Bonnie Bassler (Princeton) Part 1: Bacterial Communication via Quorum Sensing

Uploaded by ibioseminars on 27.03.2010

Hi, my name's Bonnie Bassler and I'm a professor at Princeton University
and I'm also an investigator at the Howard Hughes Medical Institute
and I'm delighted to be here to get to talk to you about bacterial quorum sensing.
So the goal of my two seminars is to try to convince you that bacteria can talk to each other
and to try to show you that what this chemical communication does
is to allow bacteria to act as enormous multicellular organisms and
accomplish tasks that they could never accomplish if
they simply acted as individuals, because they're too tiny.
And so what I thought I would do today is to give you sort of a historical tour
of how we got into this idea that bacteria can talk with each other
and then try to give you some ideas of where this field is going
and how we're trying to do something that's medically relevant
by interfering with bacterial communication.
So, to get going, the idea that bacteria can talk to each other all started
about 40 years ago when a man named Woody Hastings made an amazing observation.
So what he noticed was that there were two bioluminescent bacteria,
so bacteria from the ocean that made light, sort of like firefly light,
but what he noticed was a special property of the light.
That they only made light when they were at high cell density.
And so this is actually depicted on my first slide.
What you're looking at is a flask.
This is just a person from my lab holding a flask of a liquid culture of one of these two bacteria.
They're called Vibrio fischeri and Vibrio harveyi.
And what you can see is that the flask...or the liquid in the flask is glowing in the dark.
And that light is made by the bacteria, in this case, Vibrio harveyi.
And what Woody Hastings noticed was that at low cell density,
so when the bacteria were alone, they didn't make light.
But, when they grew to a certain cell number, all of the bacteria turned on light together.
And so the question that he wanted to know, and what we're going to talk about today is:
"How can it be that a bacterium knows the difference between times when it's alone
and times when it's in high number?"
And what's fantastic about bioluminescence is that you can just see it.
So, we're geneticist in my lab and we want to understand how these bacteria
perceive when they're alone and when they're together.
And so what we can do is to just to make mutants, turn the lights off,
look at the Petri plates and look for bacteria that are glowing when they shouldn't be
or not glowing when they should be.
And just by doing that simple experiment, we and many other groups in the field
have been able to understand how these bacteria tell the difference between
times when they're alone and times when they're together
and then initiate a signal transduction cascade,
the output of which is this beautiful bioluminescence.
So, I told you that Woody Hastings noticed this both in two bacteria,
one was named Vibrio harveyi, which is this free living bacteria that you're looking on at this slide
and the other one was its very close relative Vibrio fischeri.
So like Vibrio harveyi, Vibrio fischeri is a bioluminescent marine bacterium.
But it lives a very different lifestyle than Vibrio harveyi. Vibrio harveyi's free-living
but Vibrio fischeri lives as a symbiont in a number of marine animals
one of which is this one, which is called the Hawaiian bobtailed squid.
So what you're looking at is a squid that's been turned on its back
and dissected down the middle and I hope that you can see are these two glowing lobes
that make up a specialized light organ in the squid
and this is the place that the bacteria live.
So the way this symbiosis works is: the squid is,
this light organ is under the bottom or the mantel of the squid.
This light organ is under there and it houses Vibrio fischeri
at something like ten to the eleventh or ten to the twelfth cells per mL.
We have no idea how the squid grows the bacteria to this high cell number.
But what we know is that the bacteria are trapped inside the light organ
and in there they make a molecule that you can think of like a hormone
or a pheromone and we call it an auto-inducer.
So they make and they release this molecule
and since the bacteria are trapped inside the light organ of this squid
the molecule gets trapped inside with them
and it tells the bacteria "You're inside, not outside. So, you should make light."
And so Vibrio fischeri glows because it perceives this molecule
which helps it count its neighbors.
And so the reason Vibrio fischeri does that is because this squid feeds it.
So this light organ is loaded with amino acids and nutrients and all kinds of goodies
that makes the life of the bacterium much easier than
if the bacterium was living free-living in the ocean.
So the selection from the bacterium's point of view is that it gets fed
and gets to grow to high numbers in that squid.
Now the reason the squid is interested in this is because it wants the light from that bacteria.
So the way this work from the squid's perspective is:
this little squid, its only about this big full grown
and it lives off the coast of Hawaii, that's why its called the Hawaiian bobtailed squid
and the squid is nocturnal. So, during the day it buries itself in the sand and it sleeps
but then at night it has to come out to hunt.
And so on bright nights when there's lots of starlight or moonlight,
since this squid lives just off the coast, so just this sort of shallow, knee-deep water
since its living in this shallow water, the light from the moon and the stars
can penetrate the water that the squid lives in.
And so what the squid has developed is a shutter.
It's the squid's ink sack that it can open and close over this specialized light organ.
And so what happens is that the squid has detectors on its back
so it can sense how much moonlight and starlight is hitting its back
and then it opens and closes this shutter so the amount of light
coming out of the bottom, which is made by the bacteria,
exactly matches how much light hits the squid's back.
So this way the squid doesn't make a shadow.
So it actually uses the light from the bacteria to counter-illuminate itself
as an anti-predation device.
And so the selection for the light from the squid's point of view is that
it gets protected, for its lifetime, from predators.
So the bacteria make the light, they get fed and the squid uses the light to protect itself.
But of course this squid has this terrible problem
because it's got this dying, ten to the twelfth cells per mL culture of bacteria
in this little light organ and it can't maintain that.
And so what happens is every morning, when the squid goes back to sleep,
when it buries itself in the sand, its got a pump that's attached to its circadian rhythm.
And so when the sun comes up, what the squid does is it pumps out 95% of the bacteria.
