Hi.  I'm Colleen Cavanaugh.
I'm a professor in the Department of
Organismic and Evolutionary Biology
at Harvard University,
and the Co-Director of the
Microbial Sciences Initiative.
Today, I'm going to tell you about
the discovery of chemosynthetic symbioses.
These are intimate associations between
bacteria and marine invertebrates
that allow both partners to
colonize and thrive
in otherwise inhospitable environments.
The discovery of these symbioses
followed on the discovery of
the deep-sea hydrothermal vents
and the communities that flourish around these.
But finding the symbioses at the vents
also highlighted that
these occur in other habitats,
such as coastal sediments
that have a lot of sulfide.
So, today, what I'm going to do is
first provide you with some background on symbiosis,
on chemosynthesis
-- the basis of these symbiotic associations --
on the hydrothermal vents,
and then on my academic path through this adventure.
So, symbiosis, the term,
was coined by Anton deBary
to mean the living together of differently-named organisms.
These are associations with benefit
to at least one of the partners,
and as examples,
there are corals,
which have intracellular photosynthetic algae that feed them;
legumes that form symbioses
with nitrogen-fixing bacteria
-- so, effectively, they're self-fertilizing;
chloroplasts and mitochondria
in our cells and plants,
which evolved from free-living bacteria;
and of course, recently,
we know about the human microbiome,
there's been a lot of work in the last 7-8 years,
and the impact that your own bacteria,
those within your gut, on your skin,
and all over your body,
have on development of your immune system,
nutrition, neurobiology...
the list goes on and on.
So, symbiosis is an
incredibly important aspect of biology.
So, the common theme, here, for these symbioses,
and one of the main processes,
is CO2 fixation.
Carbon fixation is...
typically, we think of it by plants,
which is via photosynthesis.
This is taking carbon dioxide out of the air
and turning it into sugars,
and in plants you use light to fuel that operation.
In bacteria that are capable of chemosynthesis,
they use inorganic compounds,
such as hydrogen sulfide, iron, hydrogen,
and oxidize these compounds to produce the energy
that is then used to fix CO2
in the same processes as photosynthesis.
Deep-sea hydrothermal vents
are ecosystems on the sea floor.
They were discovered in 1977,
in association with the search and study
of seafloor spreading.
Essentially, these are hot springs
that are associated with volcanic activity,
along the ocean ridges.
You can see in the top photograph
that these ridges circle the Earth
like seams on a baseball.
These are areas where the sea floor is spreading,
there's a lot of volcanic activity.
Sea water percolates down into the deep ocean...
or, the subfloor...
is heated, chemically charged with compounds,
and then is forced up through other fissures,
as you can see on the bottom photo
with the so-called "black smoker",
and, around these communities,
around these vents,
are these lush communities of
tube worms, clams, mussels, shrimp,
with biomass that rivals that of the rain forest.
This totally revolutionized our perceptions of life
in the deep sea.
Typically, it was thought to be
very, very sparse, because food from the surface
-- photosynthesis --
wouldn't make it down, and this...
so, it was really, how is this working?
Bringing up samples of sea water
on some of the first cruises by geologists,
they noted that it smelled very strongly of hydrogen sulfide,
and it was proposed that bacteria
using such compounds
could be the base of the food chain here,
via chemosynthesis.
As you'll see from the rest of the talk,
in addition to free-living bacteria
supporting these ecosystems,
symbiosis is prevalent and in fact
the dominant form of carbon fixation at the vents.
So, my path to chemosynthesis
and symbiosis in the deep sea
started at the University of Michigan.
I'm from Detroit and went to Michigan,
and there was a marine ecology
offered at the Marine Biological Lab,
by three Michigan professors,
it was a Michigan course.
We had two...
we had two weeks of lectures
and then had to design our own project,
and to me it was incredible that
I learned that not everything is known,
that textbooks aren't always right,
and I fell in love with research,
and I fell in love with Woods Hole,
and managed to work out so that
I could come back after graduating at Michigan.
