Hi.
My name is Julie Huber, and I'm a scientist at Woods Hole Oceanographic Institution.
And in Part III of my talk, I want to bring together a lot of the information from the
first two talks, and combine it with a new method we developed in my lab
to study subseafloor life at Axial Seamount.
So, I ended Part II basically telling you that, through our genomic tools, we determined
that not all hydrothermal systems host the same populations, especially in the warm anoxic zone
beneath the seafloor.
And what I wanna talk to you about now is how we've really been probing that subseafloor signal
using something called RNA stable isotope probing.
And the goal of this is really to focus in on the autotrophs living beneath the seafloor
without this swamping signature of deep-seawater mixing through the crust.
So, RNA stable isotope probing is a relatively new method.
But it basically brings together the power of stable isotopes with molecular biology.
And the idea is you do an experiment where you put in a labeled substrate
-- whether carbon, nitrogen, oxygen, or, for some, others --
in our case in a bottle.
And you let an experiment run.
And then at the end of the experiment, you gather and extract the RNA,
you physically separate it, and you can look at which organisms have taken up that stable isotope.
And this allows you to identify the active microbes during your experiment.
You can look at carbon flow or interactions between different organisms.
Or in our case especially, really looking at what processes those organisms were carrying out
when they took up that label.
So, at Axial, what we did is basically collected this vent fluid, and then set up a series
of bottle experiments.
And in half of them we put a control of bicarbonate, because remember, again,
we're interested in who the active autotrophs are.
And so we're looking at the organisms that have the ability to take up CO2.
And we have both an experimental control with the light isotope and a 13C experiment
with the heavy isotope.
We then take those bottles, we add some hydrogen gas to we create the reducing conditions
beneath the seafloor, and then we incubate them at a variety of temperatures that we think exist
beneath the seafloor based on our genomic data.
We then let these experiments run for anywhere from about 9 to 36 hours.
And this actually took a number of years to work out the right timeframe for carrying them out,
because you want to catch the label when it's in the organisms that first took it up,
not look at secondary consumers, unless that's your question, which in our case it was not.
So, at the end of the experiment, we filter all of the vent fluid, we extract the total RNA,
and we put it in a density gradient in an ultracentrifuge,
and we spin it at very high speeds for a couple of days.
And what that allows is for the RNA to distribute itself based on the density,
and you can separate that light isotope from the heavy isotope.
You then precipitate RNA from each of those different density fractions, and you can make
a plot like what's shown here, with buoyant density on the x-axis and RNA concentration on the y.
We can take a zoom in on that, and talk a little bit more about what we're seeing here.
So again, on the x, buoyant density, and on the y, RNA concentration.
And what I'm showing here in orange is the heavy isotope, and in the green is the light isotope.
And you can see that this heavy isotope is a higher density.
And what that represents are the organisms that took up the 13C label in our experiment.
And what I'm mostly gonna be focusing on today is really the active autotrophs
and what we're learning about them in this part of the plot.
However, it's important to know that we in fact did a lot of different sequencing of this RNA
to convince ourselves that the experiment worked.
And so we sequenced both the light fraction, which was enriched, as well as its corresponding control,
and we did that with both experiments.
And that was to convince ourselves that what we're seeing in this heavy 13C peak
are actually the organisms that took up the label in the experiment.
So again, I'm only gonna be showing this red fraction, which is those active autotrophs
in the experiment.
So, here we have an experiment that we carried out at Marker 113, at three different temperatures:
up at 30 degrees, 55, and also 80 degrees Celsius.
Again, representing different habitats that we think exist beneath the seafloor.
You can see that the 80-degree experiments aren't quite as lovely as the other two,
and that has to do with these high temperature organisms having a higher GC content in their DNA,
which often makes these experiments a little bit... can be a little bit messy.
And so, again, in all three of these experiments, though, we do have good separation
between this lighter isotope and the heavy isotope.
And what I'm gonna be showing you in the next slide is just the sequences
from this active autotroph peak in all cases.
