There’s a party in the soil, and microbes are the VIPs. They’re feasting on the compounds that plants secrete through their roots, creating a lively zone called the rhizosphere. In this episode, biologist Jennifer Pett-Ridge of Lawrence Livermore National Laboratory has your backstage pass.
The Joint Genome Institute presents the Genome Insider podcast.
Transcript of the episode
ALISON: Hey! I’m Alison Takemura, and this is Genome Insider, a podcast of the US Department of Energy Joint Genome Institute, or JGI.
I was planting some seeds recently, and I picked up a handful of dark, rich soil. I just took a moment to ponder: who’s there? The short answer: Microbes. A lot of them. But they’re not all the same.
JENNIFER: When we say microbes, we use that really a little loosely. And these days, I define that as including bacteria, archaea, fungi. And I even include viruses in the microbes.
ALISON: Those are environmental viruses, the kind that infect other microbes. And that person you just heard from was Jennifer Pett-Ridge.
Jennifer Pett-Ridge, senior staff scientist and leader of the Environmental Isotope Systems Group at Lawrence Livermore National Lab, in Livermore, CA. (Jacob Long)
JENNIFER: Hi, I’m Jennifer Pett-Ridge from Lawrence Livermore Lab. And I am a senior staff scientist.
ALISON: And if you’ve ever wondered what the world looks like to a microbe in the soil, she is definitely the right person to ask.
JENNIFER: I think of the ball pit, right? At IKEA, where I used to take my kids and drop them off while you’re shopping. Right? There’s this huge pit, they, they throw all these kids in there. But, you know, you’ve got all these, these mineral particles that have, you know, different shapes and colors and different reactivities. And they might be holding carbon or not.
ALISON: That carbon might be sugars or saccharides from plants or proteins from dead, exploded microbes, stuff that’s sticking to the particles.
JENNIFER: And then you’ve just got these little crevices, kind of in between. And if you can imagine that the balls were the same size as kids, you know, that’s kind of what I think about in terms of the ratio of the available space, the sort of the open pore space to the solid matrix. Kind of on that scale.
ALISON: Bacteria and archaea are very small — on the order of just a micron, which is one one-hundredth the thickness of a human hair. So, the structure of soil keeps microbes kind of in the dark about their surroundings.
JENNIFER: The reality is that for an individual microbe, if it fits on one side of a soil particle, it may not be able to really sense at all the organism that’s just living on that other side of the same particle. For the most part, they are kind of stuck where they are; they’re probably in a biofilm.
ALISON: A biofilm is like a big goop of microbes, held together with all sorts of polymers including protein, fats, sugars, or polysaccharide, and even DNA that microbes have exuded. Some soil microbes can move a little bit, though. For example, if they have tail-like flagella, which helps microbes to swim around.
JENNIFER: But it’s the fungi who really move and particularly the, what we call, mycorrhizal fungi. And those are the organisms that have a long, long-term relationship with plants.
The roots of Avena fatua, also known as wild oat grass, and the soil surrounding them. This zone, the rhizosphere, influences microbial life. (Erin Nuccio)
ALISON: Mycorrhizal fungi — myco meaning fungus and rhizzal meaning root — have special branching filaments called hyphae, which comes from the Greek word for ‘web.’
JENNIFER: Hyphae that are part of mycorrhizal fungi that are connected to roots, they’re deriving carbon from the host plant.
ALISON: That carbon is basically sugars and other organic compounds, and because it’s made from photosynthesis, another word for it is photosynthate. Fungi love that stuff.
JENNIFER: And what they’re doing in return for the plant is exploring the soil. They’re looking for nitrogen, for phosphorus, even for water that they bring back to the plant in exchange for that photosynthate.
ALISON: These different jobs cycling nutrients are ecosystem services. And how microbes carry them out is what Jennifer spends most of her waking hours thinking about. In fact, she’s a leader of one of the Department of Energy’s scientific focus areas looking at the soil microbiome.
JENNIFER: So our project is called Microbes Persist, because we’re interested in how microbes affect how much carbon sticks around in soil.