And so now the bacteria are dilute and of course that little molecule,
that hormone, auto-inducer molecule I told you about, is gone.
And so now the bacteria can't perceive that molecule, so they don't make light.
But then as the day go by, the bacteria start doubling, they keep releasing this hormone molecule
and at night, they perceive it, and the light comes on exactly when the squid needs it.
And so the reason to tell you about these two, first stories is because
now we understand that hundreds of species of bacteria make auto-inducers
and talk to one another with these chemical communication systems.
So, the way we understand this in the simplest terms is that at low cell density,
so this is over here, when a bacterium is alone or in dilute suspension,
it's making and releasing these molecules that I have as these little blue dots.
But, of course, if the bacteria is dilute, the molecules just diffuse away.
But as the cells grow in number, when the population density increases,
since all the bacteria are releasing these molecules into the environment
the more cells there are, the more of this molecule there is.
And at a particular concentration of the molecule, which is indicative of cell number,
the bacteria perceive that the molecule is there
and then they all change their gene expression in unison.
And so in the case of Vibrio fischeri and Vibrio harveyi, they turn on light.
And so what you can see then is that these bacteria are acting like an enormous
multicellular group and turning on and off gene expression,
which is really changes in behaviors, on a population wide scale.
And so now we have a fancy name for this: we call it quorum sensing.
The bacteria vote with these little chemical votes, they count the votes,
and then everyone responds to the vote.
And what I hope you'll think by the end of this seminar is that this had to be
one of the first steps in the development of multicellular organisms.
So this is sort of how it works in generic terms and we also know a lot now.
In these past few years we've learned a lot about the mechanisms
that underlie these communication systems.
And so there's a couple of sort of general themes that are emerging.
And so what we now understand is that in gram negative bacteria,
they typically have systems like the one I have depicted on this slide.
So this...these ovals will be my cells
these are supposed to mean bacterial cells from here forward.
So what we know about gram-negative bacteria,
is that they have an enzyme which is called LuxI, for the inducer.
And this is the enzyme that makes the signal molecule, which on this slide are these red diamonds.
And all of these signal molecules are what are called acyl homoserine lactones.
And I'll tell you more about those on the next slide.
So what we now understand is that hundreds of species of gram-negative bacteria
all have highly conserved LuxI enzymes
that make an acyl homoserine lactone signal molecule
and that freely diffuses in and out of the cells
so the more cells there are the more the molecule there is.
And when the molecule hits a particular amount
it gets bound by a partner protein that's called LuxR for regulation.
And what happens is when LuxR binds the auto-inducer molecule
that unveils a DNA binding domain on the LuxR protein.
So this complex of the LuxR protein and the signal molecule bound
can sit at the promoters of genes that the bacteria want to express
when they're in a community and they turn them on.
And so what this allows bacteria to do is to count their numbers
and then turn on genes that are useful only when the bacteria are acting as a group,
but are not useful when the bacteria are acting as individuals.
So again to reiterate, we now know that there are hundreds of systems like this
in gram-negative bacteria. So that's how the systems work.
We also know a lot about these signals that are made by these LuxI-type enzymes.
So what I did on this slide was just put a few different species of bacteria,
these are gram-negative bacteria that have quorum sensing.
And then I have the molecule that they use to talk with next to them.
And so what I hope you can see is that all of the molecules are related.
So again these are called acyl homoserine lactones.
And so what you see is that the right hand part of all of the molecules is identical.
That's the acyl homoserine lactone or the homoserine lactone.
But this left part, see these carbon chains?
Each one is a little bit different in every single species of bacteria.
And so what that does is to confer exquisite species specificities to each of these languages.
So what I mean by that is that only the bacterium that makes the signal can respond to it
and no other bacteria can. So for example,
if we take the Pseudomonas signal and but it on Vibrio fischeri nothing happens.
And likewise, Pseudomonas is impervious to the Vibrio fischeri signal.
So these molecules fit like locks and keys with their partner LuxR proteins
and there is no cross-talk in these systems.
They are for intra-species communication
and so what we think is that this kind of quorum sensing
allows bacteria to count their siblings.
It allows them to count and talk within their own species.
So that's how gram-negative bacteria do it.
We also now know that gram-positive bacteria have quorum sensing.
And so the ideas are similar to what I've already told you
but the mechanism that gram-positive's use to communicate is a little bit different.
So here I have sort of a model for gram-positive bacterial quorum sensing.
So gram-positive bacteria use peptides as their words or as their signals.
And so the way that this works is that the peptides are encoded in genes
and they get made as a precursor protein. So sort of a big protein.
And then there's a processing system that cuts that big protein into a smaller peptide
and then that gets secreted outside of the cell by a dedicated secretion machinery.
But again, the idea is the same:
the more cells there are, the more of this peptide there is accumulating in the environment.
And in this case, when the peptide hits a critical amount,
it gets bound by a transmembrane bound protein
so sort of a sensor that connects the inside of the cell to the outside.
And when the peptide gets bound, that initiates a phosphorylation cascade.
The bottom line of which is that, ultimately, a transcription factor gets phosphorylated.
That activates it, it lets it sit on the DNA and turn on the genes that the bacteria want
to express when they're a community.
So even though the mechanism is different than in the gram-negative bacteria,
the idea is really the same. The number of auto-inducer molecules, peptides in this case
increases with cell number increasing,
and then information gets sent in to a DNA binding protein
that turns on all of the genes the bacteria need to carry out group behaviors.
So that's how these work in gram-positive bacteria.
And just like I told you for gram-negative bacteria, we also know about the peptide signals.
