for his highly cited research contributions in a wonderful event organized by Clarivate Analytics attended by a large audience in New Delhi including other awardees, scientific advisers to Government of India, and many dignitaries from Indian universities and industries.

One of his papers published in 2002 passed 10,000 citations by Web of Science and is ranked 174 out of 45,602,967 journal and conference articles recorded by Web of Science from 1900 to 2017. Kalyanmoy is the first recipient of this award. Besides this award, the Citation Awards 2017 event also gave away Research Excellence Awards to individual researchers and institutions in India for their impacting contributions.

The award citation stated “Professor Deb is recognized for research on multi-objective optimization using evolutionary algorithms, which are capable of solving complex problems across a range of fields involving trade-offs between conflicting preferences. A 2002 paper, with more than 10,000 citations, ranks among the 200 most-cited papers recorded in the Web of Science, 1900-2017, and is by far the most influential paper ever produced by an Indian scientist, as reflected by citations.”

See photos below of Kalyanmoy receiving his award.

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The phage from the local lagoon

This post is by MSU postdoc Sarah Doore

Earlier this year, I wrote a blog post about doing some hunting with a graduate class here at Michigan State University. I’m not talking about hunting in the traditional sense though, since what we sought were bacteriophages—or viruses that infect bacteria—in the local environment. We were specifically interested in bacteriophages that infect enteric bacteria, like Escherichia coli, Salmonella enterica, and Shigella flexneri.

We asked the students to come up with a location and methods they wanted to use to check for bacteriophages (“phages” for short), then did some hands-on isolation using non-pathogenic strains of bacteria in our lab. We got to look at some new phages and the students’ creativity helped us find at samples we never would’ve imagined testing before. The project was fun for the students and fun for us!

By spring, we’d already planned to repeat the activity in the same grad course and were trying to bring phage isolation to undergraduate biology labs at MSU. So, when a Nebraska high school teacher heard about the project and seemed interested in doing something similar for his science class, we brought him to our lab over the summer and showed him the ropes.

Fig 1. Jason fishing for some phage in Nebraska.

After taking him through the isolation procedure, we made a plan for how he could have his students isolate their own phages back in his classroom. We also sent Jason, one of the lab’s graduate students, to Nebraska to do a trial run and see what the classroom still needed. You can see Jason collecting a Nebraska water sample for this in Figure 1. Come fall, we sent a box full of supplies to the high school and anxiously waited for the phage hunting module to begin. We were SO curious to see where students would decide to look—and, even better, what types of phages they’d find.

On collection day, each of the 50 students got their own sterile tube and went to a place they thought would most likely have phage, or at least somewhere they thought might have interesting results. They labeled the tube with their sampling location, then brought it back to the classroom to test it for phage.

A few common themes emerged among the sample locations the students picked: pond and river water were popular choices, as were the school’s baseball and football fields. There were also some more creative ideas, like the two students who sampled their dogs’ water bowls and the one who scooped a deer’s footprint in the woods. One of my personal favorites was “pond by frog,” which is just specific enough to make you wonder: what frog? What was the frog up to? Could this frog be an unknown reservoir for enteric phages? (Spoiler: it wasn’t, and I’m oddly disappointed by this result.)

Back in the classroom, the students tested their samples to see if they had found any phage. Out of the 50 samples, 16 of these had phages in them. Most phages—11 out of 16—came from ponds. The rest of the phages came from the baseball field (3), football field (1), and grass outside the classroom (1). The students sent their samples to our lab at MSU so we could also take a closer look at these new phages.

We’re still in the process of characterizing all of them, but so far it looks like some have interesting morphologies (see Figure 2 for an example of what I’d call an interesting plaque morphology). One also has a unique host range that we haven’t seen yet. This phage infects Salmonella enterica, Escherichia coli, and Citrobacter freundii: three types of enteric bacteria that belong to the same family but are otherwise significantly different. This particular phage came from pond water near a cattail. So…maybe cattails make better homes for phages than frogs do?

Fig 2. On an agar plate like this, phages kill their host and leave a clear spot where bacteria would have grown (“the circle of death”), known as a plaque. This one has fuzzy edges and a little belly button in the middle.

Although we’ve only been doing this kind of isolation for about a year, we’ve already discovered a total of 36 new phages, some of which are pretty rare and/or have really unique qualities (like the broad host range mentioned above). We plan to keep challenging students to sample their environment as long as we have the resources to keep up with them.

