Quorum sensing

Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Quorum sensing
Today:

What is Quorum Sensing??

Sensing: Sensing is the reception of that signal. In the case of QS bacteria communicate
with each other via the production of small, diffusible molecules or signals.
Quorum: the number of bacteria needed to induce / repress expression of genes.
Quorum sensing: the ability of bacteria to monitor their own population density and
modulate gene expression accordingly (Fuqua et al 2001).
The discovery that bacteria communicate and modulate gene expression patterns in
response to bacterial signaling has changed our general perception of bacteria as simple,
single-celled organisms.

Other terms for quorum sensing seen in the literature:

Density-dependent gene regulation
Bacterial signaling
Bacterial esperanto
Bacterial communication
Bacterial gossiping
Bacterial eavesdropping

Many times an organism could benefit from knowing how many

cells are present
1st observation that individual cells worked together in the slime
mold Dictyostelium discordeum

This eukaryote transitions from a collection of unicellular

amoebae into a multicellular slug-like organism and then
into a fruiting body within its life time.
Can think of this as 3 different ecological states

Bacteria
A multitude of bacteria are stronger than a few, thus by union are able overcome
obstacles too great for few .. Dr. Erwin F Smith, 1905 (Father of Plant Bacteriology)

Pseudomonas aeruginosa in lungs

Xylella fastidiosa in xylem

Bacterial mat in Yellowstone

Why might bacteria talk to each other?

QS enables bacteria to co-ordinate their behavior and respond quickly to

environmental stimuli.

These responses include adaptation to availability of nutrients or other

environmental conditions, defense against other microorganisms, and
the avoidance of toxic compounds.

It is very important for pathogenic bacteria during infection of a host (e.g.

humans, other animals or plants) to co-ordinate the expression of
virulence traits in order to escape the immune response of the host and
establish a successful infection.

Many people think of bacteria as unicellular organisms

Stimulus

Stimulus

Stimulus
Stimulus

Stimulus

In the absence of communication, each cell has to individually sense and respond to
environmental cues

Bacteria as multicellular organisms

Stimulus

Stimulus

Bacterial signaling and quorum sensing allows bacteria

to act similarly to multicellular organisms

Discovery of the phenomenon

known as Quorum Sensing
Known many marine organisms are
capable of producing light.
Ca. 35 yrs ago while studying the Hawaiian
Bobtail Squid (Euprymna scolopes), it was
observed that it produced light in distinct
organelles.

1.5 inches

The organelles were colonized by a large

number of a single spp. of bacterium
Vibrio fischeri

Light
organelles
In vitro, the bacteria
could produce light
only at high cell
densities

Light Production by a Culture of Vibrio fischeri is

Dependent on High Cell Density

Incubate shaking

Inoculate fresh medium

with V. fischeri

At various times, record

cell density and light
production

Light
Production
(Lux)

Normal Cell
Density Where
Light Production
Begins

102

107

Cell Density (cfu/ml)

Light Production by a Culture of Vibrio fischeri is

Dependent on a Diffusible Signal

Incubate shaking

Inoculate fresh medium

with V. fischeri

At various times, record

cell density and light
production

Centrifuge and filter

to remove cells

Inoculate with V.
fischeri
Incubate shaking
Conditioned medium

Light
Production
(Lux)

Cell Density
Where Light
Production
Begins in
Conditioned
Media

102

Normal Cell
Density Where
Light Production
Begins

107

Cell Density (cfu/ml)

Conclusion: There must be something (signal?) accumulating
in the medium that causes the rapid light production

Quorum Sensing and Diffusible Signals

AHL-Mediated QS
The regulation of gene expression in response to the intracellular
concentration of N-acyl homoserine lactone signals.
Conserved mechanism of gene expression among Gram- bacteria that
allows certain genes to be expressed at high cell density.
Involves two components:
I gene: synthesis of the signal (e.g. luxI), (signal synthase)
R gene: interacts with the signal to regulate transcription
of specific
genes (e.g. luxR); (transcriptional regulator).

LuxI: encodes an AHL signal synthase

At low cell density, transcription of luxI low
Few molecules of signal made
Signal follows concentration gradient out of cell
Notice luxI is the first
gene of the lux operon
luxR is transcribed
separately

