Hi. I'm Lucy Shapiro. I'm a professor at Stanford University. I'm also
a part of the department of international studies at Stanford University.
And what I study are bacterial cells and how they work. That's going to be
the first talk that I'm going to give you. And the second talk, I'm going to
talk about why understanding bacterial cells and viruses has become
absolutely critical in understanding the way in which we live in a global village.
And I'm going to talk about in the second talk emerging infectious diseases
and how understanding what goes on inside this tiny world, the world of
viruses or the world of bacteria is absolutely vital for dealing with
emerging infectious diseases. Now my first talk as you can see from the
title is the dynamics of the bacterial chromosome, how it is organized,
how it separates during segregation and how it's coordinated during cell division.
So we're going to go into the inner workings of the bacterial cell
and try to understand how these things happen. I should mention
right before I start that the work we've done in understanding this bacterial cell
has helped us design an entirely new class of antibiotics. I'm not going to
talk about that today but it's something that is the offshoot of all work
on either bacterial cells or viruses. So now let's talk about Caulobacter.
Caulobacter is a simple bacterial cell. In fact, the name Caulobacter
"caulo" means stalk in Greek so that this is a stalk bacterium, you can
see the stalk here. What happens is that at every cell division,
which you see happening here, you have an asymmetric division.
So get a swarmer cell from one part and a stalk cell from the other.
The swarmer cell can swim away to find food. The stalk cell sits
down on a rock and waits for food to come to it and then it divides again.
This swarmer cell, which is shown here going through the cell cycle,
will first release the flagellum, which is this wavy thing down here.
These are pili--you get rid of both the flagellum and the pili.
The circle inside the cell indicates the DNA. And once you differentiate
from the cell with the flagellum, which is called a swarmer cell,
into a stalk cell, you initiate the replication of the chromosome.
As the chromosome is replicating, which is shown here, you're turning
on lots of genes, that allow you to build a new flagellum at this pole
opposite the stalk, and you turn on all the genes for cell division
and chromosome segregation. Now indicated below this cartoon of the cell
cycle are all these boxes. And these boxes indicate individual modules
that happen at this time during the cell cycle. And these modules
have many, many genes within them. So for example, flagellar
ejection means that there are like 45 genes that have to be turned on
to get rid of it and then to rebuild it. Then as you move through the cell cycle
what happens is you initiate DNA replication, flagellar biogenesis,
pili biogenesis. And all of these functions have to be coordinated
and regulated and be a part of turning on and turning off the replication
of the chromosome. So let's now ask, "Well how is this happening?"
What about the chromosome is something that we really have to understand?
So shown here is a picture of an E. coli cell. That's a bacterial cell--
very similar to Caulobacter that's been very extensively studied.
And in this particular picture, it's been exploded on an EM grid so
that you can actually see the DNA as it's released from the cell.
And it's really quite a mess. There's a huge amount of DNA and it
doesn't really look like it has any organization. But in fact, when you
understand that in this small bacterial cell that is approximately 2 microns--
very little, ten times smaller than the smallest eukaryotic cell.
The DNA is a thousand times longer than the cell if it were completely
stretched out. So we have to compact this DNA very tightly
into the cell. And remember that inside the cell you're going to have to
duplicate this DNA, make a complete copy of this great big mess
and while that's happening you have to transcribe the genes that are
allowing you to proceed through the cell cycle. So how is this all happening?
Well, one of the first things we did was first ask, "Are there specific
parts of that chromosome that sit at specific parts in the cell?"
And what I show you here is an experiment in which we have
actually lit up a particular pole of the cell. And so up in this
picture, you're going to see red dots, and you're going to see
green dots. The red dots, indicate the origin of replication. So the
bacterial chromosome exists as a circle. And in that circle, you have a
origin sequence, where you initiate DNA replication. DNA replication
in bacterial cells proceeds bidirectionally, ending at a terminus.
And what I'm showing you here is in this little swarmer cell we have
a one origin at the pole right down by the flagellum and the terminus
winds up at the other pole. So now as we begin replication and go
through the cell cycle (this is in synchronized cells) you can see that
the very first thing that happens is that the origin duplicates and a
new one goes to the opposite pole. Then as you continue through the
cell cycle, you see finally you've duplicated the termini, which means
you've completed DNA replication. But these cells are dead cells.