Although bacteriophages have been studied for over a century, and we’ve known for awhile that they’re abundant, we don’t know as much about the diversity or identity of the phages that are out there in the environment. Hopefully now we’re starting to answer that question too, and in the process we’re beginning to appreciate our own local composition of microbes.

If you’re interested in doing some of your own citizen science, we’ve got some resources for you! Maybe you’re a teacher who wants to try this in your classroom, or you know of someone who might be interested.

The protocols can all be found on the Parent lab’s website here (under “Video Protocols”). There’s a written version for the entire process of phage isolation, with videos for certain steps that are easier to understand and do yourself after seeing someone else do them. You can also follow us and/or ask us questions on Twitter @phage4lyfe.

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In search of evolutionary hotspots

This post is by MSU grad student Emily Dolson

Imagine that an alien species arrives on earth. It happens to be able to live and reproduce in any part of the world, and, over successive generations, it begins to adapt to its new environment. Among other things, it adapts to eat new kinds of food. Where in the world would you expect the first alien capable of eating papayas to be born?

Most people would probably say South or Central America, since papayas live there. When asked why they said that, there are two potential explanations they might give:

  1. The ability to eat papayas is more beneficial in regions where papayas actually grow.
  2. Regions where papayas grow are also populated by similar plants, like guavas.

The first of these explanations is based on either a misunderstanding of the question or a misunderstanding of evolution (but don’t worry, it’s a common one!). We didn’t ask where aliens that eat papayas are most likely to survive long term; we asked where the very first alien that could eat a papaya would be born. Recall how natural selection works: mutations occur randomly, some happen to be useful, and organisms that have mutations that happen to be useful usually go on to be successful. The first alien to be born with the ability to eat papayas has presumably had some sort of mutation that (in the context of the rest of its genome) gave it this ability. That ability hasn’t yet had a chance to prove to be useful or not. This alien may never even encounter a papaya in its life, in which case having the ability to eat them will have no effect. The presence or absence of papayas can have no influence on the birth of the first alien that can even interact with them.

The second explanation, however, is plausible. South and Central America are home to other tropical fruits, such as guavas. Tropical fruits have similar physical and chemical properties. If an alien can eat a guava, it probably only needs a few subtle mutational tweaks to be able to eat a papaya (assuming that papaya-eating even requires further adaptation). Once a subset of the aliens in the tropics gain the ability to eat some sort of fruit, their progeny will likely go on to be very successful in the regions where these fruit grow. These aliens will be well-positioned, in both physical and mutational space, to begin eating other fruits, such as papayas.

While aliens are unlikely to invade earth and eat our tropical fruits, the phenomenon of a population encountering a set of entirely new challenges as it moves into a new geographic region (or as the geographic region in which it currently lives changes) is common. And it’s common for these new challenges to be related to each other. It’s no accident that plants with similar fruit live in the same region; plants in the same region experience the same selective pressures and may also have a shared evolutionary history. These factors generalize across a wide variety of scenarios. Thus, if some of the challenges of thriving in a new area are easier to solve than others, the easy ones can serve as “evolutionary building blocks” for the harder ones. The presence of such building blocks, i.e. simple adaptations that provide a good starting point for more complex adaptations, has been shown in previous work to be important to the evolution of complex traits (Lenski et al., 2003).

If spatial layout does indeed impact the ease of adaptation, it would be useful to understand, both for evolutionary biology and evolutionary computation. As species shift their ranges in response to climate change, they will traverse regions where different traits are advantageous. Predicting how the positioning of these regions impacts evolution will help us predict whether the species will be able to survive. Evolutionary computation often takes advantage of evolutionary building blocks by rewarding solutions to different problems over time, but this is an imprecise art. If we could understand how to reward them differently across space to promote evolution of an overall solution, we could more easily generalize evolutionary computation to more problems.

Of course, it’s also entirely possible that these spatial effects are too small to care about. Recently, I’ve been trying to figure out whether or not that’s the case (Dolson and Ofria, 2017). Since this would be an incredibly labor intensive question to address in the lab or field, I’m using the Avida Digital Evolution platform to perform preliminary experiments on the computer. Once we know more, I’d be very interested in collaborating with wet lab biologists to see if our digital results are consistent with results from DNA-based systems.