Then I worked at the Ecosystems Center, with John Hobbie,
and during this time I was
basically in a dark room, looking in a microscope,
the whole day, all summer,
counting bacteria.
He had developed a method,
using fluorescence microscopy,
for actually being able to count bacteria in the real world
that are too small to be able to be seen
with a light microscope,
so it was a very revolutionary period of time
on the numbers of bacteria in the world.
I then went to Harvard, to graduate school.
My plan was to work on
coastal and open ocean nutrient cycling,
as this was part of the research
that I was doing when I was in Woods Hole.
So, this brings me to Harvard.
As a first-year graduate student,
I was taking a class called
Bio 255: Nature and Regulation of Marine Ecosystems.
It was basically professors...
three professors that offered it,
they talked about their own research,
and then they brought people in to
talk to the students.
And because the vents had only been discovered recently,
we had a series of talks on them,
and the first was on the ecology, by Ruth Turner,
a professor in my department,
the second was on chemistry, by Russ McDuff,
who was a chemist at MIT working on vents,
the third was on microbiology.
Holger Jannasch was from Woods Hole,
from the Woods Hole Oceanographic Institute,
I knew him there,
and he talked about the presumed microbiology,
chemosynthesis being the base of the food chain,
and a lot about sulfur bacteria, iron bacteria,
and many others.
Last, we had Meredith Jones,
the curator of worms from the Smithsonian Institution.
He came, and he was basically
a histologist and a taxonomist,
and in class he described
these tube worms, seen in this picture,
which can get up to over a meter in length.
And it turns out, very peculiarly,
they did not have a mouth of a gut.
And so the big question was,
how do they feed?
And, essentially, their smaller relatives
that could take up dissolved organic carbon
across their epidermis,
but these were so big,
it was thought the surface-to-volume ratio was much too small,
and the concentration of organic carbon at these vents
was low.
So, amazingly,
he was basically describing the tube worm
section-by-section,
through a one-meter tube worm,
and I was still awake when
he came to this slide,
and this is the so-called trophosome tissue.
This is the tissue that fills
the coelomic cavity of these tube worms.
It's a brown spongy tissue.
It was named trophosome
in previous descriptions
because it has the gonads embedded in it,
and the tissue was thought
to feed the developing eggs and the developing sperm.
So, he mentioned...
he showed this picture in the...
during the talk,
and if you look in the back
that's a millimeter ruler, so this is a regular photograph,
and he mentioned that
he saw all these crystals, and he had them analyzed,
and it turned out they were pure elemental sulfur.
It was at this point where I
jumped up and said, "Well, it's perfectly clear!
They have a symbiotic association
with these chemosynthetic bacteria
that live inside of them,
and then they feed the animal,
just like dinoflagellates,
the algae and corals, feed their corals."
He said, "No, no, we think it's a detoxifying organ."
And I said,
"Well, that's perfect, because
these bacteria can oxidize sulfide to elemental sulfur
and render it less toxic, or not toxic,
and can still fix CO2 and feed the tube worm."
So, after the talk,
I was able to convince Dr. Jones
to send me some tissue,
and that I would prove there were bacteria there.
Indeed, I thought it was a done deal,
I thought it was a given,
but I had to actually go through...
jump through many hoops to try to prove
that there were bacteria in this tissue.
Now, I might mention
I had never had a microbiology course,
and I had worked for John Hobbie almost...
well over a year-plus,
so I had a love of bacteria,
but I was looking at...
the majority of which could never be cultured,
so I was really jumping in at a basic level.
I had done scanning electron microscopy,
which is a method to
be able to look at tissues at higher magnification.
And you see on this higher...
this upper figure,
this is a scanning EM of the trophosome tissue,
which is very lobular,
and what I did was took a piece of tape
and pulled off a little bit of the surface,
and if you look closely
you'll see that it looks like...
the bottom figure, is what it is...
basically, it looked like lots of grapes to me,
large-type spheres,
and I was so disappointed
because I was used to looking at tiny little things
that were bacteria,
and I was like, "Oh, my goodness, these are huge...".