So again, these are just a couple of peptides that we know are auto-inducers
for different gram-positive bacteria. And they can be a little bit fancy.
So sometimes its just the naked peptide or occasionally they'll have moieties stuck on them.
That's what this asterisk is supposed to mean.
Or they can be cyclized. So they put a few bells and whistles on these signals.
But the bottom line is they're peptides and again, in every case,
each species of gram-positive bacterium has its own peptide.
So these are private languages that are species specific.
And so, gram-positive bacteria use this to count their own cell number for intra-species communication.
And so that's how these systems work.
And now I want to just tell you a few of the behaviors
that are controlled by quorum sensing in different species of bacteria.
So I already told you how it works for Vibrio fischeri,
this bioluminescent bacteria that lives inside this squid.
So that story you've already gotten.
There's another bacterium, Pseudomonas aeruginosa,
which is a terrible opportunistic human pathogen.
This bacterium lives in the soil and it's relatively harmless unless one has cystic fibrosis.
And so probably you know that people who have CF have a genetic mutation in their lungs
so that they can't clear or sterilize their lungs.
So you and I breathe in all kinds of bacteria but we have mechanisms to keep our lungs clean.
People who have cystic fibrosis can't do that.
And so what happens is they have an infection in their lungs
that has all kinds of different bacteria in it.
And for reasons that we don't really understand, typically a person who has CF
in his or her teens will become permanently colonized by Pseudomonas aeruginosa.
And this is the bacterium that kills them.
And the reason is because Pseudomonas has about a hundred genes
that are controlled by quorum sensing, all of which are important for virulence.
And so what happens is that Pseudomonas gets into the lungs
and it makes what's called a biofilm. That's how bacteria stick in us or on us.
So it sits down, it adheres to the lungs, and it secretes all kinds of terrible virulence factors:
proteases and hydrolases that damage the person's lung tissue.
And all of these genes are controlled by quorum sensing.
And so even though this is a terrible disease,
if you actually think about this from the bacterium's point of view,
it's a fabulous strategy for being a pathogen.
The last thing a bacterium wants to do is to get in and when it's at only a few cells
start secreting all of these toxins and virulence factors.
That's why your immune system evolved.
It evolved to do surveillance and get rid of pathogenic bacteria.
So the strategy that Pseudomonas uses
is to get in, to wait, and to count itself with these small auto-inducer molecules
and to recognize when it has the right number
that if all of the bacteria secrete these virulence factors
in unison they'll be able to infect an enormous host.
And so what I hope you can see is that even though the disease is devastating
from the bacterium's point of survival, it's a really good strategy
to wait until you know there's enough of your friends around, if I can say that,
that if you do this thing together, you will be successful.
And so that's basically how we understand quorum sensing.
Another couple of examples, Agrobacterium tumefaciens is a plant pathogen.
And this pathogen causes crown gall tumors on plants
and it's a problem in the agricultural industry.
And what it uses for...with quorum sensing is mating.
So Agrobacterium has the virulence genes on a mobile piece of DNA.
And so what happens is when some of them start infecting the plant
what they do in response to quorum sensing is to start transferring this plasmid around.
So they make the entire community more infective than it was originally.
And so again if you want to give your DNA to somebody
you want there to be a recipient there. So that should be a behavior that's social:
that lots of cells need to do it together.
And then finally, I just put one more on here.
This one is called Erwinia carotovora. This is a plant pathogen.
You've seen this one. It's the one that turns your lettuce
and your potatoes brown in your refrigerator.
And it's a lot like Pseudomonas in that what it does is it waits and it counts its cells
and it recognizes when there's lots of cells there
and then they secrete together all of their virulence factor to make
a wound in the plant. But then what it does, it also has this very insidious strategy,
because simultaneous to that, what Erwinia also does, is to secrete all kinds of antibiotics
that it has immunity to but that other competitor bacteria
in the environment won't be immune to.
So it keeps the wound for itself and its siblings
and it fights off all the other competitors by killing them with these antibiotics.
And so this list goes on and on and on.
And I hope that what you can sort of see from this list
is that all of these kinds of behaviors
are behaviors that cells need many, many cells
acting in synchrony to make the behavior effective.
And so that's what we understand about quorum sensing,
that this is the beginning of multicellularity and these bacteria are carrying out tasks
that they could never be successful at if they simply acted as individuals
because they're too small, individually, to have an impact on the environment.
So, if you remember from the beginning of my talk, I told you that Woody Hastings
discovered this business of these bacteria communicating with molecules
in two different luminescent bacteria. Vibrio fischeri, that I've told you about
and also its relative Vibrio harveyi.
And so you might recall that I told you that Vibrio harveyi is a bioluminescent marine bacterium.
It has quorum sensing, but it lives a different lifestyle than Vibrio fischeri.
It's a free-living bacteria. And so we were interested in understanding
"How does a free-living bacterium in the ocean achieve quorum sensing?"
And so we did what I told you already,
which is to simply make mutants in Vibrio harveyi,
put them on Petri plates and look for bacteria
that weren't making light under conditions they should be
or were making light under conditions that they shouldn't be.
And just by doing that very simple experiment we were able to find the genes
involved in the quorum sensing system for Vibrio harveyi.
And so this is the model we've come up with for Vibrio harveyi.
And this is supposed to be the inside of the bacterium.
This over here is the periplasmic space.
And then way out there would be the outside.
Right, so this is supposed to be the bacterial membrane.
And much to our surprise, when we started working on Vibrio harveyi,
what we noticed was that the bacterium had a completely different quorum sensing system
than Vibrio fischeri and all these other LuxIR bacteria
that I told you about in the beginning of the talk.
What we realized right away is that the bacteria made two different autoinducers.
