I received his B.S. in biology from Boston
College and went on to get a master's

degree and quality of your studies
at University of Michigan and

move from the school University
of Michigan to get his Ph D.

in medical microbiology studying with
Michelle Swanson studying Legionella and

model and other waterborne have been.

Posed with Bonnie Ballard
Princeton University to study

something in the real Colorado
where he defined targets.

Possibly tell you about today and
it's very nice work there today

research and it's already regulation of
horses that actually have been recalled.

So thanks Patrick is this
working OK can hear me.

Fantastic.

Well thank you for inviting me to
breakfast or maybe I should say I guess I

invited you I don't know how that works
but enjoyed the food it looks delicious.

I'm going to tell you today about the work
we do in my lab across the street

in Cherry Emerson so I've been
a Georgia Tech for three years and

have a nice car great of graduate
students and Patrick my postdoc and

some undergrads and we study this
fascinating bacterial behavior called

Quorum sensing which is cell the cell
communication in bacteria and

it allows unicellular
organisms to act as groups and

they do that by producing and
secrete small chemical molecules

that accumulate in proportion
to population density.

And then the bacteria respond to these and
orchestrate complex behaviors

as a group and we are both interested in
the molecular mechanisms of how they do

that how they convert extracellular
chemical signals into gene expression and

then what those genes and code for
and what behaviors they provide for

the bacterium and in particular I studied.

That process in a bacterial
pathogen called the cholera

which you might have heard about in
the news lately was the causative agent of

that outbreak of cholera in
Haiti after the earthquake.

This is a fascinating bacterium
because not only is it capable

of causing human disease diarrheal
disease but it also is an indigenous

inhabitant of marine systems so
it has this amazing dual life style.

Sile life cycle where it can
live out in marine systems and

thrive just happily when it gets into us.

It can cause disease and it gets into
us because people who are unfortunate

enough to consume contaminated food or
water.

Allow the bacteria to gain
access to the small intestine.

It can stick to the small intestine
attach their produce the cholera toxin.

Which is delivered directly into
intestinal cells resulting in hyper

secretion of ion and

water into the lumen of the intestine
resulting in the diarrheal disease.

So it's a dreadful disease it's still
common in developing countries again

where their infrastructure is relatively
poor we don't hear about it much in

the United States but it's still a major
worldwide concern and so we're interested

in understanding the molecular mechanisms
of how it senses in response to

its environment which of course all living
things have to be able to do pretty well.

The story.

I'm going to tell you
about starts in the ocean.

And it's going to end in the ocean.

So what I'm showing you here
is the bob tailed squid So

this is a fascinating organism.

And what I hope you can see are these
blue orbs on either side of the squid

on the bottom.

These are light organs and

they're colonized by a bioluminescent
Breo called the fish fry and

this is a wonderful symbiotic
relationship in which the bacteria and

the squid both benefit so this squid
lives in Hawaii in shallow waters and

burrows into the sand during the day and
at night it comes out to feed and

in the moonlight it casts a silhouette
on the sea floor and it's a lie.

As predators to track it and detected.

So this symbiotic relationship has
evolved so that these bacteria

that are in these light organs allow
the squid to counter illuminates so

it no longer casts a silhouette on the sea
floor these bacteria glow in the dark and

then and in the morning when the squid
is about to burrow back into the sand

it expels ninety nine percent
of that bacterial culture.

And the bacteria that remain in
that light organ stop glowing and

only as they double and
double in that light organ

which to us is like a flask doubling
in doubling throughout the day.

Only when they reach
high densities at night.

Does the entire culture
synchronously by a luminous and

so this is a fascinating example
of bioluminescence in the ocean.

There's lots of examples of
that now in the seventy's.

This was discovered by Woody Hastings at
Harvard the interesting feature is how

in the world do these bacteria do that.

How are they regulating bioluminescence
in response to population density and

that turned out to be the first example
of Quorum sensing that I'm going to tell

you about turns out now we
think all bacteria Corum sense.

And we're learning what the chemical
signals are language is and

what they do with it but
this was the first example.

And the way that these bioluminescent
quorum sense is that at low cell densities

they have a signal producing protein or
an enzyme to synthesize a small chemical

signal which I'm showing you here
is just a green Pentagon and

that low population densities when
there's not a lot of bacteria.

There's not a lot of that chemical signal.

So it is not high enough concentrations
to bind to its receptor.

And in the absence of ligand
binding this receptor

is unable to regulate
downstream genes which in this

case are the Lucifer ace genes as
well as it turns out many others.

At high cell densities in that light organ
bacterial concentrations could reach

tens of the eleventh permille you

get a sufficient accumulation of
that auto industry signal now and.

Portion of that cell density that
these signals bind to the receptor.

And this ligand receptor complex
is a transcription factor and

it can bind to these group response genes
and turn on use a phrase expression so

the entire culture
bioluminescence at the same time.

So it turns out that this was the first
described quantum sensing system.

The other systems described that I'm going
to show you about so they have different

bells and whistles they have different
chemical languages that they use but

the same end result is that in
response to these chemical signals.

There's changes in gene expression.