You know, I synchronized the cells. I do in situ hybridization of a labeled
probe. I want to see how these chromosomes are actually able to
move a particular locus. How does it move that origin from one
pole of the cell to the other? And let me show you the technique
that we now use to do this. Here I have a plasmid sitting in the cell.
And this plasmid has on it a lacI gene, which is a gene that codes
for a protein that can sit on a specific binding site on the chromosome.
What we do is we hook that gene up to a fluorescent tag. So it
turns on a bright light. Sitting next to it on the plasmid, I in fact have
another protein. It encodes for another protein--trpR--that sits next to
a different fluorescent tag. So one is shown in blue. The other is shown
in yellow. In the meantime, what we've done is we've inserted into
the chromosome shown here, repetitive copies of a lacO gene or
in fact a tetO gene. And these are the binding sites that we're
putting into a specific place on a chromosome so that once we
turn on these labeled tagged proteins, we can actually visualize
where it is in the cell. So down here, I show the cell. And in one part
here--let's just look at tetO. TetO is now being bound by those little
lit-up proteins that bind to TetO and we can actually see them
in the cell. And if we turn on both, and let's say we tagged two
different loci in the chromosome, we can then see two different
dots as shown down here. So given that what we started to do was
ask, "Where are these positions relative to the origin of replication?"
Here's the origin of replication. It's tagged with something yellow.
And if it's tagged with yellow, when we look at the cell, which is shown
diagrammatically here, we see that the origin is in fact closest to the
flagellum, which is what we saw when we did that other experiment.
Next, what we did, is we labeled another part of the chromosome
with a different color. And we said, "Well, where is that going to go
in the cell?" And when we looked at that in the fluorescent microscope,
we saw that it was moved away coming in this direction. Then we labeled
up yet another. And that's shown here, and that comes next.
And then we labeled yet another. And so what we're beginning to see
is a linear order of the genes on the chromosome that reflects where
they line up in the cell. This was a big surprise. It tells us that the DNA
sitting in this cell is not a disorganized mess and individual loci
probably know where they are. Now at this point we were just looking at
a few loci. What we have to do is look at lots. And so the way
in which we looked at lots is we took this mariner transposon
that had all these TetO sequences lined up on them, and we
jumped this transposon into the chromosome shown here. So that
we actually jumped in 114 different sites. One transposon jumped in
a particular site in an individual cell. So every cell has just one
transposon in a particular site. And then what we did was we mapped
and measured where each one was. And as you can see here in this
half I show little dots. And we sequenced and measured exactly
in each individual cell where that transposon was. On the other side
I have yet another group that we measured at a particular site in the cell.
And then because each one of these was tagged with a fluorescent tag
we were going to be able to see where it was in the living cell. And
here's the answer. The answer is that there is a linear relationship
between where that TetO array was put in the cell, on the genome,
and where it exists in the cell. So therefore, we now know that the chromosome
is a highly ordered structure and that every gene has a specific cellular address.
The origin is near the flagellum. The terminus is near the top of the cell
and every locus in between goes to a specific place. Furthermore,
using this technique, we're able to actually follow the movement,
let's say of the origin, in real-time in living cells.
And up here what I show you is moving now through the cell cycle and looking
quite closely at--this is the origin and then as soon as replication
starts, the replisome, which is the machine that copies DNA, is laid down
on top of it. And as soon as you initiate replication, you see that the
dot that lights up the origin travels to the other pole. And I show this
down here, and if you can watch this, here's a swarmer cell with the
origins at this pole and they are moving right down to the other end
of the cell very rapidly. And when we first saw this, we just couldn't
believe it. Remember, these cells are tiny and we're looking at them
in real-time living cells and watching a newly duplicated segment of
DNA harpoon across the cell and hit the other pole. And I show
that in three different panels here. In each one you can follow
the origin of replication as it moves to the other pole. This allowed us
to actually design a computer algorithm that watched the movement
of these origins and mapped out for us the rate of movement
of the fluorescent tag. So we were able to show that it takes
0.2 microns/minute for the new origin, which is this blue line,
to move from one pole of the cell to the other, independent of cell length.