These bright green spots are fluorescently labelled bacteria from soil collected from the surface of plant roots. For reference, the scale bar at bottom right is 10 micrometers long. (Rhona Stuart)
ALISON: It’s a fascinating question, with implications for negative carbon emissions. Instead of releasing that carbon as carbon dioxide into the atmosphere, and exacerbating the climate crisis, Jennifer’s thinking about how to keep that carbon in the ground. In this episode, we’ll hear more about how Jennifer’s getting to know the soil microbes munching on carbon: who’s there, who’s alive, and what they’re doing.
There are two major approaches that Jennifer has used to figure out who’s active in the soil. The first is Stable Isotope Probing, or SIP for short. And the second approach is looking at what genes soil microbes express. Her isotope experiments were actually really crucial to deciphering later gene expression experiments, so let’s dig into the isotope story first. Jennifer explains how a stable isotope experiment works.
JENNIFER: So, when we do a stable isotope probing experiment of a rhizosphere, what we do is we grow the plants. And those are contained in a labeling chamber. And all that is, is a big plexiglass box a couple, a couple feet in diameter, that we fill with 13 CO2. So 13 is the heavy isotope of carbon. It’s a stable isotope, it’s not radioactive. But it is typically only 1% of the carbon pool, whereas carbon-12 is the major isotope. So if we’ve added 13 C into the atmosphere, any carbon that’s fixed by the plant and any photosynthate that’s created and, and exuded through the roots, is going to be heavy and labeled in that 13 carbon.
ALISON: And thus, any organism that’s busy taking up those compounds..
JENNIFER: and growing and dividing and using that carbon to build its nucleic acid backbone is going to become literally heavier.
ALISON: Once the microbes grow with the plant for at least 4-5 days, Jennifer’s team can do a couple different things. One is to actually image the cells, so they can see who’s active.
JENNIFER: And because it, you know, you can almost imagine, it’s like, we’re shooting a sandblaster in a wall, and we raster across the surface of the sample. So, then because we’ve, we’ve collected all those particles of sand that came off the surface, we can recreate an image that is a chemical image of that cell.
Bacteria in a thin sectioned and freeze-dried soil aggregate light up using an isotope-based imaging technology called NanoSIMS. The microbes appear to have ingested glucose labelled with carbon 13, indicating they’ve been recently active. (Christina Ramon (left), Jeremy Bougoure & Marco Keiluweit (right))
ALISON: And wherever there’s more isotope, the brighter that part of the image will be, so the colors become more yellow or even white.
JENNIFER: They look like they’re glowing. The organisms that use that particular substrate. The way that we render the images, we look at a ratio of the heavy isotope to the light isotope.
And so we literally, in our images, since, against a dark background of cells who were all kind of napping or doing nothing, who’re not eating that substrate, the few that are doing whatever activity it is, you know, maybe taking up ammonium or taking up glucose—
ALISON: Both compounds that can be labelled with heavy isotopes.
JENNIFER: They’re literally glowing! They’re little bright spots in the image.
And when you kind of put that next to maybe a secondary electron image, where we have a lot of 3D kind of resolution, you kind of go and look at those same spots, you’re like, Oh, my gosh, look at that cool, little, you know, desiccated-looking raisin that’s right there. Because that’s kind of what they look like, after they’ve dried out.
ALISON: That happens all the time when they’re prepped for imaging.
JENNIFER: And you just know, that cell, it was active, it was busy that day, and took up what I fed it. We can start to really decipher what it is that those, those particular organisms were up to.
ALISON: Jennifer and her team can also start to identify the active soil microbes, by taking a soil sample, breaking up the cells and looking for DNA that’s laden with heavy isotopes.
JENNIFER: And we put it in a heavy liquid and put it in a centrifuge and spin it for five days.
ALISON: Uh, just for comparison, the sample spins that I would do were on the order of minutes. So, I was shocked!
JENNIFER: I know it takes a long time! And what happens is the heaviest the 13 C-labeled DNA fragments are going to move lower in that centrifuge tube, and the less labeled are going to be at the top. So we actually have a physical gradient that we separate out.