So the Vibrio that I work on collar
is actually bilingual we say so

it actually produces two
small chemical signals.

These are identified in the bass
the lab in the last ten years.

CA I want a collar auto inducer one
is this molecule here it's three S.

hydroxy four tried Dechen own
this molecule is only produced

by videos and only their burrows
have receptor is to respond to it.

So this bacterial signal allows for

private conversations just
among your cousins right.

The collar produces the.

So it's private conversations
among similar organisms.

They also produce though a second
chemical signal called auto industry to

which is a few ran a sealed bore
a diaster I'm showing you on the right.

Over half of all bacterial species
that have been sequenced to date have

the enzyme to synthesize this and
produce this chemical signal and

it has been shown to facilitate
interspecies communication.

So e-coli can produce auto and do sir to.

And vibrio Harvey I can respond to it and
bioluminescence as a consequence and

vice versa.

Auto industry to produce by that can
be sensed and respond by e-coli So

bacteria both have a private conversation
or a private language and a more.

Oral language and the model for why in the
world you would ever build a system like

this is that the bacteria are counting
themselves and others and

discriminating between who they're
surrounded by and you could all imagine

that we would do very different behaviors
when we're only surrounded by our family

members that we might do in public and
presumably the bacteria are able to

coordinate gene expression in response
to whom they're surrounded by and

their distinguished distinguishing between
self and others to turn certain genes on

when they're alone and
other genes on when they're in a group.

So we're interested in how that works and
what those responses are.

So for the rest of the talk I'm
going to condense down these

chemical signals into these
simple little shapes for you.

So this is the Quorum
sensing regulatory pathway.

That's like quarter to
nine in the morning.

Don't get all panicky about the pathway
I'm going to take you through it slowly

the end result again is the same is that
there's going to be chemical signals

on the outside resulting in changes
in gene expression at the bottom but

you can see that this pathway in the
organism I study is a little more complex

than the simple one.

I showed you initially.

So at low cell densities.

When those auto inducers are produced and

they're at low concentrations
because there's not a lot of cells.

There receptor unbound so in this case the
seek us sensor is the cognate sensor for

CA one and this looks P.Q. sensor in
the inner membrane is the sensor for

a I two and the absence of Ligon
these are two component histidine

proteins which means that in the absence
of their ligand they behave as kinases.

Shuttling phosphate through
a phosphorylation cascade.

So they donate phosphate to Lux you
which is a transfer ace and then

locks you donate that phosphate to locks
which is a response regulator protein.

Looks own needs to be phosphor
related to be an activator protein and

so at low so that's.

These When it's phosphor related.

It can bind to the promoter of these small
regulatory Arnie's that are the focus of

my talk we call them the quorum regulatory
Arnie's and there's four of them.

So at low so then cities these
four smaller nays are transcribed

and they participate with this are in
a binding protein called Q. and

repress a downstream target happy hour and

half hours a transcription factor it's the
it's the global regulator of the core I'm

sensing response so it's at low so then so
these when the smaller names are made.

The consequence is that
happy hour is not expressed.

And you end up getting
expression of the P.S.

or biofilm genes that promote attachment.

You get expression of the cholera
toxin that causes the disease.

I told you about and the T.C.P.
which is the Pillai

toxin Coreg you lated Pillai that
allow them to attach to the intestine.

And in happy hours absence you do
not express some other factors.

So that high so
densities everything switches.

So now you have a lot of bacteria.

You have a lot of that chemical signal
those signals bind to their cognate

receptor.

And now these receptor switch from being
kind nice is the being phosphate cases so

there's a conformational
change upon Ligon binding and

now phosphate flows up the pathway.

Luxo is no longer phosphor related that's
why show it to you in grey it's inactive

and so those smaller natives are not
expressed and in their absence

happy hours produced so now you have
the transcription factor right just like

before at high cell densities you
have the transcription factor made.

And in this case hap
are shuts off biofilm and

variance production and
you get expression of this happen.

A protease which is thought
to be a detachment factor.

So the model.

For the function of the core I'm sensing.

Pathway in vivo is that the bacteria
enter the small intestine

at low densities they start attaching and
producing variance factors and

when they reach high density auto
industry accumulation triggers release

back into the environment by shutting
off production of the cholera toxin and

factors that promote attachment and
instead producing a protease that promotes

release from the intestinal surface so
Quorum sensing in this case

allows the bacteria to transmit back
into the environment to find a new host.

That's the model.

I should remind you
though that I told you.

Cholera can exist out in marine
systems totally independent of humans.

So one of the interesting parts of
this Quorum sensing pathway that

we're studying now is not only the role of
Quorum sensing in vivo which is of course

important in meant medically relevant but
also the role of this pathway

in marine systems where they don't
have any encounter with human gut and

I'll get to those studies at the end and
that some.

The role of this in D.N.A. uptake.

All right.
So we're going to focus then on

the beginning of the talk about the
molecular mechanisms of how it is these

non-coding regulatory Arnie's can
repress expression of happen.

So what I first

want to tell you is that these smaller
names are functionally redundant.

So I told you there are four non-coding
smaller names these are in coded around

the bacterial chromosome and
interject regions.

