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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