ALISON: They then, very carefully, separate the different layers of DNA by poking a hole in the bottom of the tube, and dripping out the layers, drop by drop and hour after hour, into multiple new tubes.
JENNIFER: And then each of those layers we, we sequence. And that allows us to distinguish in the heavier layers [because the DNA is denser], they’re going to be the organisms who took up more of the label,
ALISON: i.e, the heavy isotope, like 13 carbon,
JENNIFER: in the lighter layers [because the DNA is less dense] are organisms who were not interested that day in plant exudates, or were dormant or, for whatever reason, their DNA was there, but they were not active.
ALISON: So, in a project with the JGI, Jennifer and her team used this technique on organisms hanging out near the root, the rhizosphere.
JENNIFER: And we just took all of that DNA that was in the heavy fraction, and got it sequenced.
ALISON: Because DNA from different organisms is all mixed together in these samples, the data they got back was a jumble.
JENNIFER: But you can then use bioinformatic tools that the JGI is very expert at, to actually, to pull together the individual genomes of the organisms who would have contributed to that sample.
ALISON: And with the genomes, Jennifer’s team could now say, ‘These are the organisms that are really active and they’re chowing down on those exuded plant compounds.’
Then, Jennifer used that laundry list of microbes to inform her next experiment. She and her team, with then-postdoc, and now-Lawrence Livermore staff scientist Erin Nuccio leading the charge, asked a question: What microbes are active in distinct sub-environments of the soil? For instance, which are active next to dead roots, and which are active next to live ones, in that zone called the rhizosphere?
Erin Nuccio, staff scientist at Lawrence Livermore National Lab, hard at work isolating rhizosphere samples from switchgrass grown at UC Berkeley. (Kateryna Zhalnina)
JENNIFER: That community that surrounds the rhizosphere, that’s just having a huge party, you know, from all the different kinds of exudates and organic acids and amino acids and different kinds of compounds that the, the root is sloughing off and exuding. So that, that party is, it’s changing, who’s there. But, you know, if you go look at who’s in the rhizosphere, in a three-week-old plant root, versus a 12-week-old plant root, they’re really remarkably different communities.
ALISON: So the idea of the experiment was who was actively expressing genes, or making messenger RNA, or mRNA transcripts, in those different environments. And then to also find out what those genes even were.
JENNIFER: Because that’s a great clue to what they’re doing. Now, it’s not the end of the road, because if you’ve taken biology recently, you know that those transcripts have to be translated and made into a protein and the protein has to be active. So, there’s still a number of steps. But if an organism is expressing a particular transcript at a very high rate, that’s a good indication that there’s a reason; they’re responding to some sort of environmental cue.
ALISON: So, Erin set up the experiment.
JENNIFER: She had an annual grass called Avena barbata growing in soil from California.
ALISON: This plant, Avena barbata, is everywhere in California, and it’s also known as wild oats.
JENNIFER: And we were able to grow it over its whole lifespan, about 12 weeks, and very carefully dissect out the soil that’s immediately next to the roots.
ALISON: They had a spectrum of conditions going; some samples had living roots, some had decaying dead roots, and some samples had both.
JENNIFER: The point is that all of those situations are different, unique habitats with different substrate – or we call that food – availability for the microorganisms.
ALISON: And they anticipated that the microbial activity would be really different between these different conditions. So they ran the experiment, collected the soil, extracted the RNA and gave that to the JGI to sequence.
JENNIFER: And we, in the meantime, had created this kind of— I don’t know, I’m gonna call it crazy, but it’s really amazing — hodgepodge database of different genomic resources,
ALISON: All were genomes from soil.
JENNIFER: And those came from single-cell sorted genomes, they came from a couple of really deep metagenomes…
ALISON: Deep metagenomes are DNA sequences from lots of organisms that were sampled all together. And, for the database, they also used those isotope-labelled genomes from microbes that had eaten heavy carbon and gotten their DNA labelled.
JENNIFER: We took all of these kinds of different genomic data and created a database of, you know, here’s everything we know about the genomes of the organisms that live in this soil.
ALISON: Their database was key to understanding the mRNA transcripts that they sequenced.
JENNIFER: We were able to map those transcripts back to this great database..
