Quorum sensing in plantassociated. S. Brook Peterson Parsek Lab UW Microbiology

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1 Quorum sensing in plantassociated bacteria S. Brook Peterson Parsek Lab UW Microbiology

2 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

3 Luminescence in Vibrio fishceri Vibrio fischeri Euprymna scolopes

4 Luminescence is induced at high cell densities LUCIFERASE ACTIVITY OPTICAL DENSITY (600 nm) TIME (hours) Hastings and colleagues.

5 Spent supernatant from dense cultures induces early luminescence production Eberhard, J. Bacteriol.

6 The activator in spent supernatant: N-(3-Oxohexanoyl)-L-homoserine lactone

7 The mechanism of signaling in V. fischeri = AI luxi synthase luxr activator - low cell population densities - diffuse environment (seawater)

8 The mechanism of signaling in V. fischeri = AI luxi synthase luxr activator induce lux gene expression (and others) - high cell population densities - confined environment (squid)

9 Why is light production linked to cell density? Surface seawater Blue= DAPI stained bacteria Green= Added microspheres for size comparison (5 μm) (Smith et al., ODP Technical Note) GFP-tagged V. fischeri colonizing squid light organ (Nyholm et al, PNAS)

10 Moonlight Counterillumination Shadow Shadow

11 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 Ann. Rev. Genetics)

12 LuxI-type synthases produce a variety of fatty acyl-hsl signals C4-HSL 3hydroxy-C4-HSL 3oxo-C6-HSL C8-HSL 3oxo-C8-HSL >300?? species use fatty acyl-hsl signaling ~30 different signal structures described New diversity: first aryl- HSL found in R. palustris O O O 3oxo-C12-HSL HO 7-cis-3hydroxy-C14-HSL N H p-coumaroyl-hsl

13 Other kinds of signals: Ng and Bassler, Ann. Rev. Genetics

14 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)?

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16 Virulence factors: sneak attack by Diagram to illustrate Erwinia carotovora PCWDE= plant cell wall degrading enzymes PDR= plant defense response Mäe et al., MPMI.

17 Evidence for sneak attack: Mäe et al., MPMI.

18 Testing sneak attack with transgenic tobacco Plant expressing expi, making C8-HSL Inoculated with 2 X 10 6 CFU Erwinia carotovora Vector control plant Mäe et al., MPMI.

19 QS-regulated antimicrobial production Violacein in Chromobacterium violaceum wt ΔbtaR2 Bactobolin of Burkholderia thailandensis Phenazine antibiotics of Pseudomonas chlororaphis Carbapenem of Erwinia carotovora

20 Hypotheses to explain benefit of QSregulation of antimicrobials: Minimizing cost/benefit ratio: [antimicrobial] Killing threshold ADAPTATION? METABOLIC COST? Cell #/time

21 Does exposure to a low dose of a QS-regulated antimicrobial induce increased tolerance in a sensitive species?

22 Rhamnolipids of Psuedomonas aeruginosa kill Bacillus subtilis Both species are saprophytes, can colonize plant rhizosphere. No addition Rhamnolipids P. aeruginosa rhamnolipids (Xavier et al. 2010, Mol. Microbiol.) CFU/ml B. subtilis Time (h) An et al., submitted.

23 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

24 B. subtilis adapts in response to a low dose of rhamnolipids 10 9 Log CFU/ml Control Non-pre-exposure Pre-exposure time post inhibitory dose (h) An et al., submitted.

25 Hypotheses to explain benefit of QSregulation of antimicrobials: Defense of newly liberated resources: Antimicrobial competitor competitor competitor

26 QS-regulation to coordinate disease progression

27 QS regulation of extracellular polysaccharides- Pantoea stewartii WT ΔesaR ΔesaIR ΔesaI von Bodman et al PNAS

28 Regulation of EPS in P. stewartii Minogue et al Mol. Microbiol. EPS

29 QS and EPS affect attachment of P. stewartii WT ΔesaR ΔesaIR ΔesaI EPS gene mutants

30 P. stewartii QS mutants in planta Reduced dissemination: Koutsoudis et al PNAS

31 P. stewartii xylem colonization WT WT ΔesaI ΔesaIR

32 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?

33 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

34 Acyl-HSL addition to Medicago truncatula - 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) Mathesius U. et.al. PNAS 2003;100: by The National Academy of Sciences

35 Extracts of M. truncatula seedlings contain QS signal mimics Gao et al MPMI

36 Quorum quenching = signal degradation

37 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).

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

39 B. cereus lactonase mutant in competition with C. violaceum C. violaceum wt C. violaceum QS- + B. cereus wt + B. cereus lactonase mutant + B. cereus wt + B. cereus lactonase mutant

40 B. cereus lactonase mutant in competition with C. violaceum C. violaceum CFU B. cereus/ml Starting CFUs of B.c. and C.v B. cereus B. cereus wt aiia B. cereus aiia (pad123) B. cereus aiia (pad123-aiia)

41 B. cereus lactonase mutant in competition with C. violaceum C. violaceum + C. violaceum cvii CFU B. cereus/ml B. cereus B. cereus wt aiia B. cereus aiia (pad123) B. cereus aiia (pad123-aiia) Starting CFUs of B.c. and C.v.