It turns out that you need to
delete all four of the smaller

nays to see any sort of
phenotype if you have a single

smaller in a present on the chromosome you
get a standard quantum sensing response.

And that's depicted in
this particular figure.

So this is showing this is
a plasma carrying debris or

Harvey I one of those bioluminescent
organisms carrying it's so

you can monitor the amount
of light produced over time.

Per cell.

So we're measuring.

The amount of Lucifer as
a measure of light Purcell and

we're measuring it with increasing
optical density and so this.

This curve here for the wild type strain
we call this a lux curve because what

happens is we come in in
the morning to the lab.

We've grown a culture overnight just like
in The Squid and it's at high density so

it's bioluminescent.

That we dilute it one to one thousand just
like when the squid expels the culture and

now the culture just has a gradual the K.

in the residual genes present in the
cytoplasm as it's dividing and dividing So

we see a deep decrease in
bioluminescence by many logs and

then when you reach a critical
threshold concentration.

When the culture is that sufficient
densities you get a dramatic induction of

bioluminescence in response
to those are the inducers So

this is simply a we call this
a lux curve for a while type.

If you delete that lock so
Gene I told you about

that controls the smaller in A is what was
observed is that your constituents for

bioluminescence you're stuck
at high cell densities

because that response regulator
happy hour is always made.

It's never being repressed.

So you get this constituent phenotype.

You get that same phenotype
it turns out if you delete

this on a chaperone call each If Q..

But what you should notice here
with the black symbols is that.

If you delete any three of the smaller and
A's and

simply leave one of them on the chromosome
you still generate a dense the dependent

phenotype suggesting again that
they are functionally redone.

You only need one and
only when you delete all four

the recapitulate this constituent
of phenotype So it turns out.

That that feedback loop I'm showing
you here is responsible for

this behavior and that when you
have a single small R.N.A. present.

You have feedback in that
you get a little extra.

Expression which allows you to up
regulate the remaining smaller ne so

there is this functional redundancy
is due to this feedback loop but

the main reason I told you that
is because this allows us then

to study the smaller names without having
to necessarily worry about four of them

simultaneously on the chromosome but
we can study a single small R.N.A. and

look at its ability to regulate its target
which in this case is that happy hour.

Transcription factor.

So how are these smaller
names then thought to work

on their downstream target these are
noncoding Arnie's and the regulatory and

the mechanism is based on models developed
in E. coli by Susan goddess MN at the N.H.

and others and the model is the following
that these smaller unease which

are proximately one hundred base pairs in
length fold into some predicted structure.

But they have a particular region that
allows them to base pair and form and

are earning earning
a duplex with an M.R.A.

that has sufficient D.N.A.
sequence identity for pairing to occur.

And that pairing occurs right over
the right of his own binding site so

that when the smaller
names are made in our case

density they base pair with
half hours message and

prevent for I was normal access so
that no half hour protein is made.

That high school densities when
the smaller names are absent

have parts messages free to be
translated and you get half our protein.

So we were interested in
testing this hypothesis.

So we used first some computational
methods to look at the predicted

structure of the smaller And so
this is simply an Folau rhythm

to look at the lowest free energy
structure predicted structure

of the smaller name is this does not
necessarily reflect the native structure.

And I'm showing you just one of
the smaller name is this is actually Q.

our number two.

But each of the smaller names folds
into a very similar structure.

And I'm highlighting in green
what we think is the business.

And this.

This molecule so
Helix One helix three and Helix four.

Are relatively stable stem loops and
they're thought to be structural and

perhaps eight in
association with each If Q.

which is that are in a binding protein
I told you about whereas helix three.

Is one hundred percent conserved among all
sequenced vibrio that have been studied so

far so we have about twenty five
examples of sequence of embryos

many cholera strains that are Harvey
either all of them have a similar Quorum

sensing system and
all of them have multiple smaller Ney's.

And so we have about one hundred twenty
examples of these small Arnie's and

in all cases this twenty one
nucleotide sequence is invariant

one hundred percent conserved.

And what I'm going to show you is that
that same twenty one nucleotide sequence

was predicted to associate with each M.R.
in a target.

So we were interested then studying
the contribution of these nucleotides

to binding to its message.

We focused initially on these first
six nucleotides because as you can see

there predicted to be unpaired and

therefore likely to participate at
minimum in the initial interaction with

the message without having the need
to restructure the smaller ne.

We're interested in looking
at this structure as well but

of course if we start changing the
nucleotides in the structure we not only

change nucleotide sequence but
we change predicted structure as well.

So we focused on these
first six nucleotides.

Not only were we able to use these
computer algorithms to look at

the predicted structure of the earning
itself but you can use algorithms like

Target R.N.A. and Arnie up to look
at the predicted interactions.

So these algorithms will
take a sequence genome and

predict what targets the smaller name
might interact with or you can feed them

your own M R N A and ask them to look for
the predicted interaction interface and

so again I'm showing you that
same twenty one nucleotide see.

On the top in the five prime
to three prime orientation and

then the predicted interaction
with the message on the bottom.