And so we named them in the order we found them
Autoinducer-1 and Autoinducer-2.
Autoinducer-1 is an acyl homoserine lactone.
So it's one of these run of the mill quorum sensing autoinducers
that's typical of gram-negative bacteria.
And actually there's a picture of it on my last slide.
The second autoinducer, autoinducer-2, was clearly not a homoserine lactone.
So homoserine lactones have very standard biochemical properties.
They all sort of behave the same biochemically.
And it was very clear to us that autoinducer-2 was something different.
And I'm going to spend a lot of time telling you about autoinducer-2 as we go along.
Each of these autoinducers was detected by its own sensory system.
So autoinducer-1 was detected by a protein that we named LuxN
and two proteins called LuxP and LuxQ work together
to detect and send the information in from autoinducer-2.
So LuxN and LuxQ and all of the other colored proteins that I have in this slide
are what are called bacterial two-component signaling proteins.
And these are highly conserved proteins in all kinds of species of bacteria.
And they're all set up the way that I have on this slide,
which is that they have a transmembrane domain that connects the inside of the cell
to the outside of the environment.
And so these transmembrane domains are responsible for detecting
things that are happening on the outside.
In the case of today's talk what they're detecting are these autoinducers.
And then that information gets sent into the inside of the cell
by a phosphorylation cascade that always involves histidines and aspartates.
The way that this signaling system works is that at low cell density,
so when the autoinducers aren't there, these sensors, LuxN and LuxQ, are kinases.
They auto-phosphorylate and the phosphate gets transferred
through this circuit to a protein called LuxU and then finally to LuxO.
And LuxO's job is to turn off luciferase.
And luciferase, which are the genes and the enzymes that make light,
is down here. It's called LuxCDABE.
So there are five enzymes, C, D, A, B, and E, that are involved in making light.
So at low cell density, the system shuts light off.
But then at high cell density, so when these autoinducers accumulate,
and they get bound by their sensors, it flips a switch.
And the sensors change from being kinases to being phosphatases.
So phosphate flows backwards through the system
and that dephosphorylates LuxO and releases it to allow luciferase to be expressed.
And so the bottom line is that at low cell density light is turned off
and at high cell density light is turned on.
And that matter and the way the bacteria detect those two situations
is by whether or not these autoinducer molecules have been built up.
And then I want you to also notice that there's another protein
which I refer to, named LuxP, that's involved in autoinducer-2 detection.
So I just want to remind you that LuxP is the primary binding protein for autoinducer-2
and it interacts with LuxQ to send the signal.
And LuxP looks like a family of proteins that are typical in bacterial periplasms
that all look like sugar-binding proteins.
This protein looks like the ribose binding protein of E. coli.
And we're going to come back to that in a few minutes.
OK, so, by this complicated system information comes in to turn on or off light.
And, so, we were curious at this point why Vibrio harveyi would have two autoinducers.
So I spent the beginning of my talk telling you about Vibrio fischeri
that has this one autoinducer and uses this LuxIR system.
What's the point of having two autoinducers feeding into the system?
And of course we didn't know the answer to that.
But, what we thought was the only way the bacterium can get more out of this kind of circuitry
is if those autoinducers encode different pieces of information.
Right, if they encode the same kind of information, then two isn't better than one.
And, so, we wanted to explore that idea.
And, so, what you know already is that these autoinducers are on the outside of cells.
And, so, what one can do is just grow up cells, spin the bacteria out of the culture,
collect the cell free culture fluids and test them for autoinducers.
And so what we did is we just got every bacterium we could get our hands on.
We grew cultures of them up, spun them out, and we collected the supernatants.
And then we put them onto Vibrio harveyi strains
that could only turn on light in response to autoinducer-1
or could only turn on light in response to autoinducer-2.
So these were engineered strains that could respond to one or the other autoinducer.
And what we found in that experiment is that there was never another bacterium
that made an activity that turned on light through this first system.
But nearly every bacterium that we tested made an activity
that turned on light through the second system.
And, so, our interpretation of that result is that
these two autoinducers are two different languages.
Autoinducer-1 is the species specific language for Vibrio harveyi.
You'll recall that that's a homoserine lactone.
Only Vibrio harveyi makes that.
This second autoinducer we could show was broadly made in the bacterial kingdom.
We could find hundreds of species that made this activity.
And, so, what we thought then is that maybe this is the language of inter-species communication.
This is the bacterial trade language.
Because if you think about how bacteria really live in the wild,
they live in complicated mixtures with hundreds or thousands of other species of bacteria.
And if we end up being right in the quorum sensing field that this is about counting,
it's not good enough to only be able to count your own siblings.
There has to be a mechanism for taking into account other species of bacteria in the environment.
And that's what I'm going to try to convince you that autoinducer-2 is about.
So to get proof of that, what we did was made mutants that couldn't produce autoinducer-2
in several different species of bacteria,
including Vibrio harveyi, E. coli, Salmonella, lots of different bacteria.
And then we cloned the gene that was involved.
And what we found was that indeed, it was the same gene in every case.
And so we named that gene luxS.
And so what you can do probably know that all these bacterial genomes are sequenced
so you can take your gene and plug it into these public databases
and then find out who has that gene.
And what we found is that, sure enough, at last count there were about
500 bacterial genomes that are sequenced
and more than half of those genomes contain a highly conserved luxS enzyme.
And so what I did was to simply put a smattering
of a few of the bacteria that are in the genome database
that have this luxS gene. And so what's important about this list
is that it is a who's who of clinical pathogens.
And so you remember that I told you with autoinducer-1 signaling
many bacteria use quorum sensing to turn on virulence.