ALISON: And that allowed them to determine who the organism was expressing these genes. And where!
JENNIFER: And that is, to our knowledge, the first time that that had been done in soil, certainly in the rhizosphere. But even in soil, it’s, it’s really rather rare to take that approach.
Avena barbata grows in an enclosed chamber with carbon 13-enriched carbon dioxide to track down metabolically active soil microbes in the rhizosphere. (Jennifer Pett-Ridge)
ALISON: Erin created what she called guilds of organisms. It’s a term used in ecology, but it goes way back. Think of medieval times, like 500 years ago: guilds of craftsmen, like glass workers, blacksmiths, or merchants. They’re groups with specialized abilities, just like microbial guilds.
JENNIFER: Right away, when we looked at the data, there were clear patterns in both time and space, that, you know, these organisms all seem to be really, really active and expressing these kinds of transcripts, early when the plant’s growing, whereas, you know, these other group of transcripts only seem to show up very, very late as the plant was starting to senesce.
ALISON: Some microbes were really only found next to living roots, and some only next to dead ones.
JENNIFER: So it really, it allowed us to take, this kind of big gamish of organisms and start dividing them up into categories, different guilds that appear to have a sort of speciality in terms of the way that they relate to their substrate.
ALISON: These substrates are different organic compounds, like cellulose, xylan, pectin, starch, glycogen, and other kinds of sugars. They’re sort of like different jelly bean flavors. But for microbes. And they don’t eat all the flavors; they specialize.
JENNIFER: And that’s kind of the way we think about these microorganisms who— they might even be, In a phylogenetic or taxonomic sense, very closely related, but they appear to differentiate and create different niches for themselves or inhabit different niches by carrying out different roles.
ALISON: JGI’s sequencing helped uncover these results which are published in The International Society of Microbial Ecology, or ISME, Journal. And that work has had a big impact on Erin’s career trajectory and funding.
JENNIFER: Around the time that that article was published, she put in a proposal to the Department of Energy’s early career awards program. And she was awarded one of those very prestigious grants, and that’s going to fund her for five years to look at more rhizosphere processes.
ALISON: But that’s not the end of the story for the RNA dataset that Jennifer and Erin created with JGI. The next chapter was, unexpectedly, about soil viruses.
JENNIFER: I’ll be really honest that when we wrote our original JGI proposal, we didn’t have viruses on the mind at all.
ALISON: Someone else did, though.
JENNIFER: A graduate student who worked with Mary Firestone and Jill Banfield realized, wait a minute, there are these things called RNA viruses,
ALISON: i.e. viruses that use RNA instead of DNA to encode their genome,
JENNIFER: and we know almost nothing about them in soil. But if they exist, they’ll be in your samples. They’ll be in your dataset. So Erin shared the dataset with him.
ALISON: And that ‘him’ was Evan Starr. He found all sorts of sequences from RNA viruses.
Evan Starr looked for the signatures of RNA viruses in the soil data unearthed by Jennifer’s team. Evan’s now a field application scientist with Insightful Sciences. (Courtesy of Evan Starr)
JENNIFER: And because of the kind of unique CRISPR tools and tools that the Banfield lab has developed, he was actually able to determine who the likely hosts are for these different viruses.
ALISON: CRISPR is a microbial immune system that leaves chunks of viral DNA in a microbial genome. So it’s possible, through those sequences, to link a virus to its microbial victim or host.
JENNIFER: He was able to show that, Oh, these are ones that likely parasitize fungi, and then there are another group, the Leviviridae, who infect Proteobacteria. And they seem to be really changing in their abundance along with their hosts, which is just a whole— again, it goes back to the kind of ecology questions that we love to kind of structure our work with. We think of predators and prey being part of a kill-the-winner scenario. Once a population of prey becomes very, very abundant and is winning, they are, you know, more attractive for predators to come along. The viruses, you know, in some way act, just like all predators do. They are targeting large populations. And so we see these kind of cyclical nature of the abundances of some of the viruses that we identified.
ALISON: The team found hundreds of RNA viral species. It kind of boggles the mind, that there’s still so much to discover.