LuxI

LuxR

luxR luxICDABEG

AHL= N-acyl-homoserine lactone

As cell density increases, there are more cells

making AHL

LuxI

LuxR

luxR luxICDABEG

LuxI

LuxR

luxR luxICDABEG

LuxI

LuxR

luxR luxICDABEG

Level of AHL increases

LuxI

LuxR
LuxI
O

LuxR

O
N
H

luxR luxICDABEG

O
O

luxR luxICDABEG

LuxI
O

LuxR

LuxI

LuxR

O
N
H

luxR luxICDABEG

luxR luxICDABEG
LuxI

LuxI

LuxR

LuxR

luxR luxICDABEG
luxR luxICDABEG

Until a threshold
concentration of signal is
reached intracellularly

LuxI

LuxR
LuxI
O

LuxR

O
N
H

luxR luxICDABEG

O
O

luxR luxICDABEG

LuxI
O

LuxR

LuxI

LuxR

O
N
H

luxR luxICDABEG

luxR luxICDABEG
LuxI

LuxI

LuxR

LuxR

luxR luxICDABEG
luxR luxICDABEG

AHL binds, dimerizes LuxR,

the transcriptional regulator

LuxI

LuxR
LuxR LuxR

LuxI
O

LuxR

LuxR
LuxRN
H
O

luxR luxICDABEG

luxR luxICDABEG

LuxI
O

LuxR

LuxI

LuxR

O
N
H

luxR
LuxR
LuxR luxICDABEG

luxR luxICDABEG
LuxI

LuxI

LuxR

LuxR
LuxR LuxR
LuxR LuxR

luxR luxICDABEG

luxR luxICDABEG

LuxR binds to promoters,

activates transcription of the
lux operon

LuxI

LuxR
LuxI
O

LuxR

O
N
H

luxR
luxICDABEG
LuxR LuxR

O
O

luxR luxICDABEG
LuxR LuxR

LuxI
O

LuxR

LuxI

LuxR

O
N
H

luxR
LuxR
LuxR luxICDABEG

luxR luxICDABEG
LuxI

LuxI

LuxR

LuxR

luxR luxICDABEG
LuxR LuxR

luxR luxICDABEG
LuxR LuxR

The lux operon is transcribed,

and luciferase is produced

LuxI

LuxR

Luciferase

LuxI
O

LuxR

O
N
H

luxR
luxICDABEG
LuxR LuxR

Luciferase

luxR luxICDABEG
LuxR LuxR

LuxI
O

LuxR

LuxI

Luciferase

LuxR

Luciferase

O
N
H

luxR
LuxR
LuxR luxICDABEG

luxR luxICDABEG
LuxI

LuxI

LuxR

Luciferase

luxR luxICDABEG
LuxR LuxR

LuxR

Luciferase

luxR luxICDABEG
LuxR LuxR

Luciferase produces light

Light

Light
LuxI

Light

LuxR

Luciferase

LuxI
O

LuxR

O
N
H

luxR
luxICDABEG
LuxR LuxR

Luciferase

luxR luxICDABEG
LuxR LuxR

Light
Light
LuxI
O

LuxR

LuxI

Luciferase

Luciferase

Light

N
H

luxR
LuxR
LuxR luxICDABEG

Light
LuxI

LuxI

Light

Light

LuxR

LuxR

luxR luxICDABEG

LuxR

Luciferase

Luciferase

Light

luxR luxICDABEG
LuxR LuxR

luxR luxICDABEG
LuxR LuxR
Light

Light

Positive feedback regulation

luxI

luxC

luxD

luxA

luxB

luxE

luxG

Note the 1st gene in the operon

Activation of lux operon also produces more AHL synthase
Which makes even more AHL signal
Which causes an exponential increase in signal and luciferase
Which ensures maximum gene expression

Many Gram negative bacteria produce N-acyl-homoserine lactone signals

By 1991 more quorum sensing systems were discovered

A. tumefaciens

E. caratovora

P. stewartii

P. aureofaciens

R. leguminosarum

R. solanacearum

Quorum Sensing (QS) Controls Phenazine Gene Expression

in the Biological Control Bacterium Pseudomonas chlororaphis
strain 30-84

o
o

PhzR
o

PhzI

(+)

(+)

phzI phzRphzXYFABCDO

QS enables the production of phenazine

antibiotics

Fluorescent Pseudomonas Species

Fluorescent Pseudomonas Species

Phenazines Required for Disease Control

30-84

Pierson & Thomashow. 1992. MPMI 5:330-339

Pathogen inhibition

30-84

Phz

Restored

Pierson & Thomashow. 1992. MPMI 5:330-339

Persistence in natural soil

Phz+

6
4

Ph
m zut
an
t

30
-8
4

Log CFU/G Soil

10

Phz-

2
0
0

Plant/Harvest Cycle

5
Mazzola, Cook, Thomashow, Weller, and
Pierson. 1992. AEM 58:2616-2624

Using Reporters to Study Ecological

Interactions
Construction of a Quorum Sensing Reporter

kan
phzI

phzR

phz operon

Strain cannot produce phenazine by itself: white

In the presence of QS signals it produces phenazines: orange

Can Different Species of Bacteria

Communicate via QS?
~8%

Positive communication
indicated by the orange halo
phzI

phz X Y F A B C D O
phzR

Developed an quorum sensing reporter and

demonstrated that cross communication occurs
Pierson EA, Wood DW, Cannon JA, Blachere FM, Pierson LS III. 1998. Molec. Plant-Microbe Interact. 11:1078-1084.