You could see that that prediction
again included overlap with

the ribozyme binding site.

And you could see one of the important
features of these smaller bacteria and

these are a lot like micro Arnie's and
you carry out a systems.

They have these mismatches so
one can't simply scan a genome and

look for complementary sequence to
this twenty one nucleotide region and

what I'm going to tell you I don't show
you in this particular presentation but

we have now five or
six targets of the smaller Anees.

The same twenty one nucleotide
sequences involved in pairing.

And yet
in each case the mismatches are different.

So this is the real challenge for
us and it's not

unique to the bacterial world is trying
to find the targets of the smaller names.

Particularly when the rules you learn
from one particular interaction

don't necessarily apply to
the other interactions.

So again what we did here is we took
a computer algorithm called R.N.A.

up and we use that algorithm to predict
the consequence of changing these

particular nucleotides and predict the
consequences to pairing of its target and

then we use those predictions
to do our experiments.

So what I'm going to show you
that is an eco lie experiment.

So remember I told you we can get
away with using a single smaller and

looking at its control on a particular
message and so we do that by

treating Nicolai as if it were a test
tube which of course it's not and

what we do is we have two different
plasmids we have one plasmid where we

can express the smaller in a way and then
in two we call that we introduce another

plasmid which harbors a half hour G.F.P.
translational fusion.

So it has the predicted pairing region and

instead of monitoring that half
hour itself we're monitoring green

fluorescent protein expression as
a readout for small and a function.

And so what I'm showing you that
over here is happy expression.

And.
I'm showing you on the bottom

the different smaller Ney's or
happy hours that we included

on the plasmids as well as
the in silicon predictions.

So when Happy Hour is expressed
in the coal law alone we get

the maximum level of G.F.P. expression.

When we introduce in now the plastic
carrying the smaller NE We

get a predicted free energy of that
interaction using this algorithm.

And we see that we get
repression as expected.

Right.
Our interpretation is that the small army

is pairing with message and
preventing translation.

That's what we're testing.

If we look at the predicted interaction
it's you don't really need a computer

algorithm to look and
see that if you change that single

egg you should have no consequence to
pairing right so we actually did that.

The prediction of course is that there's
no change in the binding and we see that.

That smaller name is still
able to fully repress.

And that computer algorithm then predicted
that of these six particular nucleotides

the most important nucleotide
was that this position here.

And so what we did is we changed the C.
to A G..

We call that the C thirty gene
mutation the prediction was that it

would have a major effect on
free energy of binding and

what we see is that we lose
all of our repression.

As predicted.

So again you should be able to then
also make the mutation on happy hour.

Alone and
see the same effect in the prediction

is that again it would disrupt
the binding and we see no repression and

then the real proof of the punch is
that you should be able to restore

the interaction by combining
the two mutations right.

And the prediction was in fact
that we would restore pairing.

And sure enough we do so now we have the
mutation both on the smaller on a side and

both on half our side.

We restore repression consistent with not
only is there an interaction there but

the interaction is exactly
where we think you.

Is between those two nucleotides.

So this was an eco like sperm and.

Of course what we wanted to do next.

Then is in vitro test actual binding of
the smaller an eight to its message and so

this is the work that Patrick did in
the lab Patrick who introduced me.

So he in vitro synthesised the smaller
Ney's happy hours message and

purified their collars each If Q.
Arnie chaperone or

on a binding protein and
that a chick you protein as I told you.

Is predicted to bind to our Nase and

facilitate the interaction of
the two Arnie's with one another.

And that's why deletion of Q.
from the chromosome resulted in

the same phenotype as not
having smaller names at all.

So he tested that in vitro by measuring

binding of a radial labelled smaller
an eight to the half part message and

so this is a gel shift experiment where
the smaller A is radio labeled again he's

using just a single smaller and
A The same one as in vivo and

you can see that when we add increasing
amounts of unlabeled happe our message.

And in this case he's using about for
an animal or of the smaller NE and

you can see when we add enough of the
message up to six hundred forty Manimal or

so a vast molar excess we end up getting
a shift in indicative of a complex or

an interaction between the two.

However if he includes H.F.
Q. in that reaction.

The shift occurs at much lower
concentrations of half arsed message and

so we can actually plot those and
calculate the K. D.

or effective binding constant and we can
see that we get an increase of about

fifty fold in the K.T. and these numbers
are consistent again with the E. coli

literature on the contribution of H. If Q.
to a smaller in a M.R. any interaction.

So this was pleasing that H.
If Q. indeed was aiding in

allowing the smaller Nantz target
to associate and then he used that.

System then to test those mutants
those mutations we had made and

shown in vivo played a role and he was
testing whether the IN VIVO phenotypes

we saw were due to actual binding defects
between the smaller name its target.

And so now he's only looking at
a single concentration of happy hour.

So this is should be digital
we're just looking for a shift or

no shift and in this case.

When we have just the wild type smaller
than a alone you see that it migrates

this far.

When While type R.'s
provided we see the shift.

However if that happens or

has that single base permutation that
have in vivo consequence we see no shift.

The mutated smaller in a still
migrates at the same position.