So, in this plot what I'm showing you is data that I showed you in Part II of the talk,
that first plot over here, which is just the taxonomy of all the messenger RNA transcripts
that we detected from that in situ filter fluid.
And what I want to do is then compare that to the three experimental conditions,
just to say, which organisms were active and taking up the label in our experiment?
And so, what I hope you can see is that when we compare what was happening
at the point of sampling to our three bottles, they're quite different.
All the colors are present in that metatranscriptome, representing the diversity of organisms
in this environment, and in our experimental conditions we see less colors.
And so, basically, fewer organisms took up the label under these different temperature regimes.
So, at both 30 and 55 degrees, we have a group of bacteria called the epsilonproteobacteria,
which took up the label.
In this case, these organisms tend to oxidize sulfur.
And here we have organisms that both reduce sulfur and nitrate.
We also see some methanogens popping up, here, at 55 degrees.
At 80 degrees, we have a different group of methanogens within the archaea.
Again, these are high-temperature autotrophs.
And interestingly, this purple bar, here, these are actually a heterotrophic archaea.
These experiments we probably ran a little bit too long, and so we're starting
to see those secondary consumers come up and eat the autotrophs.
And of course, if that's something that you're interested in, sort of those trophic cascades,
this is a great method to get at it.
In this particular example, we weren't, but it's definitely set off some future experiments
that we're gonna be doing.
So, we can then dig in to those transcripts that we've created from these different experiments,
and say, well, how are they fixing carbon?
How are they gaining energy?
So, in this example, I'm just showing different genes for five different known carbon fixation pathways,
which are shown here in the different colors.
And then, similar to the first part of my talk, this is the metagenome,
representing all the potential,
this is the metatranscriptome, right here, representing all the genes that were turned on when we took our sample,
and then our three experimental conditions in the bottles.
And what you can see is all five of these carbon fixation pathways are present in the metagenome,
meaning the potential for it exists in all of the... all of the samples.
However, when we look at the metatranscriptome, you can see that only four of the five
were turned on at the time of sampling.
And then, in our experiments, there is even less diversity.
So, at 30 degrees, it was only the reductive TCA cycle, versus down here at 80 degrees,
where we have methanogenesis turned on.
And so we can really hone in on which organisms, which... are using what pathways under
these different thermal regimes.
We can also look at how they're gaining energy.
And again, this is similar to the diagrams I showed in Part II, where we have the metagenome
and the metatranscriptome.
And then the three temperature conditions.
And again, looking at oxygen, nitrogen, sulfur, hydrogen, and methane.
Again, what you can see is in that first column, almost all the bubbles are present.
In the second, less are present.
You can really tell what's going on in situ.
And then within our experiments, even fewer.
And again, what this is showing is certain organisms use certain metabolisms under
different environmental conditions.
And we can take a look at what's actually happening in our bottle versus what happens
in the... in the environment, because obviously we have major experimental artifacts here.
But by comparing it to the natural sample, we can see, for example, that a lot of organisms
are using these genes related to oxygen.
But in our bottles, we pretty much got rid of oxygen, and so organisms are switching
to anaerobic metabolisms.
We see a lot of organisms doing nitrate reduction at these lower temperatures.
And as we move into some of the higher temperatures, we see a lot of methanogenesis going on,
and this maps really well back to what we see in the environmental samples.
So, now we know who's taking up the label, we know how they're fixing that carbon,
and we know the energy sources they're using to do it.
So, I've just showed you one example from one place on the seafloor.
And in work that we still haven't published, we've done these experiments
at a variety of vents across Axial, and comparing them.
And I just wanna show you one example.
This is the data I already showed you from Marker 113,
where you have these high-temperature methanogens who took up the label.
And if we compare that to the two other vents that I introduced in Part II, Anemone and Marker 33,
you can see they look completely different.
And so, at this really high temperature range, as we had... as was suggested by our metagenomic data,
there are different populations of high-temperature organisms living beneath the seafloor
carrying out autotrophy.
So, at the Anemone site, it's this group of high-temperature bacteria within the Aquificales.