Now, it has been thought for many, many years--because we really didn't
know but it seemed like a logical thing to say--that this great mass
of the bacterial chromosome duplicated as a mass and a mess
and was attached to the membrane of the cell and the chromosomes
only got pulled apart by the growth of the cell. This turns out not to be
true. The copying of the DNA and then the movement of the segments
as it's being copied occurs simultaneously and is independent
of the growth of the cell. And that again is shown here. So now
what I've done is just to help you get through this in understanding
exactly what I've said. This shows the dynamics of bacterial chromosome
segregation. So we start here in G1 phase with our organized chromosome,
our origin, our terminus, and this yellow dot is showing a particular locus.
And let's follow these. So as this swarmer cell releases the flagellum (and it will
grow a stalk in a minute), what happens is the replisome gets formed on this part
of the cell. That's that green stuff. And replication begins and as you move,
what happens is that the replisome is spewing out DNA in both directions. The origin
has been harpooned to the other end of the cell and as you spool out DNA
at its pole, it separated pulling to the two poles. Now if you follow your little yellow dot here
as this guy is duplicated, what's happening is it is moved to the incipient new cell
at exactly the same place, like a mirror image of where it was before and then
cell division happens and we're back to the beginning so that when this cell divides
the top and the bottom will look very similar. But what could be doing this?
So it was thought of for many, many years that the bacterial cell was too small
to require proteins that help things move. It was too small to have particular
protein complexes in specific places. Its DNA was not wrapped up in a nucleus.
So people thought, "Well the cell is so little that proteins and pieces of DNA
could be anywhere in the cell in milliseconds by diffusion." In fact, this is not true.
Bacterial cells have proteins that control movement. They have localization of
complexes at different places in the cell. And one of the candidates for
helping this happen is an actin-like protein. Now actin has been well known
for many, many years in eukaryotic cells being involved in cytoskeletal functions
and movement. Bacterial cells have an actin homolog. And that's called MreB.
So we decided to investigate how MreB might help or function in the cellular
movement like moving that origin. The MreB actin--this one is showing the polymerization
of that actin in another kind of cell. Down here it shows in fact how this actin is
organized almost in all bacterial cells, including caulobacter, and it's organized in a spiral.
All the way down the length of the cell. And so in order to study this particular
protein and how it might work, we of course made mutants, knocked it out.
The cells change shape. They become sort of round balls and then ultimately die.
So the cell really needs this. But I wanted something that would immediately
inactivate that actin in some way. So what we did was go--well first of all we read
the literature and found that a group of people led by Dr. Wachi in Japan had
discovered a small compound (we'll call it A22) that when applied to E. coli,
which is usually a rod, causes it to form a sphere. Now Wachi didn't know
how this compound worked, but since we know that if you make a mutation in MreB,
in that actin, you get cells to ball up and form a sphere, we figured, "Well let's
collaborate with him and try to find out how this works." So here's the compound.
It's easy to make. It's a very simple compound and the way in which we determine
how this works, is we carried out a screen for mutants that are resistant to this
little compound A22. And we found, as you can see here, lots of them and
we then showed that of these 20 alleles all of them were able to grow
in the presence of A22, which means they're resistant. And then we proved
that this was true by replacing the mutant gene with the wild type gene
and you go back to sensitivity. And then we prove it in fact in a wild type background
with the mutant gene. By sequencing exactly where these genes exist on the
chromosome, we were able to show that each and every one of those 20 alleles
that are resistant to MreB exists in the ATP-binding domain of the actin monomers.
Therefore, A22 is a specific inhibitor of MreB function. Now using this inhibitor,
we were able to ask, "Does the origin move if we've knocked out the function of MreB?"
But first, we had to prove that MreB does not disrupt DNA replication or initiation.
And in fact it doesn't. A control is using a compound hydroxyurea that does
stop it, but if you treat it with A22, it does not. So now we're ready for the experiment.
And here's the experiment. What you do, as I told you before, if you start with
a swarmer cell (the green dot is the origin), as the swarmer cell differentiates into a stalk
cell, you have the separation of the origins. One remains at this pole, the other goes
to the other side. Then you go through the cell cycle and you wind up with the
same organization of the chromosome in both. Now here's the experiment.
What we did was we added A22 to these swarmer cells, and asked, "What's going to happen?"