It cannot bind to while
type our message but

the combination of the two
together results in a shift so

absolutely consistent with the IN VIVO
results in the cold light.

So up to this point.

Then I have shown you the interaction
of the smaller target any coal but

of course any coal lie when they're both
under control of plasma is we've totally

removed all the biology we claim
we're interested in studying which is

quantum sensing right I'm a biologist
I want to study quantum sensing So

what we did is we took all of
the information we had gained in vitro and

unequal and
moved it on to the cholera chromosome.

And we did it in this order because doing
e-coli experiments is pretty simple.

Right.
We have to plasmids we make

some mutations to move them onto the
collar chromosome was not trivial because

first we had to delete all four indulge
in a smaller names from the chromosome.

Then we had to lock their real
cholera in a condition where

the smaller names are always expressed so
we use an illegal of luck.

So on the chromosome where the spark tape

has been mutated to a glutamate or
who made to a spark tape and

this effectively locks luxo in an active
phosphor related confirmation.

So luxo deforest seventy mutant.

It's constituent of Lee
producing a small Arnie's so

we delete all the smaller
names we make a lock.

So do you for seventy.

Then we introduce back on to the
chromosome either our smaller name we want

to study and the illegal of
the half our message we want to

study put them on
the chromosome under there and

dodginess control in single copy and
that's what I'm going to show you here.

So first what we want to do is simply look
at how part protein levels in cholera.

And so these are western
blots with our anybody using

the same mutations we
told you about already.

This is the hap are banned here and
this is a control Lane And so

what you see here is that in
the absence of Happy Hour we

see no hap are protein we
can see the wild type or

the mutated happe are protein However when
the smaller an A is now are expressed in

cholera we see a near elimination
of happier protein as expected.

However if we make the mutation single
nucleotide mutation in the smaller an A or

the message we abolish repression and

the combination of the two
restores repression.

So indeed the smaller names can fully
prevent translation of half arsed message

by base pairing.

And then we're interested in looking
at the consequence the phenotypic

consequence of now disrupting
the quantum sensing response so

we monitored expression of the P.S.A.
for a happy and Cami a transcription.

And also the phenotypes controlled by each
of those genes and I'm going to show you

those data now and you should just
focus on the pattern of expression.

So for this V. P.S.

gene which is the Vibrio polysaccharide
Gene what you'd expect to see since it is

repressed by half hour is a pattern
that looks like this little smiley face.

So you should see maximal expression
when there's an interaction and hap R.

is prevented from being translated
you should see that it decreases.

When the interaction doesn't occur and
you re store it with the two mutations so

we see this pattern of expression for

of the polysaccharide transcription
we see the exact same pattern for

biofilm formation so the two columns here
that represent maximal polysaccharide

expression we see the production of this
floating biofilm It's called pellicle for

cholera at the broth air interface and you
see it in the third and fifth test tube.

We also looked at the production
of virulence factors which

are again repressed by half hour and so
since they are also repressed You should

see a similar pattern the smiley face and
we see that again for

a which is a transcription
activator of the virulence genes.

And we actually monitored cholera
toxin secretion into the medium and

we can see the same pattern that you
only get cholera toxin production.

In the third and
fifth bar just as we expected.

And then if we look at the next two
targets now notice that these are happy

are activated targets.

So you should expect that the pattern
of regulation will be opposite so

we should see frowns right.

And so that that's exactly what we see for
happen.

A transcription we see maximal expression
when the interaction does not occur.

And we can actually monitor
secreted pretty sick tippity

as a measure of happy
transcription as well.

And then the final phenotype we measured
was this competence gene or D.N.A.

uptake that's going to
be the rest of the talk.

That's why it led you in this direction.

You should see again that this is a frown
or repressed when the interaction

occurs and we can see that at
the level of transcription.

And then we can actually monitor D.N.A.
uptake or transformation frequency.

So we're monitoring the ability
of cholera in response to

quantum sensing molecules to
take up extracellular D.N.A.

from its environment and that's what the
remainder of the talks are going to be on.

So what I've shown you.

And is that in response to auto
inducers these smaller nays

are regulated in response to density and

that their ability to bind to happy hour
affects the Quorum sensing response and

it turns out that there are hundreds of
genes controlled by half are the reason

I'm showing you these are these
are the ones that most people study which

are the interesting genes involved
in disease and attachment and

I'm going to talk to you for the rest of
the time now about this competence gene or

Cami A which is involved in D.N.A. uptake.

So I told you a little lies so
far which is just that this quote

I'm sensing pathway indeed regulates
this competence genes but in addition

to core I'm sensing there's another
extracellular signal that's required.

And that signal is chitin.

So all the Vibrio is live out
in marine environments and

chitin which is what is used
to compose crab shells and so

plankton Molds is a great source
of carbon for those organisms and

re environments that can
figure out how to eat it.

So if you can have
a breakfast of Titan you're

doing pretty good right
because you can eat shells.

So it turns out all that Rio's have
multiple quite in a sense to break down

cotton into its constituent and
that's a tilt and

so it turns out that to monitor
competence or D.N.A. uptake.

We actually have to grow cholera.