And then at Marker 33, a different group with... which is in the same phylum, and these organisms
tend to use hydrogen, sulfur, and nitrate.
And you can see that there are a few methanogens coming up at Marker 33 in our experiments,
but they really thrive at Marker 113.
Another cool thing that we've been able to do is kind of compare these experimental results
with some of our genomic data from Part II.
And in this case, I'm comparing it to those metagenomic bins that I talked about,
where you could really focus in on populations that were... are active in situ, or in the environment.
And again, we saw... just without doing any experiments, we saw that methanogen populations
were very active at Marker 113.
And that's what's shown in our SIP experiments, in our RNA-SIP experiments.
And again, two different populations of these Aquificales were present in the other two vents,
and that is also reflected in both the SIP and the RNA...
and the metagenomic and metatranscriptomic data.
So, by having both the omics data, in and of itself, as well as the experimental system,
we're really able to constrain who is doing what and under what conditions.
What we're doing now is we're trying to take all this data together and put together
kind of an ecosystem model for how autotrophy works in Axial.
And there are a lot of other components to this model that I haven't brought
into the discussion today.
This is really just focused on those primary producers.
But we've also been looking at their potential rates, we've been looking at the viruses that kill them,
and we've been also trying to culture a lot of these microbes in the laboratory
so that we have model systems to study and constrain a lot of these environmental parameters.
And so, what I'm just showing here are... is the plume above the volcano,
all the way over here, and then the three vents, and kind of the dominant metabolisms
and organisms carrying them out across this temper... temperature regime.
So, we're bringing together both the SIP and the omic data to make these types of models
about how life works beneath the seafloor at Axial Seamount.
So, to wrap this up, both Part II and Part III, our omics data shows us that we
have different taxonomic and functional profiles across these vents.
They're quite stable from year to year, despite things like deep-sea eruptions.
And we're not sure what maintains that stability.
But we think it is definitely linked to the geochemistry, as well as the physical structure,
of each of those sites.
Our genomic binning data shows that we have very vent-specific populations,
especially deeper, in the warmer parts of the subseafloor.
And our RNA-SIP data also showed those same differences in those active high temperature groups,
as well as different metabolisms at each temperature within a vent.
So, even though all we're collecting is maybe a 25-degree fluid coming out of the seafloor,
we see a huge range of metabolisms and temperatures that these organisms can exist at
beneath the seafloor.
So, this combined approach is really helping us to define subseafloor primary production
at Axial Seamount.
So, this RNA-SIP work and genomic work, it's really kind of a deep dive into details.
But since this is Part III, and the end of all parts, I just wanted to remind you of
some of the big take aways from this type of work.
First is that the discovery of deep-sea ecosystems, the hydrothermal vents in particular,
really changed the way we view life on our planet.
And it's actually opened up even more possibilities about how we could think about life beyond Earth,
potentially on ocean worlds such as Enceladus, which is Saturn's moon,
or Europa, which is Jupiter's moon, both of which are known to have an ocean.
Another big take away is that this very fundamental reaction between seawaters...
seawater and rocks creates diverse energy sources that microbes can use, not only to gain energy
but also to support these chemosynthetic ecosystems.
And in thinking about all the possibilities, I'm quite certain we have not discovered
all the possibilities for the types of energy sources that microbes can extract to support ecosystems.
The third point is that the subseafloor is really a global oceanic habitat.
It's everywhere there is a seafloor, which is the entire ocean.
And the dominant organisms in those environments are really these amazing microbes,
who put up with an awful lot to thrive in this environment.
And finally, the deep sea is, you know, one of the most unexplored corners of our planet,
as is most of the ocean.
And even though it's mostly out of sight, it should not be out of mind.
So, with that, for Part III, I just wanted to thank the agencies that funded this RNA-SIP work.
In particular, the Moore Foundation, NSF, and the Schmidt Ocean Institute.
And thank all the great people who worked on this project to develop this technique
in a pretty challenging place.
And with that, I just wanted to say thank you.