Hello, my name is Richard Losick and I'm a professor at Harvard University
and this is the second part of a three part presentation on
the developmental biology of a simple organism.
In this part, I'll be telling you about multicellularity in a bacterium,
the spore-forming bacterium Bacillus subtilis.
Bacteria have traditionally been thought of as solitary creatures
that go about their business on an individual basis
but increasingly we've come to recognize that bacteria can form
complex, multicellular communities known as biofilms.
I'm going to tell you about biofilm formation in Bacillus subtilis
where you'll see that the ability to produce a complex community
is linked to the process of sporulation.
The first topic that I'll cover is the formation of these communities
and a visualization of spore formation taking place in aerial structures.
Then I'll tell you about how these communities are held together,
what's the basis for their architecture?
And you'll see that there's an extracellular matrix
that glues the cells together, kind of the mortar for building these structures
and that the synthesis of this matrix is governed by
an intricate regulatory circuit that I'll describe.
And then finally, we'll look into a biofilm and we'll see that it resembles a tissue
with different kinds of cells in different places and with dynamic changes
in which different kinds of cells either grow or diminish
in their relative abundance in this tissue-like structure.
Well, that B. subtilis was capable of forming architecturally complex communities
was missed for many years for the following reason.
In the many years since this bacterium was discovered,
it's been worked on in the laboratory and, as a consequence,
over time, it's inadvertently become domesticated.
And so the standard laboratory form of bacteria don't form robust, multicellular communities.
It's as if we've bred this out of the bacterium over time.
Here, for example, is a colony of B. subtilis on a plate
and you can see it forms a relatively unstructured colony.
And here is a culture...a standing culture of cells in which the bacteria
have collected as a very thin film at the surface of the culture.
But once again, not much architecture there.
But now if we go back to a wild strain of the bacterium
we see something that's dramatically different.
In the colony you can see a rich architecture with thick veins
of cells and other kinds of structures
and likewise, at the air liquid interface, a thick mat of cells known as a pellicle forms
that also has a distinctive architecture with all kinds of detailed features.
Let's look more closely at the biofilm structure and in particular
I'd like us to focus on the outer edge of the colony where you can see
that there are aerial structures that rise up from the surface.
I'm going to show you next one of these aerial structures in a cross-section view
with cells that harbor a fusion of the LacZ gene for beta-galactosidase
to a promoter that's under the control of a sporulation transcription factor.
So we'll be able to see where spore formation is taking place in this aerial structure
by staining the structure with a chromogenic dye
that turns blue when the beta-galactosidase product
of the reporter is produced.
And as you can see there's a striking, intense blue staining near the tip, the aerial tip
of this surface of this structure that rises from the surface.
And as I go on, I'll argue later that it might be appropriate to think of these structures
as fruiting body-like structures that perhaps have been selected in evolution as...
for the purposes of dispersal of spores and I'll further suggest
that sporulation in the context of this community
depends on the very formation of the biofilm.
Let's look even more closely at this aerial structure
by scanning EM...by scanning electron microscopy.
And what you can see is that the biofilm consists of long chains of cells
that are cemented in parallel fashion to each other.
Long chains of cells, each one is glued to each other.
The glue that holds all these chains of cells together is known as the extracellular matrix.
The bacteria export a matrix material that cements the chains of cells together
so that the architecture can be built. This matrix consists of a polysaccharide
or a so-called exo-polysaccharide and a specific protein.
The polysaccharide is produced by enzymes encoded
within a large operon known as the eps operon,
for exo-polysaccharide operon. And the protein is encoded
with an operon that's referred to as the tasA operon.
Let's consider how these two operons are turned on under the right circumstances
that lead to the formation of the multicellular community.
This is mediated by an elaborate regulatory network that involves fully six regulatory proteins.
So on the right are the two operons, the one for polysaccharide synthesis
and the other for the protein and circled are the six regulatory proteins
that govern the expression of these two target operons.
This looks bewilderingly complex but, as I hope to show you
at its heart it has a simple logic.
So to simplify things, first of all let me get rid of the regulatory protein on the right
and let's just focus on the remaining five proteins;
SinI, SinR, AbbA and AbrB and Spo0A.
SinR and AbrB are repressor proteins, they each cross-repress both target operons.
So SinR represses the polysaccharide operon and the protein operon.
Likewise, AbrB contributes to the repression of the polysaccharide operon and the protein operon.
So two different repressors help to hold both of these matrix operons off.
In order for the operons to be de-repressed,
we need to antagonize the action of these two repressors.
So let me first convert the names of those two proteins into repressors,
the red repressor and the blue repressor, to make things look simple.
And now let's consider the SinI and AbbA protein. What are they doing?
Well, SinI is an anti-repressor that binds to its respective repressor to inactivate it.
Likewise, AbbA is an anti-repressor that binds to its repressor to inactivate it.
So let's convert SinI and AbbA into anti-repressor and anti-repressor.
So now you can see that there's a simple logic here
in which two parallel pathways of repression and anti-repression govern the expression
of the two operons that are responsible for matrix production.
This redundancy probably helps to ensure that the matrix genes are held totally silenced
until the right time and the right place for a community to form.
How does this whole system get going?
Well that's the role of the master regulator Spo0A.
Spo0A is a master regulator both for sporulation
as we saw in my previous presentation on sporulation
and it's also the principle regulatory protein
that's responsible for triggering biofilm formation.
Spo0A turns on the gene for both anti-repressors:
the SinI anti-repressor and the AbbA anti-repressor.
So, when Spo0A becomes activated, that leads to the production of two anti-repressors.
The two anti-repressors then bind to and inactivate their respective repressors,
and that finally leads to de-repression of the two operons that are responsible
for matrix production.