And so what we wondered was that if autoinducer-2 and luxS end up being this generic bacterial language
and bacteria are using this to control virulence, one can start to think of making
anti-quorum sensing strategies that could be used in a broad range of different bacteria.
And so the field now is trying to do that in an effort to make new kinds of antibiotics
that no longer kill bacteria but just make them so that they can't count one another
and can't turn on virulence. And so what's interesting in that realm is that all of these pathogens
and if you look at that list its many, many more pathogens,
have luxS. They all make autoinducer-2; we showed that.
And what's amazing about the list is that this list
is both gram-negative and gram-positive bacteria.
So unlike in the acyl homoserine lactone and the peptide quorum sensing systems,
we think that autoinducer-2 is a much more ancient language
that arose before the split
between gram-negatives and gram-positives.
So now we knew that all these bacteria made autoinducer-2,
they all had this highly conserved gene and enzyme LuxS.
And so the questions for us at this point was: What does luxS do in producing autoinducer-2?
What is the autoinducer-2 molecule?
And then, what do all these other bacteria do in response to autoinducer-2?
Obviously they don't turn on bioluminescence. These aren't bioluminescent bacteria.
And, so we wondered, do they use this molecule as information?
And so that's what I'm going to finish this part of the talk by telling you is first:
what is autoinducer-2 and what is luxS's job?
And then what these other bacteria are doing with this new molecule?
OK, so, the first thing: What's the autoinducer and what's LuxS?
So, remember, I've already told you that the bacteria give you this gift.
They put these molecules on the outside and so lots of people, you know,
we included, have been purifying autoinducers from cell free culture supernatants of bacteria.
And so we had been trying to do that with autoinducer-2, sort of doing traditional biochemistry
and we could never purify this molecule.
And we didn't understand at the time why we couldn't get it
but the point was, we couldn't purify it.
And so we thought, "OK, we can't use traditional biochemistry to get it
but can we use what we've learned from these genomes to just figure out,
to infer what luxS does just by looking at where it's placed in these genomes?"
So probably you know that bacteria put genes that work in pathways
nearby one another, usually in operons.
And so what we did was to just look over these genomes and ask:
"Could we find any pattern to where luxS was in these different genomes?"
And in a couple of cases, indeed, we could.
And so I've just put on this next slide the case that we've found for Borrelia burgdorferi,
which is the bacterium that causes Lyme disease.
What we noticed was that LuxS was in a three gene operon in the Borrelia genome
and in a few other genomes.
And the operons were always set up like this. There were three genes.
One was called pfs, the second one was called metK and then luxS was right next door.
And so we didn't know what luxS does, but in fact, pfs and metK have known functions.
And they work together in a biochemical pathway.
And so we wondered, "Couldn't luxS work in that pathway with pfs and metK?"
And so what has been known about pfs and metK for almost 50 years now
is the following: those two enzymes work in S-adenosyl methionine utilization.
So maybe you know that S-adenosyl methionine or SAM is a very important substrate in cells.
It's involved in methylating different substrates.
So SAM is involved in putting methyl groups on DNA and RNA and proteins.
So you can't make any of those things unless you have SAM.
So it's a really important, ancient molecule.
And so the way that that works is that methionine is converted into S-adenosyl methionine
by that first enzyme we noticed which is called metK.
And then S-adenosyl methionine or SAM does its job.
It puts a methyl group on all these different substrates.
And so what the cell gets from that is the thing it wants,
like DNA or RNA, but it gets, unfortunately, a bi-product which is called S-adenosyl homocysteine.
And this is incredibly toxic because it feed back inhibits all of these methyltransferases
that are putting the methyl groups on the different substrates.
So what happens is that every time SAM methylates something a molecule of SAH is produced
and the cells have to get rid of that because of its toxicity.
So the way that they decrease toxicity is with this second enzyme I told you about, pfs.
So pfs is a nucleosidinase that takes adenine off of SAH and makes S-ribosyl homocysteine.
And so that relieves toxicity.
And it's been known since the early 60's that S-ribosyl homocysteine gets converted by
some unknown enzyme into homocysteine
and this funny molecule, 4,5-dihydroxy-2,3-pentanedione.
that nobody knows what it does.
And so what we wondered when we saw that metK and pfs were involved in this pathway
and there was a step that didn't have an enzyme associated with it
we wondered, "Well, couldn't that be luxS?
That what luxS's job is to do is to carry out this last step in this pathway."
And so to prove that was the case, what we did is we cloned and purified the luxS protein.
We put that protein in a test tube with the molecule S-ribosyl homocysteine,
let it react and then we filtered out the products.
And what we got from that was homocysteine and autoinducer-2.
And so indeed this is the pathway for making autoinducer-2.
And this final product that nobody had ever known what it was, is in fact, autoinducer-2.
And so this has always been considered a salvage pathway in bacteria.
They have to detoxify to get rid of this SAH
and doing it this way they get two useful things: they get adenine at the first step
and they get homocysteine at the second step.
But we think the real reason this pathway has evolved is because what they're trying to make
is this funny molecule which I'm trying to tell you
is a molecule involved in interspecies communication.
OK. So now I'm going to show you the biochemistry that luxS does.
So this is the reaction. So this is the molecule S-ribosyl homocysteine.
And the enzyme luxS's job is to just cut that in half
into homocysteine, which gets recycled, and then this molecule that I told you about
which is called 4,5-dihydroxy-2,3-pentanedione.
So we knew we could do this experiment and we knew we had autoinducer-2 activity in it.
So the guess was that this is autoinducer-2.
And so what we did to prove that was we carried out big reactions.
Right? Then we filtered out the protein
and then we took what was on the bottom of those reaction mixtures to the chemistry department
and did mass spec and NMR and techniques like that to try to find this molecule.