The... basically, the scale bar at the bottom is
10 microns,
so they're about 2 or so,
and I was used to looking at 0.4 micron bacteria.
I did... I talked with Holger Jannasch at WHOI
(Woods Hole Oceanographic Institute) about this,
and he did point out that symbionts...
first... oh, let me show you...
first, I did find a couple rods,
so it was promising...
a little, but only a couple.
I then talked with Holger Jannasch and he explained that
many times, bacterial symbionts,
when they're intracellular,
they lose their cell wall,
because they're osmotically protected,
and so they can have very weird shapes
or even just balloon to spheres.
So, I had hope.
In addition, we looked...
where the blue arrows are
show what looked like delineations of the host cells,
and that we inferred...
we were testing whether they were actually
intracellular or not, as well.
So, another line of evidence
that I went after, next,
was examining whether these spheres
had DNA,
and to do this I used
epifluorescence microscopy by using a stain called DAPI,
and this brings me back
to my work with John Hobbie .
So, I thought, if these spheres had DNA,
then I would have another line of evidence.
And indeed, as you can see in my notebook,
these are how I did all of my microscopy,
which is by drawing,
you can see these spheres,
and I had notes that they're about 1-2 microns... 2 microns...
and that they stain with DAPI,
indicating that they have DNA.
So, this is another line of evidence.
I also, to control...
to check the size,
took a scraping of my cheek cell,
which you see in the lower side of this notebook page,
and it turns out that the nuclei were
about 10-12 microns,
so I had another line of evidence that these things,
the "grapes", were bacteria.
But some of the more definitive work
came with transmission electron microscopy.
This is where you take thin sections through tissue,
and it actually shows the ultrastructure
of the internal organelles and organs,
and, indeed, these looked like bacteria.
So, the bacteria are labeled "b"
in this slice through the trophosome,
and they have a typical size and morphology,
and if you look at the panel on the right,
they have the typical cell envelope of Gram-negative bacteria
-- those inner two lines --
and then an outer membrane
that's inferred to be that of the host,
indicating that they are within the cytoplasm,
but separate.
So, taken together,
all of these data, along with work by other researchers,
which had shown that there are
enzymes associated with chemosynthetic bacteria
in the trophosome tissue,
and other data,
we concluded that this is
a symbiosis between these giant tube worms,
and that they form associations
with sulfur oxidizing chemosynthetic bacteria.
They have not yet been cultured.
They fall within the gammaproteobacteria,
determined by DNA sequencing.
And there has been lots of research on these,
which I hardly suggest you look at.
In general, they're shown to have
many, many adaptations to this symbiosis,
which include, of course,
being mouthless and gutless
-- they're being fed internally --
but also biochemically.
That red you see in the plume is a hemoglobin,
and it's unique in that it binds
sulfide and oxygen in separate locations.
Our hemoglobin is poisoned by sulfide,
but here they manage to deliver both compounds
to the bacteria, separately.
So, given this sort of symbiosis,
and the benefits accrued to both
-- it's a nutritional symbiosis,
the worms get internal nutrition,
they're able to live in areas
that they don't need photosynthetic food,
and the bacteria get rich...
I have, with my bacteriocentric point of view...
benefit because they
get compounds for energy generation
from both anoxic and oxic environments.
At vents, the sulfide is produced geothermally,
but the oxygen is in the overlying seawater.
And the... by colonizing an animal,
coevolution, adaptations...
the animal is bringing the sulfide and oxygen to these bacteria.
Given the benefits for both,
I would predict, and did,
that these would be everywhere,
not just at vents, but in sediments also...
like, muds, that have sulfide.
So, you can imagine my delight
when a paper was published in May,
only two months after I started work on the tube worms,
entitled "Gutless Bivalves".
They must have chemosynthetic symbionts,
they were mouthless and gutless,
and they live in sulfide-rich muds.
And there was a conjoiner in Woods Hole,
right off the coast.