Well, what happened really surprised us. DNA replication started and you make two
new little origins, but nothing moved to the other end. And as you went through
the cell cycle, but of course you were not separating the chromosomes--you didn't
divide the cell. And using a cell sorter--FACS--we were able to show that we indeed
had just duplicated the DNA. So we had two chromosomes but they had not
separated. This tells us now that MreB, actin, is important for the separation
of the newly replicated origin of DNA replication. We then went on and
showed that MreB binds to some structure at that part of the cell--at that part
of the origin. Now what I've shown you is we have coincident DNA replication
and segregation. How does the cell use this to coordinate all of its different functions?
And it does so in two different ways. So I'm going to tell you two very short
stories. One way it does it is it controls the spatial regulation of where you put
the cell division site. Now let me show you how this works. So what I'm showing
you here is another cartoon of the cell cycle. Shown in green is something called
the FtsZ tubulin, which is a protein that is able to go to midcell and
contract the cell to carry out cytokinesis. And as you can see here, it's in the
center of the cell. Cell division happens, and when it finishes, you still have some
of this FtsZ left at that pole. So now let's look back and look at this swarmer cell
that has just resulted from cell division and what we have here is the FtsZ left
at the pole; at the other pole is the origin. And bound to that origin is a newly
discovered protein, which we call MipZ. So we have MipZ bound to this origin. We
have the FtsZ tubulin at the other side of the cell. And when the origin with its
MipZ moves to the other part of the cell, then the FtsZ monomers go to midcell.
So the question that we're faced with is, "Might MipZ directly inhibit FtsZ assembly at
the correct cell site?" So here's what this thing looks like. What I show you here is a
piece of DNA in which we have the origin shown here, and a protein called ParB lines up
all the way around the origin. And then this MipZ protein forms by binding to ParB
but now very tightly so that it in fact forms some kind of gradient. And when
we look very closely, using the fluorescent microscope labeling either the ParB
with GFP or in fact the MipZ protein with YFP, you can see that the ParB is very
tight at the poles, whereas the MipZ forms a gradient with the least expression
at midcell. So now, what we did was we purified MipZ. We purified the FtsZ tubulin
and what I show you here is that in the absence of MipZ, the FtsZ monomers
form these long polymers. This is just a higher magnification. But if we then
add MipZ to it, these big long monomers get very small and curvy and they're
non-functional, telling us that MipZ is an inhibitor of the polymerization of the
FtsZ tubulin that will carry out cytokinesis. With this information, we can now
draw a model. So here is a cartoon of the Caulobacter cell. This work was done
by a postdoc in the lab, Martin Thanbichler and this little blue dot here is the
origin of replication. Nothing is happening yet. This is a cell that has just differentiated
from a swarmer to a stalk cell. At the other pole of the cell is FtsZ, waiting to
do something. Here at the origin we have the ParB, and we have all this MipZ
sitting on it. Now as soon as the origin is duplicated, that ParB-MipZ complex
lines up on that as well. So we now have it on two. And as it moves across
the cell and as the MipZ hits the FtsZ at the other pole, it moves away
to the region of lowest concentration. Therefore, the movement of these
origins from one pole to another using an inhibitor like MipZ allows you to
polymerize FtsZ at the place in the cell where there's lowest concentration of
inhibitor and that is the center of the cell. Now the second
story I'll tell you, and it's really the last story I'll tell you, is that another
way that the cell integrates these various functions--integrates DNA
replication and segregation with the progression of the cell cycle--is to
control the master regulators, to control the expression of the master
regulators. And it does this by using an epigenetic mechanism, which is
DNA methylation. So if you have a chromosome of Caulobacter, then a
swarmer cell, there are sequences in the DNA--GATC--that get methylated on
the adenine. And when you start the cell cycle, you're methylated both on
Watson and Crick--on both strands of the DNA. Once you begin replicating though
the two newly replicated chromosomes are only hemi-methylated.
They are methylated where they started, but the new one is not. And that's
going to be the epigenetic control mechanism that's used. So what I
show here is the genetic circuitry that is controlled by three master regulators.
And how that allows you to move through the cell cycle. The three master
regulators are a protein called DnaA. This is made first during the cell cycle.