On crab shells in
the presence of Titan and so

what I'm showing you here is that not only
does cholera respond to auto industry

molecules by this elaborate
phosphorylation cascade.

But in response to cotton you
get activation of another

regulator called T. fox.

It's a regulator of transformation
that's why it's named as such and

in the presence of Titan it was shown
that collar up regulates genes for

utilising chitin as Keitany says but
in addition what was observed and

this was by Mike arrays in two
thousand and five by the school Nick.

Is that you also saw up regulation of
some competence genes involved in D.N.A.

uptake.

And this is really fascinating I love
this paper because it was known since

two thousand and
one when they sequence the cholera genome

that it looked like it had all of the
machinery required for taking up D.N.A.

right transformation is this classic
studies by Griffith in one thousand twenty

eight that showed that you can
transform bacteria into a pathogen

if you give it cell extracts
from a strain that has genes for

virulence factors this was a classic
experiment that identified D.N.A.

as the molecule of inheritance in the one
nine hundred twenty S. turns out cholera

was thought in two thousand and one that
it should be able to do this too but

no one could get that to work in the lab.

And what school MC connected was that

perhaps the missing signal that we weren't
including in our laboratory microcosms was

the presence of that cholera has
evolved to live out in the ocean.

And eat in so in fact if you grow
them in the presence of cotton

you actually can document D.N.A.
uptake and he showed that.

And importantly what he showed is that you
need a functional Quorum sensing system to

do that as well.

So we began studying this pathway as well
because our interest is this side of

the pathway and the shooting and
the school and lab work on the cotton side

and so what we actually do is
we do assay is with crab shells.

So this is our Titan do simple system so
it's a twelve well plate.

So my son and
I go to the cat farmer's market and

gorge ourselves on blue crab and
then I simply bring in the shells for

my graduate student to
use in her experiments.

She breaks them up into
little pieces little pellets

they're autoclave they're sterilized.

We add artificial seawater
that's been autoclave and

we grow a biofilm on the surface
of that crab show and what we do

to monitor whether they can take up D.N.A.
is to artificially introduce.

Into the cholera chromosome a can
of mice and resistance marker.

Now of course naturally the reason

we think bacteria might take
up extracellular D.N.A.

is to acquire new traits that if they can
acquire a piece of D.N.A. horizontally.

That and codes for genes that give them
new function that provides them some

fitness advantage in particular
environments so to monitor D.N.A. uptake.

We simply add in a nice reporter
can of mice and resistance gene and

that allows us to then play the bacteria
in the presence of can of mice and

count the number of transform and
that have successfully taken up the D.N.A.

we provided.

So this is just genomic prep we
just use a standard genomic D.N.A.

isolation kit and this chromosomal D.N.A.

is one hundred percent identical to the
wild type collar a D.N.A. except that it

carries a can of mice and resistance gene
that we introduced at a particular locus

we introduce that in fact that the lack
of the locus So when they take up D.N.A.

They not only become can of mice and
resistant they become black minus and

we can actually record that
on the plates as well.

So we use that then to monitor D.N.A.
uptake.

And so what I'm showing you here
is we're monitoring competence or

Cami a gene expression on the top.

And we're measuring that in
a variety of mutants to document

that these auto inducers are important for
regulating D.N.A. uptake.

So in the white bars here are conditions
under which we don't in induced this T.

fox.

So when we measure competence gene
expression we do a little trick.

Instead of growing them on the cotton
we have and do simple T. Fox gene.

So we don't have to grow these on
the crab shell we can simply add

I P T G to induce T. fox in the absence
of cotton and so when we don't turn on T.

Fox expression you see no commie
expression you can see that for

the wild type strain we
get maximum competence.

However if we delete the gene for

synthesizing auto inducer
to we see a decrease we.

See a further decrease when we delete seek
USA the auto industry once in face and

we see a lower level when neither auto and
do serious produced.

These are the locked mutants.

I told you about so luck so
mutants always Corum sense.

They always take up D.N.A.
and Happ are mutants can't turn on

Kami and so
we get minimal expression and a T.

Fox mutant behave just like wild
type because we've deleted the teeth

from the chromosome but we've added
back that to the Fox inducible system.

Then we actually measure transformation
frequency on the crab shells like I told

you about.

And so now the signal is actually chitin
as well as these two extracellular auto

inducers and we can see a similar pattern
where we get a decrease in transformation

efficiency as we remove the auto industry
we get the best expression in the strain

that always squirm senses and the strains
that can't make either Happy Hour or

T. Fox We get no transforms and

again we're counting just the number
of canned resistant colonies and

we're measuring that as a percentage of
the total number of colonies present.

So we also measured the effect of purified
auto industry because those reviewers

asked us to do this in our manuscript and
we said we would love to do that.

So we took purified auto and do Cers and
now we add them back from the outside

to document that they
are sufficient to see the response.

And so on.

Now depicting over here that we've deleted
both auto industry since they says so.

Cholera can't make its own auto into Sears
and instead we're providing them exogamous

leaf from the top and
you can see that we get the same.

Graded response and competence gene
expression the maximal expression when

both are provided exogamous Li and
a decrease with one the other or neither.