OK, now let's look in more detail at the biofilm.
We've just considered in some detail the regulatory pathway that's responsible
for turning on the synthesis of the matrix.
So some of the cells in the biofilm, at least, are responsible for matrix production.
But now let's consider what other kinds of cells might be present in the biofilm.
As we saw earlier, spore formation is also taking place in the biofilm.
And there are also vegetative cells that are capable
of motility that are present in the biofilm.
So, what we're going to do is take one of these biofilms, cut it in half,
and then look at it from the side by confocal microscopy using three different fluorescence reporters:
one to a gene under sporulation control; another under the control of the matrix producing pathway;
and a third under the control of genes involved in motility.
So each of these reporters represent three distinct kinds of cells:
sporulating cells; matrix producing cells; and motile cells, motile, vegetative cells.
OK, so if you look now at the bottom you can see that matrix producing cells in red
and sporulating cells in green have distinct locations.
They occupy distinct positions in the biofilm. The sporulating cells are near the top
as we saw in that fruiting body-like structure
in the light micrograph that I showed you at the beginning.
And the matrix-producing cells are underneath.
Now let's consider the position of motile cells.
The sporulating cells for comparison, once again, are near the extreme top and the matrix...
the motile cells are near the extreme bottom.
So motile cells, matrix cells and sporulating cells occupy three different regions of the biofilm;
with the motile cells near the bottom, the matrix-producing cells in the middle, and
the spore-forming cells near the top. So we can begin to think of the biofilm as
a kind of tissue that's composed of different kinds of cells that
occupy different positions in that tissue.
But this is a dynamic tissue because the relative proportion
of these different kinds of cells changes over time.
And I can illustrate that for you with a simple experiment
in which we take these biofilms harboring these fluorescent reporters
and separate all the cells from one another, disassemble the matrix
so that the cells are separated and then we use a fluorescence activated cell sorter
to measure the relative proportion of the three cell types over time.
Let's first consider sporulating cells. So the arrow marks increasing time up to 72 hours,
and the axis that runs from left to right represents increasing fluorescence.
As you can see, sporulating cells appear as a distinct population of high fluorescence
only at about 48 hours into the process.
If we look instead at matrix-producing cells, a peak of matrix producing cells
appears between 12 and 24 hours and then diminishes somewhat over time.
Then, finally, let's consider the motile cells.
The motile cells are most abundant at the beginning
and then they gradually diminish over time and become less and less
abundant by 72 hours into the process.
So gene expression is dynamic in the biofilm.
Cells occupy distinct positions.
Cells of different types and their relative abundance in the biofilm changes over time.
Now, finally, let me come to what I think might be the most interesting finding
concerning having more than one cell type.
As we've seen, spores are produced near the top of aerial structures.
And it's appealing to imagine that perhaps these are primitive fruiting body-like structures
that perhaps have a role in spore dispersal.
Well, is the process of spore formation not only associated with
multicellularity, is it actually dependent upon it?
And we can ask that question by looking at sporulation gene expression
or the process of sporulation both in a wild-type biofilm
and a biofilm that's mutant for matrix production.
Actually, a mutant that can't make a normal biofilm.
And then we can use the fluorescence activated cell sorter to measure the
proportion of sporulating cells in the wild-type and in the mutant.
And that's shown in this slide here. You can see that in the wild-type case in blue
there's a distinct peak of cells, a distinct sub-population of cells
that are undergoing sporulation, that are expressing sporulation genes.
But when we look at the expression of the same sporulation gene in a matrix mutant,
well, that peak almost completely disappears.
In other words, in the context of the biofilm, spore formation
is substantially dependent on the formation of an architecturally complex community,
on the production of the matrix and the formation of these structures.
So, that plus the earlier result that making a biofilm, like sporulation,
are both under the control of the same master regulator, Spo0A
leads us to speculate that this has an important biological significance.
And that in nature, spores are formed not in the form of individual cells on their own
at least not all the time but are sometimes produced in the context of
complex multicellular communities in which sporulation
is itself coupled to the process of assembling the community.
Lastly, let me point to the future on an important challenge that awaits us
in the years ahead. Even though we think we understand quite a bit
about the regulatory network that governs the production of the matrix
we're a long way from understanding how matrix-producing cells assemble
macroscopically into the elaborate architectures that I've been showing you.
Well, this issue comes into even sharper relief when you consider the following finding.
My collaborators and I have been collecting wild strains of Bacillus from around the world.
And the striking finding is that frequently these different wild strains
each exhibit their own distinctive architecture.
You can see that in this slide.
Here is a collection of different, very closely related strains of Bacillus subtilis
yet each one exhibits its own distinctive architecture.
Consider this one at the top or this one over here, or this one down near the lower left.
They have their own distinct morphotype.
Surely, this must be dictated by genetic differences between these two strains,
unknown genetic differences and in principle it should be possible to identify
the genetic differences between these strains that give rise to these different morphotypes
and thereby obtain a clue into the larger challenge of understanding
how morphogenesis is controlled, how a multicellular community with a distinctive
architecture is created by the expression of genes involved in the formation of the biofilm.
Finally, it's important for me to emphasize that all of the work that I've told you about
has been a wonderful collaboration between my laboratory
and that of my good friend Roberto Kolter at the Harvard Medical School
on the Cambridge side of the Charles River.
The individuals that have been involved in this story
that I've told you are Dan Kearns, Win Chai, Frances Chu and Anna McLoon.
And on the Harvard Medical School side of the Charles River in the Kolter lab
the individuals who have driven this project forward are Steve Branda, Dani Lopez,
Claudio Aguilar, Hera Vlamakis and Ashlee Earl.
Thank you very much.