And it controls about 40 genes. When this is turned on, and it's turned on
just for a specific time in the cell cycle, it then turns on the expression of
another master regulator GcrA, which controls about 50 genes. Then later
in the cell cycle, GcrA turns on one of the promoters of the third master
regulator CtrA, and CtrA controls about 95 genes. So you have a cascade
of master regulators that control all the functions that allow you to proceed
through the cell cycle. And what I'm going to show you is that a gene that
encodes an enzyme, that actually puts those methylation sites on the DNA
is a critical event in making this a cyclical regulatory pathway. And here I show
you that the first thing that gets turned on is DnaA, then what happens is GcrA
is turned on, then CtrA is turned on, and when it finishes that, CtrA turns on
this DNA methyltransferase, which comes back--is turned on at the completion of
DNA replication to label up all of the loci. So you go back to a fully methylated
chromosome. The critical question is with respect to DnaA in fact is what turns on
this cascade? How do you get that DnaA to be turned on? And this was quite
a surprise. It turns out that if you look at the chromosome, we have the origin
at one part, the terminus at the other. The DnaA gene sits right near the origin.
This CtrA gene sits on the other side and a little further down. Now when you
begin the very beginning of replication, the origin and the whole rest of the
chromosome exists in the fully methylated state, so you're methylated on
Watson and Crick. In the fully methylated state, this promoter for the DnaA gene
is able to be red. There are methylation sites in that promoter and it can
only be red in the fully methylated state. However, the CtrA gene shown here can
only be red if it's hemi-methylated. And the cell uses the methylation state of
the chromosome as a clock to tell you when to turn on DnaA and when to turn it off,
when to turn on CtrA and when to turn it off. So at the very beginning when
it is all fully methylated, you can transcribe the DnaA gene but you cannot
transcribe the CtrA gene. Once replication is initiated and you copy the DnaA
gene, then you get two copies that are each hemi-methylated. Remember
I told you that it can only be transcribed if it's in the fully methylated state.
So as soon as you get two copies that are hemi-methylated, DnaA is not
transcribed. Bingo--it's off. But it's already done its thing. It's allowed
DNA replication to start. It's controlled genes that make up the replisome
and copy DNA and it's done its thing. Then what happens is as you continue going
through the replication of the chromosome, you pass through the CtrA gene.
That goes from a fully methylated state, where it can't be transcribed, to a hemi-
methylated state, where it can be transcribed. And then it's turned on. So in fact
what I've now shown you is that the passage of the replication fork controls
methylation state and activity of two promoters of these critical global regulators
that allow the progression of the cell cycle. So then to give you a summary of
what I have shown you--this again shows you the familiar cell cycle that we've
been talking about. And I've shown you that origin movement--and we've watched
the origin move--is dependent on MreB actin. A protein complex coordinates
DNA replication and the positioning of the cell division site--that was that MipZ
protein that forms a gradient. Replication and segregation are simultaneous
and finally that whole transcriptional regulatory network is coordinated with
DNA replication via the methylation state of the chromosome, an epigenetic
mechanism. So what I have shown you is that all these various modules
that are turned on and off through the cell cycle are all hooked up and coordinated
with the act of replicating the cell's DNA. So I would just like to thank the very,
very talented bunch of students and postdocs in the lab who have done the work
I've spoken about--Martin Thanbichler did that very pretty work on the MipZ.
Justine Collier on the DnaA regulation. Ann Reisenauer on the control of CtrA.
Natalie Dye and Zemer Gitai, who's just left the lab to start his own at Princeton,
have worked on the MreB actin. Patrick Viollier and Rasmus Jensen on the
organization of the chromosome. And I would like to state now that a lot of our
work is done in collaboration with a lab that is really a physics lab. And this other
lab is led by Harley McAdams and he's a physicist. He's also a professor in the
Department of Developmental Biology at Stanford and his students all get their
PhD's in physics or electrical engineering. And we work by coordinating our labs
completely so that when his students, who are physicists and engineers, start their
thesis work they do it in the same lab with all the geneticists and biochemists
who are working in my lab. So at this point, we have a completely interdisciplinary
crew and it has opened up new areas of investigation, which I don't believe
could've been done without that. So with that, I'd like to thank you very much.