JENNIFER: The diversity of RNA viruses that we found is amazing. And that’s even given the fact that we know that this is probably just the tip of the iceberg. There’s probably far, far more that were there that we just didn’t identify, because the sequences weren’t of good quality, or, in a lot of cases, we just don’t have excellent databases to map to. So we, we might find a sequence but not know that it’s an RNA virus, just because there’s nothing homologous to it in that database.
ALISON: Remember how Jennifer said the rhizosphere is like a party going on? Well, with the discovery of the RNA viruses, it’s like…
JENNIFER: …each of the individuals who’s at the party is actually containing another 10 individuals inside of them, or hiding behind them.
And that’s kind of where we are, right now in terms of viruses. It’s just showing us that the interactions in soil are just so much more complex than we really ever realized.
ALISON: And Jennifer and her team hypothesize that those predator-prey interactions, where viruses cause their hosts to burst or lyse, are critical for the soil carbon cycle.
JENNIFER: We think a lot of the lysis that occurs, that’s driven by both DNA and RNA viruses, is creating compounds that then have the ability to be sorbed to mineral surfaces and stored or or lost, but they’re, they’re accelerating the turning of the wheel as it were, of the soil carbon cycle.
ALISON: Now, Jennifer and her team are starting to turn their attention to what’s happening on a slightly bigger scale. They’re looking at gene expression and isotope labeling in, for example, bacteria who eat other bacteria, and they’re also looking at protists, which are single-celled eukaryotes.They hope that looking at these other kinds of interactions will give them more clues to the microbial ecology happening beneath our feet.
JENNIFER: We’ve always known that there’s a food web. But when we start to create networks, based on our gene abundances, you know, it just looks like this massive spider web of interactions. And, we just know— it’s going to take some serious, maybe, machine learning to really deconstruct, what the effect is of all those interactions. It’s just absolutely fascinating how much biology is going on in soil.
ALISON: This episode was directed and produced by me, Alison Takemura, with editorial and technical assistance from Massie Ballon, Ashleigh Papp, and David Gilbert.
Genome Insider is a production of the Joint Genome Institute, a user facility of the US Department of Energy Office of Science. JGI is located at Lawrence Berkeley National Lab in beautiful Berkeley, California.
How can you identify active microbes in soil? Start off with an environmental sample of them (top right of the illustration) and split it into two treatments, keeping one unlabelled (treatment 1) and labeling the other (treatment 2) with heavy isotopes. Then with molecular biology and sequencing techniques, scientists can determine metabolically active members of the soil microbial community. (Victor O. Leshyk)
A huge thanks to Jennifer Pett-Ridge, senior staff scientist at Lawrence Livermore National Lab, in Livermore, CA. She leads Livermore Lab’s Environmental Isotope Systems Group, and also heads a U.S. Department of Energy Scientific Focus Area to understand mechanisms in the soil microbiome. She also recently completed a project with JGI as part of the Emerging Technologies Opportunity Program. The project succeeded in automating steps involved in processing samples from stable isotope experiments, which will be a big help to scientists interested in getting started with this technique. And JGI now offers this technology to researchers!
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That’s it for now. See ya next time!
Additional information related to the episode
- Jennifer Pett-Ridge’s project proposal summary: Unraveling the Rhizosphere Carbon Cycle
- Jennifer Pett-Ridge’s US DOE Scientific Focus Area project: Microbes Persist
- Facilities Integrating Collaborations for User Science (FICUS) proposal call
- The Emerging Technologies Opportunity Program (ETOP)
- Webinar: Stable Isotope Probing (SIP) technologies at EMSL and JGI
- Genome Insider Podcast: Episode 2: Role of Viruses in Releasing Greenhouse Gases? (2/2), featuring Gary Trubl, a postdoctoral fellow with Jennifer Pett-Ridge, talking more about isotope labelling experiments
- Two papers Jennifer Pett-Ridge and her team published on soil microbial communities:
- US DOE Early Career Research Program
- The JGI Annual Genomics of Energy & Environment Meeting
- Our contact info:
- Twitter: @JGI
- Email: jgi-comms at lbl dot gov