1: Ice Nucleation Control

inaZ
phzI

phzR

Ice

Ice

Ice

Ice

Ice

Ice

Ice
Ice

Ice

Ice

Ice

phz operon

Ice

Ice

Ice
Ice

WT for QS: Ice+

Ice

2: Ice Nucleation Reporter

kan
phzI

Ice

inaZ
phzR

Ice

phz operon
Ice

QS Reporter: Ice+ only when QS

inaZ

kan
phzI

phzR

phz operon

+
unkI

1: Ice Nucleation Reporter + Unknown

Ice

Ice

Ice
Ice

Ice

Ice

Ice
Ice
Ice

Ice

Cross-Communication
The demonstration that signal produced by one strain
of bacteria could alter the expression of QS regulated
genes in another strain of bacteria on plants altered
the model of QS regulation.
It is the quorum of bacterial signals (does not have to
be of isogenic origin) that the bacteria sensesnot the
number of isogenic bacterial cells.
Need to consider all the signal producing members of
the community when thinking about the regulation of
QS mediated traits and ultimately the ecology of
interactions in mixed communities.

Non- AHL signal molecules and Quorum sensing

PQS ( P. aeruginosa Quinolone Signal ) 2- heptyl - 3 hydroxy - 4 - quinolone

Small peptides in Gram Positive bacteria.

Universal signal molecule AI-2 (Gram negative and Gram positive), considered to be
a universal bacterial language Bacterial esperanto

Quorum Sensing
Requires the production of a diffusible signal
Mechanism of gene expression that allows specific
genes to be expressed at high cell density.

Quorum sensing gene regulation in different bacteria

QS involved in biofilm architecture development.

(Subject of future lecture).

QS involved in virulence gene regulation in various plant and animal

pathogens.

QS regulates ~ 5% of the genes in cystic fibrosis pathogen P. aeruginosa.

QS involved in Ti plasmid conjugal transfer in Agrobacterium.

QS also involved in various physiological processes like bioluminescence,

rhamnolipid production, exopolysaccharide production, protease
production, phenazine antibiotic production etc.,

Many of these secondary metabolites are involved in microbe-microbe and

microbe-host interactions

AHL-mediated regulation of virulence important in many

pathogenic bacteria
~ 5% of genome (350 genes)

P.aeruginosa
Cystic fibrosis patients
B. cepacia
P. stewartii
Plant pathogens
E. caratovora

These bacteria control expression of most pathogenicity genes via Quorum sensing
One Hypothesis:
A pathogen does not want to tip off its host of its presence until it has reached
a population size able to compete
Once that size is attained, activation of pathogenicity genes overwhelms the
hosts defense systems resulting in disease
If the bacteria had acted as independent assasins rather than as an army the
immune system would have wiped them out Dr. Bassler

Exploiting quorum sensing for antimicrobial therapy

We know QS regulates the virulence in many
pathogenic bacteria.
Can we exploit this fact and look for quorum
sensing inhibitors for antimicrobial therapy??
Strategy I : Quorum quenchers inactivating AHL
signals already identified:
AHL lactonases (aiiA from Bacillus sp)
AHL acylases (aiiD from Ralstonia sp)

Strategy II : Blocking the AHL receptor

protein by using other AHLs
orhalogenated furanone compounds
from marine algae (Delisea pulchra)
Mechanism??

CrossCross- communication
communication using
using Negative
Negative signaling?
signaling?

Lawn of 30-84I
(PhzI-)
Good Neighbors
Positive communication
occurs with ~8% of the
strains
Bad Neighbors
Negative Communication
occurs with ~7% of the
strains
Lawn of 30-84 (WT)

The downside to quorum sensing

1
Youre
stepping on
my flagella!
Buzz off!
Move over!

Concluding Remarks
Quorum Sensing is a
mechanism whereby
unicellular bacterial cells can
act as a multicellular
organism and respond
quickly to environmental
stimuli on and within plants.
It is important to develop your
model of the plant biome to
include the exchange of
signals and other secondary
metabolites on and within
plants.

AI-2

Mimic
s

AH
Ls
o

January 27: Assigned Reading: Dulla and Lindow

Are QS bacteria on plants sensing population size or
something else?
Use 2 reporters:
1. QS reporter
2. To identify the introduced population; introduced
bacteria produce a reporter that enables you to
distinguish between introduced bacteria and normal
leaf colonists.

Assignment:

The review must assess (one paragraph each)

1. the background and objectives,
2. methods and results,
3. discussion and significant findings, and
4. provide your opinion of the paper.
Be ready to talk about the paper. The review is due by the end
of the day Friday.