And what was interesting during this experiment is that we could find lots of homocysteine
in these reactions but we could never find this molecule.
So even though we knew the biochemistry,
we had one enzyme and one substrate in a test tube,
we couldn't find the product.
And the reason is because this molecule, this pentanedione, is incredibly reactive.
What happens is that as soon as it gets made, it starts cyclizing
into family of molecules that are in a fast equilibrium, all of which are derived from this precursor.
So you get many molecules and they're all sort of the same thing
because these rings are just opening and closing.
And so what we thought then is that one or some of those molecules
must have the autoinducer-2 activity,
but we still didn't know the structure.
And so actually, I have to say that we were a little discouraged by that because
here, we could do the reaction in one step, but we still didn't know what this elusive molecule's structure was.
And so we knew we couldn't get it from this because these molecules are opening and closing
really fast and they're all turning into one another.
And so the question was: how could we figure out which was the molecule that carries the activity?
And so what we decided to do is to just go back to what we knew about how
autoinducer-2 activity was detected. And you'll remember that I showed you
this cascade for how the information from these autoinducers comes into the cells.
And you might recall that I pointed out that there were two proteins
involved in autoinducer-2 detection, LuxP and LuxQ.
And what I slipped by you earlier in my seminar
is that this protein, LuxP, is a periplasmic protein that looks like the ribose binding protein.
And what I just showed you on this slide,
is that LuxS actually takes the ribose moiety of S-ribosyl homocysteine
and opens it up. But when this molecule closes back into a ring
it looks a little bit like ribose because it was made from ribose.
And so that started to make sense to us, that there should be
a protein that looks like it can bind a ribose-like molecule involved in autoinducer-2 detection.
So what we decided to do then was to take this protein, and purify it.
And get it to reach into this in vitro mixture that has all of these molecules
that are made from the pentanedione. We could get it to reach in there
and pull out the rearranged moiety that had the autoinducer-2 activity.
So sort of like an affinity purification.
Of all this mix of molecules that were rearranging in solution, just let LuxP
reach in there and grab the one that had...that is autoinducer-2.
And so we did that experiment in collaboration with a colleague
at Princeton University; a professor names Fred Houston, who's a crystallographer.
And so what we did was to crystallize LuxP
in the presence of autoinducer-2 and solve the structure.
And in fact that turned out to be pretty easy to do
because these periplasmic binding proteins are very well studied
and they all look sort of the same. So this is our crystal structure of LuxP.
And all of these proteins, including LuxP, look sort of like clam shells.
They lock down on the middle to a sugar that's at the interface between the two domains.
And our protein was no different. It looked like this clam shell
and there was a big space in the center where a molecule could bind.
And when we did a little closer up version of this,
where we just get rid of all the electron density from the amino acids,
what you're left with is more or less a skeleton of the crystal structure
and right at the ligand binding domain, we could see a lot of electron density
indicating that the autoinducer-2 molecule was there.
And so to prove that that was correct what we did was we just heated the protein up to
about 50 degrees and let this stuff fall out
and if you put it in a tube with Vibrio harveyi, they turn on light.
So indeed, this was the autoinducer.
So we could zoom in a little bit more to try to figure out
what was the molecule that was sitting there in the active site.
And so in this slide what you're looking at is the amino acids from LuxP
that coordinate the ligand. And I hope what you can see are two rings.
And this was a surprise to us because from the biochemistry
I showed you a couple of slides ago, when that pentanedione molecule folds up
there's only five carbons in that.
And we could account for those five carbons from this bottom ring.
So this was the folded up pentanedione that we knew about.
The problem was there were all these other atoms on the top.
And we were confused by that because we didn't know where they came from.
So to prove in fact that this was the correct molecule what we did was a mass spec of the protein
bound to the ligand and then we did this gentle heat denaturation so that the ligand falls out
and we took the mass of the protein again.
And the difference in mass is 194 which is exactly what this molecule weighs.
And so what we thought then is that LuxS, and we know this now,
every LuxS makes that linear pentanedione and then it folds up into this ring structure
but they're still very reactive because these rings have a lot of oxygens on them
which are highly reactive. So we thought, "OK, something must add to the top across those oxygens
to make this double ringed molecule that has a molecular weight of 194.
And the surprise was this atom right here.
We thought that should be carbon because that would connect the ring
and that would make a molecule of 194.
The problem was that chemists didn't like that idea because it's very well known
that carbons are very unstable if they have four oxygens on them.
And so what we found out later was, in fact, that's not a carbon atom.
What it is is a boron atom.
So it turns out that boron is very abundant in the ocean.
Right? And remember, this is a marine bacterium.
And boron also loves to add across cis-hydroxyl groups that are on rings.
And so what we found out, in fact, was that the real signaling molecule
is this one that I'm showing you here,
that has a boron on the top of that ring.
And to prove that that was the case we did boron NMR. You can look for boron 11.
And showed that, sure enough, this is the Vibrio harveyi molecule and it has boron in it.
And so that was actually a surprise because boron,
even though it has a very well studied role in chemistry,
it doesn't have a very well defined role in biology.
But in fact there's lots of it the ocean
and so this molecule could be made spontaneously
when that pentanedione gets released from the bacteria.
And so now, in fact, even though this molecule doesn't look like the biochemistry
that I showed you from the LuxS reaction, in fact, we can account for everything that's happening.
What we know now is that every LuxS protein makes this molecule, which we call for short DPD,
for dihydroxy pentanedione. Every LuxS makes that linear pentanedione.
But that molecule, as I told you is very reactive, and so it starts cyclizing.