And that again is in the presence of T.
Fox induction.

But we can also then sprinkle in those
auto industry into our quite an ass.

Say in the crab shells and
we see a similar pattern of expression we

get maximum transformation efficiency
when both are provided and

we see a decrease and we see the minimal
amount of one hundred fold less

when neither auto industry is provided.

So we thought this was kind of cool but

we thought the most interesting feature of
the Quorum sensing system I described for

you is that the Vibrio auto
inducers remember I told you there.

Bilingual.

Those auto industry molecules are produced
by members of the genus not just by

the species.

So all their burrows produce
CA I want auto into service and

all that produce two or most of them
do and have core I'm sensing systems.

So we're wondering whether auto
industry is produced in a complex

biofilm might be able to induce D.N.A.
uptake for cholera.

Because right now we're growing these
model species biofilms on our crab shells

so to me this is how many colleges this to
me is ecology but for the ecologist this

is not hard core ecology I mean I'm
growing these things in the shell in

a twelve well plate monoculture So
we kick that up just a tiny bit and

what we do is multi species biofilms
where we grow a very a cholera strain.

That can make its own auto and
do searchers and grow it in combination on

the biofilm with another vibrio species
that can produce the chemical signals for

it and we think that this more
likely mimics natural biofilms

that are going to be mixed species in
composition rather than modern species.

And so what I'm showing you here
then is that cholera responds

to interspecies auto into Cers and
promotes D.N.A. uptake in response.

So now the auto inducers again are not
being provided by cholera itself.

They're not being added in sprinkled in
from the top as purified auto industry

molecules but they're being provided
by another vibrio grown in that

same microcosm environment and
we're measuring transformation.

Quincy.

This is our control over here.

So again this is an auto
industry fission strain.

When grown in coal culture with
a cholera strain will take up D.N.A.

maximally if that cholera donor
produces both auto into serious and

then we see the same decrease as before.

When that strain produces one the other or
neither.

We can also call culture in
the presence of its cousin Harvey I And

Harvey I in this ass say that is
communicating with cholera and

inducing D.N.A.
uptake by its pathogenic cousin.

So in this case the real
Harvey I we have again four

different strains that we
have in the lab a strain

of Harvey eye that makes both auto
into series one the other or neither.

And you see the same pattern.

And then we got some other vibrio
strains from some colleagues and

we are showing you just a few examples
here this is the Perry hemolytic us that

actually makes both auto
industries like I told you and

we get a pretty good
transformation frequency.

However vibrio Fisher I It turns out is
one of these rare videos that makes auto

industry two only and
not auto industry one we see a decrease.

So we think that this demonstrates
that cholera can not only sense and

respond to its own chemical
signals to take up D.N.A.

but signals produced by other
members of the consortium.

And what we're interested in
doing now which we haven't yet

demonstrated is that these experiments.

Again I told you we have
extracellular D.N.A.

that is collar a specific D.N.A.
its color is D.N.A..

And what we love to document is that in
response to auto inducer chemical signals

produced by your cousins is cholera
able able of taking up D.N.A.

from its cousins right.

Interspecies D.N.A. uptake in response
to interspecies Autum inducer signaling.

And that's what we're working on as
one of the things Future Directions.

So then this is the video choleric or

I'm sensing system we think this
complexity is this beautiful.

And we're interested in studying the
components that allow this court unaided

response to these.

Extracellular chemical signals.

I spent most of the talk telling you just
about this little repressive arrow that

the small army is actually
repressed up are but

it turns out that these smaller
names have lots of targets.

So I identified.

A couple of years ago that these
small armies can not only repress

certain genes but activate others
they again do that by base pairing.

But they do that by it's called anti
antisense activation which is they base

pair with a message in a region that opens
up the rabbits on buying site rather than

including It turns out that the smaller
names actually repress their own activator

So else in the lab is studying the rules
for each of these interactions.

Turns out that the smaller Ney's we found
some additional targets both activated and

repressed and Patrick is working on
one of those turns out that these

interactions between this kite and uptake
system and the Quorum sensing system.

I have depicted them as these
are straight arrows implying that

those are direct interactions but
that is not necessarily the case.

So just as this pathway
has multiple steps.

A B. C. D. E. F. each of these arrows
can have multiple steps as well in this

molecular geneticists we love finding new
arrows and pathways that's what we like to

do Rays make connections so
Elena is actually working on a project and

it's identified that this step is in fact
at least three steps here she's identified

new members of that pathway so
we're pretty excited about that.

So hopefully what I have been able to
communicate with you is that bacteria

talk to one another.

Using small chemical signals that we call
I went to Sears that Quorum sensing plays

a role in collars association
with the human host but

also a role in the environment.

That H.F. Q. dependent smaller in a M.R.

in a base pairing interactions
coordinate the response.

That in the presence of Titan cholera
can take up extracellular D.N.A.

in response to auto industries
that we provided from.

Of locations either
purified self produced or

produced by others and that ought
to induce or is derived from other

members in this complex multi-species
biofilm can promote that D.N.A. uptake.