I had already planned to be in Woods Hole that summer,
working on another project,
but I completely switched
and moved down,
and I had another fortuitous event,
and that is that Holger Jannasch's
microbial diversity course,
at the Marine Biological Lab,
was focusing on sulfur, because of the vents,
and he had the sulfur microbiology dream team,
which included, of course,
Holger, Gijs Kuenen,
Yehuda Cohen and Jan Gottschal.
And, effectively, I moved in to learn about sulfur bacteria.
And, as you can imagine,
I was trying to figure out how to find
a 1-micron, 2-micron sized bacterium
inside of a 1.5-cm long animal.
It didn't have elemental sulfur as a clue where to look.
But, fortunately, Gijs Kuenen pointed out that
rubisco, the CO2 fixing enzyme of the Calvin cycle,
which is unique to plants and autotrophic bacteria,
could be used as an assay for localizing
what tissue bacteria might be in.
And, indeed, the gill tissue of these animals
had very high activities of rubisco,
as you can see in this graph.
And indeed, with transmission electron microscopy,
I was able to show that
there are rod-shaped bacteria living
within the gill epithelial cells,
and a close-up shows that they have
the typical Gram-negative cell envelope,
and these, too, are included in what appears to be
a host membrane.
Direct counts of these indicated that
there are 10^9-10^10 bacteria per gram of tissue,
and they also have enzymes
associated with sulfur metabolism,
and fix CO2 depending on various sulfur compound additions.
So, we concluded, indeed,
that this also was a symbiotic association
with chemoautotrophic sulfur bacteria.
Since then, we've done a lot of molecular characterization.
They are gammaproteobacteria
and, while there are strain-level differences,
it appears to be a monoculture,
so, again, a very specific symbiotic association.
The discovery of chemosynthetic bacteria
in the tissues of Solemya
also helps explain a rather peculiar behavioral aspect.
They produce Y-shaped burrows
and hang out right at the apex of the Y.
This had been suggested to either be
a refugium from predators
or perhaps they were gardening bacteria
within the tubes.
However, the symbiosis
explains why they would do this.
They're pumping oxygen from above,
from the overlying seawater,
and sulfide from below,
in the sulfide-rich sediments.
So, they're creating the habitat
that the bacteria need,
and flowing the sulfide and oxygen over their gills.
The bacteria fix CO2,
feed the animal;
the animal is feeding the bacteria.
So, here we had two examples,
one at the vents
and one in reducing sediment,
just coastal sediments,
and this indicated to me,
indeed, that these must be everywhere,
and it turns out that all of the major macrofauna
at the deep-sea hydrothermal vents
have chemosynthetic symbionts.
This includes the bivalves,
the vent mussels
and the giant clams,
and also shrimp,
which, instead of having intracellular bacteria,
actually have them on their surfaces
as epibionts.
Further, when I and many researchers
around the world
looked at other organisms
that live in sulfide-rich muds,
we found these symbioses with oligochaetes,
with nematodes,
and with colonial ciliates.
And, indeed, the picture that looks like a rope,
that is a nematode,
and all of the filaments on top
are actually the bacteria,
so again, these are on the outside.
So, this hopefully
demonstrated that these chemosynthetic symbioses
are extremely widespread in nature,
they occur in habitats
at the interface of oxic and anoxic environments,
such as the vents and sediments,
and all I've told you about here is the discovery phase.
There has been a lot more research continuing on these various...
various symbioses,
ranging from genome sequencing
to looking at optimal growth at deep-sea pressures
and chemical gradients.
And I'll just point out, again,
as a bacteriocentric,
given the remarkable metabolic breadth of microbes,
I predict that there will be many, many
other symbiotic associations,
not just with chemosynthetic bacteria,
but with others that have
diverse metabolisms
that would be advantageous
to animals and other eukaryotes
that cannot achieve these sorts of processes.
And, on that note,
as I challenge my students,
both in class and in the lab,
expect the discovery!
Thank you.