And in the case of Vibrio harveyi, this important ring is formed,
and you have to notice that this is a chiral carbon with the oxygen up and the methyl down.
So this ring gets formed. That gets hydrated spontaneously. There's a lot of borate in the ocean
which adds across these two oxygens to make the final signaling molecule
that we found in the crystal structure.
And so what we know is that all of these reactions happen spontaneously
based on the precursor molecule that the bacteria make.
And, in fact, we can now make this molecule DPD.
We can put into a test tube and we can watch all of this happen.
And so we're very confident now about how
the Vibrio harveyi...what molecule Vibrio harveyi
uses for quorum sensing, autoinducer-2 quorum sensing.
But once we figured this out it actually gave us another puzzle.
Which is, as I've told you, there's a lot of boron in the ocean,
so maybe this kind of a molecule makes sense for Vibrio harveyi.
But I've also told you that hundreds of species of bacteria make and use autoinducer-2
and there's very little boron in terrestrial environments.
And so what we wondered, "Is autoinducer-2 one word, this one,
or is it a family of words that are all made from this precursor?"
So for example, this could be someone's autoinducer,
or this could be another bacteria's autoinducer.
And the other thing that I alluded to a minute ago is that there's this chiral carbon.
So when this molecule folds up, oxygen up, methyl down, there's another pathway
which is the opposite stereochemistry that could be across the bottom of this chart.
So there's many molecules that are possibilities.
And we wondered, "Are bacteria using all of these other molecules as autoinducers?"
And so we wanted to try to do experiments to get at that
and of course the one thing that we had learned from this
is we couldn't just purify molecules from cell free supernatants
because all of these are present. And so what we had to do is a trick similar to one
that I've shown you which is to get a sensor from some bacteria,
get it to reach in and pick out the molecule it likes.
And so, what was lucky was at the time that we were working on the chemistry of autoinducer-2
we were also trying to answer another question that I alluded to earlier
which is: What do other bacteria use autoinducer-2 to do?
What genes do they turn on and off?
And so we had started to work on E. coli and Salmonella, enteric bacteria.
And simply, we were doing genetic screens to ask, "What are the genes
that are controlled by autoinducer-2 in E. coli and Salmonella?"
And we found a number of genes that autoinducer-2 regulates
but what's important for today's talk is that we found this operon
that we named Lsr for LuxS Regulated operon.
And this operon is in the E. coli and the Salmonella genome
and it's annotated as the ribose-like transporter operon.
So what the Lsr operon encodes is what's called an ABC transporter.
And ABC transporters are widespread in different organisms
and their job are to bring molecules from the outside in.
And so they all sort of look like this one.
What they have is a B component which is a binding protein
that binds some ligand and takes it to the channel. Right?
And delivers it and it comes across into the cell and the energy is supplied by the A component
which is an ATPase. And so we found that autoinducer-2 induces the genes
that make this transporter operon and this protein LsrB
which is a binding protein, looks just like LuxP from Vibrio harveyi.
It looks like a ribose-like binding protein.
And so, of course, what molecule do we know that looks like ribose?
Well, it's autoinducer-2.
And we had already shown that the way E. coli and Salmonella respond
to autoinducer-2 is that they put it out. Then they bring it in with this transporter
and then they respond to it on the inside.
And so, of course then the guess was that the molecule that LsrB
would be binding in E. coli and Salmonella was their autoinducer-2.
And so, exactly analogous to what I told you about a few slides ago,
We crystallized this protein, the Salmonella LsrB protein, with the ligand
and solved the structure. And what we found was that
as expected, the protein looked very similar to LuxP and again,
the ligand was in the ligand binding site.
And so we could just solve the structure and what we found was that, indeed,
the Salmonella molecule and the Vibrio harveyi molecule are different molecules.
So now what I have done is to put the two molecules side by side.
And so you'll remember that the Vibrio harveyi molecule is this double ringed molecule
that has this boron moiety at the top of the ring.
The Salmonella molecule, what you can see is that it doesn't have that boron ring.
And that makes sense to us because there is no boron in your gut.
There's hardly any boron there. Salmonella would never find the molecule that looked like that.
So the first thing that's different is that Salmonella's autoinducer-2 doesn't have boron,
which makes sense given it's a terrestrial environment
and Vibrio harveyi is a marine environment.
And the second thing, which is a little more subtle, that you have to notice
is that this is that chiral carbon; oxygen up, methyl down in the case of Vibrio harveyi.
In the case of Salmonella, the stereochemistry is the opposite; methyl up, oxygen down.
But in fact, even though these molecules look different, we can connect them really easily.
And so, you'll remember the chemistry that I showed you about Vibrio harveyi
is that all the LuxS make this pentanedione and then by these reactions I showed you
and borate addition, you get the Vibrio harveyi signal.
In the case of Salmonella, remember the other 50% of the time
that the DPD molecules cyclize,
remember 50% of the time they're going to cyclize with the methyl up and the oxygen down.
That's important for Salmonella. That molecule gets hydrated
and this is the molecule that we find in the Salmonella ligand binding pocket.
And so now we know only two autoinducers that are made
from this precursor molecule, autoinducer-2.
And so of course, we're still looking to find if
this one's an autoinducer or this one or this one.
Right? But that we haven't done yet. But even though we don't know
the complete lexicon of how these bacteria use the information in that molecule to talk,
we do understand the principle for how interspecies communication works.
Because all of these molecules are freely rearranging in a very fast equilibrium
Salmonella can talk to Vibrio harveyi and likewise Vibrio harveyi can talk to Salmonella
because their molecules rearrange in between the sender and the receiver.