Fig 1. Evolutionary hotspots across all eight environments. Background colors indicate which set of resources are present in each location. Polygons show the location of hotspots for the evolution of the ability to use each resource (legend indicates which resource corresponds to which line-type).

In Avida, I created eight different environments with different patterns of resources across space. I then let 100 different populations of digital organisms evolve independently in each environment. Within each of these runs, I found the location of the first organism with the ability to use each resource in each run. From these data, for each resource and each environment, I determined which regions appeared more often then we would expect to see by chance. These regions are “evolutionary hotspots.” Sure enough, each environment (except the control, which had all resources everywhere) had at least some hotspots (see Figure 1). In some environments, most of the hotspots overlap. In others, they are largely in different regions of the environment.

Fig 2. The sequences of environments experienced by the ancestors of the first organism to be able to use the XOR resource in one of the environments. Each color represents a different environment, and the length indicates long the lineage stayed in that environment for. Notice that many bars appear to end in a somewhat similar sequence of environments.

I’m now working on trying to predict why hotspots are where they are. Surprisingly, a number of seemingly obvious explanations (e.g. the number of resources present, the presence or absence of specific resources, and local diversity) do not appear to explain the pattern. Currently, it appears that the sequence of environments that a lineage experiences over evolutionary time may be a key variable (see Figure 2). I’m looking forward to understanding more soon!

References:

Lenski, R. E., Ofria, C., Pennock, R. T., & Adami, C. (2003). The evolutionary origin of complex features. Nature, 423(6936), 139-144.

Dolson, E. and Ofria, C. (2017).  Spatial resource heterogeneity creates local hotspots of evolutionary potential. In Proceedings of the 14th European Conference on Artificial Life. Vol. 14. pp. 122 – 129. MIT Press.

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Uncovering the function of host-associated microbial communities

This post is by MSU grad student Connie Rojas

Microbes colonize every surface of their hosts. Once established, they do not live in isolated patches, but instead form highly regulated, structurally and functionally organized communities, termed ‘microbiota’. Due to the interplay of the host’s immune system with its microbiota, many members are commensals or mutualists, performing functions critical for host health and physiology. In the human mouth, resident microbiota secrete antimicrobials and enzymes that contribute to oral health. In the mammalian gastrointestinal tract, microbiota synthesize vitamins, and supply the host with energy released from the fermentation of indigestible carbohydrates. In the human vagina, members of the microbiota produce lactic acid, which maintains a low pH environment thought to protect against infection. However, despite the explosion in microbiome research, we know very little about the additional functions microbes are performing within their hosts. We also do not know whether or how they have affected the behavior and evolution of their hosts.

The host generally maintains a stable microbiota. Stability ensures that beneficial symbionts and their associated functions persist over time. Host regulatory mechanisms like physical barriers, mucosal antibodies, and immune systems work to promote the growth of certain microbes, exclude others, and keep the microbiota in check. Furthermore, because some microbes are functionally redundant and can substitute for one another, community function can be retained despite shifts in composition. In fact, alterations to the function of these naturally occurring communities have been implicated in diseases like inflammatory bowel syndrome, type 2 diabetes, bacterial vaginosis, and colorectal cancer. Nevertheless, it is unknown the extent to which fluctuations in host and microbial environments, and resultant variation in the microbiota, can be afforded before these changes become detrimental. Years of research show that microbiota are often host species- and niche-specific, and across hosts, the microbiota varies with a myriad of factors like diet, age, antibiotic use, habitat, season, and environmental stressors. However, it is very likely that numerous other factors are driving variation in microbiota composition and function among hosts, and determining how this variation affects host phenotype is a key line of inquiry.

I study the gut microbiome of spotted hyenas! Photo Credit: Lily Johnson-Ulrich

My research seeks to understand the stability, composition, and function of the microbiota at various body-sites and elucidate the socio-ecological traits of hosts influencing its structure. While most microbiome research is conducted in humans and mice, typically within the context of host health and disease; I study these questions in wild spotted-hyenas (Crocuta crocuta). Due to their complex social behavior, hyenas are an excellent model system to explore how microbiota both influence host behavior and respond to host ecology.

