Quorum sensing in plant-associated bacteria

S. Brook PetersonParsek Lab

UW Microbiology

Outline

• What is quorum sensing?• QS in plant associated bacteria

– What traits are regulated by QS?– What benefits does QS-regulation provide?

• Response to QS– In plants– In other bacteria

Luminescence in Vibrio fishceri

Vibrio fischeri Euprymna scolopes

0.01

0.1

1

10

OP

TIC

AL

DE

NS

ITY

(600

nm

)

1

10

100

1000

LUC

IFE

RA

SE

AC

TIVI

TY

0 2 4 6 8 10

TIME (hours)

Luminescence is induced at high cell densities

Hastings and colleagues.

Spent supernatant from dense

cultures induces early luminescence

production

Eberhard, 1972. J. Bacteriol.

The “activator” in spent supernatant:

N-(3-Oxohexanoyl)-L-homoserine lactone

The mechanism of signaling in V. fischeri

luxIsynthase

luxRactivator

- low cell population densities- diffuse environment (seawater)

=AI

The mechanism of signaling in V. fischeri

luxIsynthase

luxRactivator

- high cell population densities- confined environment (squid)

induce luxgene expression(and others)

=AI

Why is light production linked to cell density?

GFP-tagged V. fischericolonizing squid light organ (Nyholm et al, 2000. PNAS)

Surface seawaterBlue= DAPI stained bacteriaGreen= Added microspheres for size comparison (5 μm)(Smith et al., 2000. ODP Technical Note)

MoonlightMoonlight

Shadow

Counterillumination

ShadowShadow

Quorum sensing:

“The term "quorum sensing" describes the ability of a microorganism to perceive and respond to microbial population density, usually relying on the production and subsequent response to diffusible signal molecules.”

(Fuqua, Parsek and Greenberg. 2001. Ann. Rev. Genetics)

LuxI-type synthases produce a variety of fatty acyl-HSL signals

C4-HSL

3hydroxy-C4-HSL

3oxo-C6-HSL

C8-HSL

3oxo-C8-HSL

3oxo-C12-HSL

7-cis-3hydroxy-C14-HSL

>300?? species use fatty acyl-HSL signaling

~30 different signal structures described

New diversity: first aryl-HSL found in R. palustris

NH

OO

HO

O

p-coumaroyl-HSL

Other kinds of signals:

Ng and Bassler, 2009. Ann. Rev. Genetics

QS in plant-associated bacteria

• How might QS benefit a plant pathogen, commensal or mutualist?

– What traits would benefit a population but not an individual cell?

– Does the specific plant-associated niche make a difference (i.e. leaf surface vs. rhizosphere vs. intracellular)?

Virulence factors: “sneak attack” by Erwinia carotovora

• Diagram to illustrate

Mäe et al., 2001. MPMI.

PCWDE= plant cell wall degrading enzymes

PDR= plant defense response

Evidence for sneak attack:

Mäe et al., 2001. MPMI.

Testing “sneak attack” with transgenic tobacco

Vector control plant

Plant expressing expI, making C8-HSL

Inoculated with 2 X 106 CFU Erwiniacarotovora

Mäe et al., 2001. MPMI.

QS-regulated antimicrobial production

Phenazine antibiotics of Pseudomonas chlororaphis

Carbapenem of Erwinia carotovora

wt

ΔbtaR2

Bactobolin of Burkholderia thailandensis

Violacein in Chromobacterium violaceum

Hypotheses to explain benefit of QS-regulation of antimicrobials:

Cell #/time

[ant

imic

robi

al]

ADAPTATION ?

Killing threshold

• Minimizing cost/benefit ratio:

METABOLIC

COST?

Does exposure to a low dose of a QS-regulated antimicrobial

induce increased tolerance in a sensitive species?

Rhamnolipids of Psuedomonasaeruginosa kill Bacillus subtilis

• Both species are saprophytes, can colonize plant rhizosphere.