And so for example, we have done experiments to show if you put a lot of boron
in this reaction mix, all of the equilibrium goes to the Vibrio harveyi molecule.
Vibrio harveyi's quorum sensing response turns on and Salmonella is inhibited for quorum sensing.
Likewise, one can chelate all of the boron out of the medium and then the equilibrium
shifts to this way and Salmonella starts chit chatting and Vibrio harveyi can't talk.
So even though we don't know all of the molecules involved,
we understand that the reason interspecies works through this interconverting molecular system
is because this precursor is common for all of these bacteria and so, in fact,
I could talk to you and you can talk to me because they depend on these fast equilibrium reactions.
And so that's the state of affairs.
We're still trying to find more of these autoinducers that different bacteria use.
And so now I've told you two of the three things that I said.
What LuxS does: LuxS makes this pentanedione.
What is autoinducer-2? Well, we've started to understand that for
a few of these species and we think that there are more molecules involved.
But we understand how they talk between species.
And then the last thing that I said I would tell you is: What are other bacteria doing with autoinducer-2?
And so this is just a list of some of the bacteria
that different people in the field have started to study.
And so what you can see, this is just the name of the different species of bacteria
and then on this side are the genes that different colleagues of mine
have found that are controlled by autoinducer-2.
And so if you actually tried to read this list, what you can see
is that in most every case that's been studied, autoinducer-2,
just like peptides and homoserine lactones, controls biofilm formation and virulence.
And so it appears that the quorum sensing really is a big player.
Both intra-species and interspecies quorum sensing
are big players in controlling virulence and biofilms.
And so where the field is moving now is for people to either take their favorite autoinducer-1
or generically, to take autoinducer-2 and try to inhibit the synthases or
inhibit reception of those molecules to get new kinds of antibiotics.
So, when one works on an autoinducer-1,
the hope is to make a species specific antibacterial therapy.
And of course the hope for autoinducer-2 is that we could get an inhibitor that controls virulence
in all kinds of bacteria because you'll remember, again, both gram negative bacteria
and gram positive bacteria use autoinducer-2 quorum sensing to communicate.
And so that's sort of the practical way the field is going.
But I think we've learned a lot about chemical communication and also about
how bacteria have developed multicellularity.
And so to sum up, I hope what you've learned through this first part of the talk
is that bacteria can talk to each other and their languages are chemical.
They don't use words and sounds the way we do, they use chemicals.
We know that beyond being able to talk to one another,
that they can have multiple languages. And so at a minimum,
by having an autoinducer-1 and an autoinducer-2, what we now understand is
that bacteria can speak both within and between species.
So they have intra- and inter-species communication.
We also now know that by just having those two molecules,
what that means, we would argue, is that bacteria can distinguish self from other
by using a private language and a community language.
And what we think, then, is, of course, that's what happens in your body.
Its not like your kidney cells get all confused with your heart cells every day.
And that's because they use different hormones or different chemicals
to communicate and let these different organs in your body carry out different functions.
Again, bacteria have been on this earth for billions of years.
We think that they invented the idea of distinguishing self from other.
The other thing that we know is that there's many more molecules to be discovered.
This field is only 10 or 15 years old. We're at the very beginning of it.
It's a really exciting time. What I hope you've learned from today's talk
is that, indeed, bacteria can distinguish self from other,
but autoinducer-2 is a generic molecule.
So it doesn't say who the other is. And we know when bacteria make
complicated, architected communities like in biofilms
in fact, these are very ordered structures, you know, with one species next to another
next to a third. They're not willy-nilly.
And so we believe, the only way bacteria can get that is to have molecules that say
who the other guy is. And that's what this field is hunting for now.
And I think that's what you'll be hearing about in the next few years.
And then what I've argued and I hope that you think is an interesting idea
is that quorum sensing let's bacteria be very similar to higher organisms.
Bacteria don't act as individuals. Often times they need to act as a community, in synchrony.
And by communicating with chemicals, this allows bacteria to carry out
traits that are very similar to those carried out by higher organisms
because they've established this way to control gene expression instead of over a single cell,
over the entire population. And, of course, along with learning about these first things,
we now understand that there's a lot of opportunities for novel biotechnological applications.
For example, to make, as we've discussed, to make new therapies, new antimicrobial therapies.
Also, people would like to put these anti-quorum sensing molecules in plastics.
If one knew one was going to go to the hospital and get a catheter
that they'd get infections. If you could embed these in paints or plastics or implants
or the saran wrap that wraps up your meat at the grocery store it would be really great if
we could just use these anti-quorum sensing molecules to fight against pathogenic bacteria.
And at the same time, of course, we now know that we are covered with bacteria.
Bacteria live in us and on us all the time and they're playing an active role
in keeping us healthy. So we'd also like to look for pro-quorum sensing molecules,
molecules that make quorum sensing better in beneficial bacteria...
that help to fight off invaders.
And so we're looking for both agonists and antagonists of quorum sensing molecules
for novel biotechnological purposes.
And the final thing to do is to make a confession
that I'm not so smart. These bacteria have already figured this out.
They have a billion year head start on us.
What the field has been learning which is really exciting in the last couple of years
is that there's all kinds of natural anti-quorum sensing strategies
happening within the bacterial populations themselves.
So there's bacteria that eat each other's auotinducers.
There's bacteria that cut each other's autoinducers in half.
They eavesdrop. They cheat. They free-ride. They're doing all the kind of things that
we'd like to do and so we think that we can get a lot of the hints for how
to make these strategies just from the bacteria themselves.
So, thank you for listening to this talk.
The second half will be a much more practical talk but that's the beginning
and I hope you think, by now, that bacteria can talk to each other.
And, again, I'm Bonnie Bassler from Princeton University.