Spotted-hyenas are large, social carnivores inhabiting much of Sub-Saharan Africa. They live in large groups, called ‘clans’, which are structured by linear dominance hierarchies, where an individual’s position determines its priority of access to resources. Their societies are also characterized by female dominance, male-biased dispersal, and a high degree of fission-fusion dynamics, such that individuals move freely among subgroups several times per day. Hyenas are reared in communal dens for the first 9mo of life, are weaned at 12-18mo old, and reach reproductive maturity at 24mo, although most females do not bear young until they are at least 36mo. In my current research, I am investigating how host factors like social dominance rank, group membership, and patterns of association affect hyena microbiota structure and function. Do individuals of varying social ranks differ in the stability and functional potential of their microbial communities? Are certain microbial genetic pathways lacking in one group vs. another? How similar are the communities, in terms of composition and function, of hyenas that associate very closely? Once again, can this variation have implications for host phenotype?

About to collect body-swabs and other biological samples from a hyena, after darting

We use next-generation sequencing technologies, mainly 16Sr RNA sequencing to profile the taxonomic composition of the microbiota, and metagenomic sequencing to characterize its function. From shotgun metagenomic data, it is also possible to infer microbial community dynamics. Populations of microbes, like members of any ecological community, cooperate and compete with each other, break-down and synthesize metabolites, and adapt rapidly to ever-changing environmental conditions. A recently developed mathematical framework by Sung and colleagues (2017) reconstructs community metabolic networks from metagenomic and available metabolic data. In these networks, the nodes represent major taxa, and the edges, which are color-coded, represent interactions (gray: cooperative; red: competitive; see Figure below). Metabolites that are imported and degraded by a species are shown in purple, and those that are synthesized and exported are in blue. I hope to use this framework to identify, for example, the molecules that are important for hyena gut community function, and evaluate whether the microbial community is dominated by competitive or cooperative interactions. Changes in community function and dynamics in response to extreme fluctuations in prey abundance (e.g. arrival of migratory wildebeest) will also be assessed this way.

The human gut microbiota metabolic community-level network constructed by Sung and colleagues (2017). Also, Connie’s dream figure.

Despite the interesting questions being asked, and the multitude of research on diverse host-microbiome systems being conducted, we still have a long way to go as a field. The things we do not know are too many to list. But I hope that revival of innovative culture-based techniques, advances in single-cell genomics, and development of more encompassing bioinformatics tools can help address the many existing gaps in our knowledge.

Reference

Sung et al. (2017). Global metabolic interaction network of the human gut microbiota for context-specific community-scale analysis. Nature Communications 8: 1-12.

 

 

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Judi Brown Clarke receives Sequoyah Fellowship medal

We are proud to announce that BEACON’s Director of Diversity, Judi Brown Clarke recently received a Sequoyah Fellowship medal making her a lifetime member of the American Indian Science & Engineering Society (AISES). The Fellowship is named in honor of Sequoyah, the great Cherokee Indian who perfected the Cherokee alphabet and syllabary in 1821, resulting in the Cherokee Nation becoming literate in less than one year. In this spirit, AISES Sequoyah Fellows are recognized for their commitment to the mission of “excellence in STEM” and to the entire American Indian community. They bring honor to AISES by engaging in leadership, mentorship, and other acts of service that support the students and professionals in the AISES family.

Judi Brown Clarke receiving a Sequoyah Fellow Medal at the 2017 AISES National Confernce in Denver, CO.

In addition, BEACON co-sponsored an Alaska Native (Haida) pre-college student who presented her summer research project about “Divergent Evolution of Shipworms and Clams,” and an undergraduate summer intern who presented her research “Curriculum Development Coupling Traditional Ecological Knowledge with STEM.”

Left to Right: intern Sarah Peele, Sonia Ibarra, Joseph Hilliare, Nevaeh Peele, Wendy F. Smythe, and Lauren Smythe.

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Beneficial bacteria in Oz

This post is by MSU grad student Colleen Friel

Pea fields (with yellow-flowered canola growing in the background) near Esperance, WA.

Increasing agricultural sustainability is an important research focus in the face of climate change, rapid population increase, and growing food insecurity. Synthetic nitrogen fertilizers have fueled a huge boom in agricultural productivity in recent decades, but they come with serious environmental consequences. Fertilizer manufacturing requires large fossil fuel inputs, and fertilizer runoff poses environmental and public health threats such as eutrophication of waterways and marine ecosystems, destruction of coral reefs, and algal blooms. For example, runoff from farms along the Mississippi River has led to a dead zone in the Gulf of Mexico that is 8,776 square miles, or roughly the size of the state of New Jersey.