P. aeruginosa rhamnolipids

(Xavier et al. 2010, Mol. Microbiol.)Time (h)

105

107

109

0 12 24 36 48

No additionRhamnolipids

CFU

/ml B

. sub

tilis

An et al., submitted.

Does B. subtilis respond to a subinhibitory does of rhamnolipids?B. subtilis + subinhbitory dose of rhamnolipids

B. subtilis + no rhamnolipids

Incubate in PBS

Resuspend in fresh buffer, add inhibitory dose of rhamnolipids

Measure B. subtilis survival over time

B. subtilis adapts in response to a low dose of rhamnolipids

5

6

7

8

9

10

0 2 4 6

time post inhibitory dose (h)

Log

CFU

/ml

Control Non-pre-exposurePre-exposure

An et al., submitted.

Hypotheses to explain benefit of QS-regulation of antimicrobials:

• Defense of newly liberated resources:

Antimicrobialcompetitor

competitorcompetitor

QS-regulation to coordinate disease progression

QS regulation of extracellular polysaccharides- Pantoea stewartii

WT

ΔesaR

ΔesaIR

ΔesaI

von Bodman et al. 1998 PNAS

Regulation of EPS in P. stewartii

EPSMinogue et al. 2005 Mol. Microbiol.

QS and EPS affect attachment of P. stewartii

WT ΔesaR ΔesaIR ΔesaIEPS gene mutants

• Reduced dissemination:

P. stewartii QS mutants in planta

Koutsoudis et al. 2006 PNAS

P. stewartii xylem colonization

WT

ΔesaIR

WT

ΔesaI

Responses to QS-plant and bacterial

• Can plants detect and respond to acyl-HSLs?

• Do plants manipulate QS?

• Can non QS-bacteria manipulate or interfere with signaling by their neighbors?

A model plant to measure response to acyl-HSLs: Medicago truncatula

• Legume, close relative of alfalfa

• Forms symbiosis with N-fixing, QS-bacterium Sinorhizobium meliloti

• Roots readily colonized by saprophytes with QS

Mathesius U. et.al. PNAS 2003;100:1444-1449©2003 by The National Academy of Sciences

- Accumulation of 150 proteins change by proteomics

- ~66% of changes were dependent on particular acyl-HSL signals (C16- vs. 3oxoC12-HSL)

-May influence auxin balance- auxin responsive elements are increased

control acyl-HSL treated

GH3-GUS (auxin-responsive)

Acyl-HSL addition to Medicago truncatula

Extracts of M. truncatula seedlings contain QS signal mimics

Gao et al. 2003 MPMI

“Quorum quenching”= signal degradation

Bacillus cereus and quorum quenching

• Organisms from B. cereus group degrade acyl-HSLs via lactonase activity (encoded by aiiA).

• B. cereus and antibiotic producers commonly inhabit the same environments (soil, plant roots).

Does the acyl-HSL lactonase of B. cereus influence its competitiveness?

B. cereus lactonase mutant in competition with C. violaceum

C. violaceum wt

+ B. cereus wt+ B. cereus

lactonase mutant + B. cereus wt+ B. cereus

lactonase mutant

C. violaceum QS-

B. cereus B. cereusaiiA

B. cereusaiiA

(pAD123)

B. cereusaiiA

(pAD123-aiiA)

CFU

B.ce

reus

/ml

+ C. violaceum

wt

10

10

10

10

10

5

6

7

8

9

Starting CFUs of B.c. and C.v.

B. cereus lactonase mutant in competition with C. violaceum

B. cereus B. cereusaiiA

B. cereusaiiA

(pAD123)

B. cereusaiiA

(pAD123-aiiA)

CFU

B.ce

reus

/ml

+ C. violaceum + C. violaceum cviI

wt

10

10

10

10

10

5

6

7

8

9

Starting CFUs of B.c. and C.v.

B. cereus lactonase mutant in competition with C. violaceum