As I told you where you have a bunch
of current directions that we're all

excited to work on which is that
identifying additional targets so

Patrick has indeed identified an
additional target that he's working on now

to find those base pairing requirements
this is the work of graduate student also

in the lab identifying those connections
between the Quorum sensing pathway and

the chitin utilization pathway and so

Alaina has performed some high throughput
genetic screens to find additional members

of that regulatory cascade
characterize quantum sensing in and

and natural competence among
environmental isolates so all this work.

I just showed you is all
based on one clinical Islip

of cholera that every lab works on I mean
we all have the same experience which is

we work on one strain or one organism or
one cell and say this is how it works for

everybody right and of course that's not
the case we have a sequence genome of

Jim Watson that doesn't mean we know
how every human behaves right so

we are going now into the environment and
we've harvested environmental isolates of

cholera or characterizing them for
the regulatory pathways we've defined for

this one isolate and
of course the rules are all different and

we're interested in how they're different
how they can actually accomplish these

tasks even though it looks like they're
defective in some parts of the pathway.

And that's the work of a master
student in the undergraduate We'd

love to document interspecies D.N.A.

uptake and so Elaine is working on that
and developing some assays to do that and

monitor D.N.A. uptake in a complex
biofilm So this is some future work that.

To undergraduates are working
on now instead of relying

solely on kind of mice and
resistance as a monitor or a method for

measuring transformation we're interested
in using visualization techniques for

example G.F.P. fluorescents to monitor
live D.N.A. uptake by either come.

Focal microscopy are used to flow
cytometry to actually document in

real time the ability of
bacteria to take up D.N.A.

and not rely on waiting for twenty four
hours to play it for and I act resistance.

So this is the members of the lab that
I told you about Patrick and also so

their work was just published about
a month ago on the smaller nay.

Work and then Elaine has worked just got
accepted last week on the induced D.N.A.

uptake.

The other members of the lab I talked
about briefly what they're working on

we have this summer.

Also an undergraduate and
a teacher in the lab developing.

Teaching activities centered around
bioluminescence and bacteria and and

cells to be used in Mr top fifth grade
classroom and I want to thank of course.

Roger and Taylor who are in this
building helped us with all the Q.

and smarmy work and of course for
funding to do the work.

Thank you very much.

Thanks for the questions.

Yes So

you were like

Yeah

that's a great question.

We we weren't sure about that but
we by multiple assaye see that that

looks like it's better at repressing and
so we are interested in studying that I

mean there is certainly some play in the
when you measure bioluminescence changes

threefold are not terribly significant but
in that particular case for

multiple phenotypes not only transcription
changes we see that looks like it's more

repressive So we're interested
that yes in understanding whether.

In that particular case.

Get stronger binding we haven't measured
binding in that case whether that is real.

It's a good point.

Yes.

Yeah.

So that's a fantastic question so
one of the reasons that Quorum sensing

has gotten a lot of people excited is
because in lots of bacterial pathogens

they regulate virulence gene expression in
response to their auto into Cers Now a lot

of those pathogens pseudomonas being
the best example uses quantum sensing to

up regulate variance Cian expression in
this is why the montra was that quantum

sensing is gang behavior when they get in
a group they launch their attack in that

they don't want to do that when they're
in their bodies at low numbers cholera is

the oddball that it actually uses
quantum sensing to facilitate release.

Now our model is that that's because
collar is a self limiting pathogen and so

it never stays in your body forever it
clears whereas pseudomonas in the C.F.

long sticks and when it quantum senses
it produces more virulence factors and

more biofilm to attach better.

So in both cases or in many cases
there are medicine all chemists and

their natural products experts looking for
inhibitors or

augment years of the pathway in the case
of pseudomonas you'd want an auto inducer

disruptor right something that
prevents the auto in his or for

binding in the case of cholera the drug
itself would be the molecule and

so there is definitely an interest
in my old advisor body best there is

his heavy duty into identifying
ways to augment Quorum sensing

systems in a variety of bacteria as a way
of developing new anti-microbial agents.

Absolutely.

You know I'm a bit of school.

One of them was amazing.

LAURA.

You know my own views you know so
I knew of those.

So how is more than one hundred bucks and

this way she made fortunes of so

much of it.

Pointedly for all the world.

I mean I guess one of the big
questions when you look at the quantum

sensing pathway is you know why in
the world would you ever engineer

a system where you rely on others
signals to Module A Your response and

you know we we can come up with.

Mechanisms we don't understand
that because potentially and

we think that in the Quorum sensing
world out there in real systems.

It's not happy communication among buddies
it's they're fighting each other they're

eco like it's auto industries
that actually takes them in and

degrades them so there's.

There's eavesdropping and
there's communication and

there's battling going on and how I think
it's probably an arms race all the time.

There are there are other bacteria like
the Silis in and soils that produce lacked

an ace is the breakdown the auto industry
is that and semantically degrade them.

So how they synthesize
all that information.

I mean I don't understand that
it's incredibly complex and

of course the environment is changing
all the time I mean the teeth brushed

if you push that biofilm off and starts
from scratch again it's fascinating to me.

Great thank you very much thank.