One alternative source of nitrogen comes from the interactions between a subset of plants called legumes and specialized soil bacteria known as rhizobia. This interaction begins when plants growing in low nitrogen environments release a chemical signal from their roots. If this signal is detected by a compatible rhizobium, the rhizobium releases its own signaling molecules that initiate morphological changes in the plant root. Root hairs curl to encompass the rhizobium, and the plant houses the rhizobium in special organs called nodules. Inside the nodules, rhizobia convert atmospheric nitrogen (N2) to ammonia (NH3), which plants are able to use. In exchange, the plant supplies its rhizobia with sugars from carbon fixed during photosynthesis.

There is a wide range of outcomes in this interaction. Due to incompatibilities in signaling and other molecular events required for successful colonization, not all rhizobia can form nodules on all legumes. In addition, some rhizobia-legume combinations can form nodules but fail to fix nitrogen after nodulation. Evolutionary theory predicts that there is strong pressure on the rhizobia to cheat in a “tragedy of the commons” situation: since a single plant usually is colonized by a number of different rhizobial strains in the wild, each strain faces pressure to dedicate resources to its own reproduction rather than to nitrogen fixation for the plant. This trend is predicted to lead to a total breakdown of the mutualism, where all rhizobia fix little to no nitrogen for their plant hosts. However, this is not the case, suggesting that plants are able to control their rhizobial partners in some way and prevent this “cheating.” This might arise in the form of plants being able reward more effective rhizobia, punish less effective rhizobia, or select for more effective rhizobia during colonization.

The ability to select for more effective rhizobial strains is very important for the application of biological nitrogen fixation in agriculture. When rhizobia are used in agriculture, seeds are usually inoculated with a single rhizobial strain that has been shown to fix large amounts of nitrogen in combination with a given crop plant. However, this inoculant strain is frequently outcompeted by native rhizobia that are able to form nodules on the crop plant but fix little to no nitrogen for their host. Some legumes have demonstrated the ability to pick out the strains of rhizobia that effectively fix nitrogen for them, while excluding ineffective strains. However, not all legumes are very good at this, and we do not know what mechanism plants use for this purpose.

Making friends with the locals.

I spent the summer of 2016 at the Centre for Rhizobium Studies (CRS) at Murdoch University in Perth, Western Australia (WA) to study how plants select for effective rhizobia. Clovers are commonly used as forage crops in WA agriculture, since they are able to tolerate the challenges facing growers in WA, including low rainfall and acidic and sandy soils. The Mediterranean clover Trifolium purpureum and the South African clover Trifolium polymorphum are two such species. Both species have an effective native rhizobial partner that nodulates and effectively fixes nitrogen. If you inoculate one plant with the other’s effective rhizobia, the rhizobia will form nodules but will not fix nitrogen. If the plants are inoculated with a mixture of the two strains, the plant will pick out its effective strain and only form nodules with that strain, even if it is outnumbered 100:1.

To explore how the plants are doing this, we made bacterial mutants in which the gene for one of the proteins necessary for nitrogen fixation was deleted. We then inoculated these mutants onto the plants to see whether they were able to detect the ability to fix nitrogen, or if they used another marker to determine if a strain was an effective partner. We are also testing how the rhizobia strains react to the signals sent out by the plant and how well they are able to grow on the root systems of the different plant species. This will tell us if the the nodulation patterns we see are being determined by competition between the rhizobia rather than selection by the plant.. Understanding how plants select effective rhizobia can help make commercial rhizobial inoculants more efficient.

Planting Lebeckia in a field of nonwetting soil.

While I was in Australia, I traveled throughout much of WA, going 500+ km north of Perth to West Binnu, WA and going 700+ km southeast to Esperance, WA. During these expeditions, I assisted with field work for ongoing projects at the CRS. These projects ranged from assessing the negative effects of various pesticide treatments on rhizobial colonization in pea crops to planting field trials of the newly domesticated crop Lebeckia, which can grow in very sandy and unique nonwetting soils that plague parts of WA. It was an amazing experience to be able to interact with growers across such a large part of the country and learn how different agriculture is in Australia compared to the US. The landscape, flora, and fauna were shockingly different, and it was shocking to be able to drive for hours without seeing much evidence of humans. It was fascinating to learn how agriculture has adapted to the heat, low rainfall, and challenging soils present in WA. I look forward to using the knowledge, techniques, and relationships I developed in Australia to inform my work and increase our understanding of legume-rhizobia interactions.

 

 

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