Quorum sensing in plant pathogenic bacteria

1. Quorum Sensing in Plant Pathogenic Bacteria

2. Content
• Introduction
• History
• Types of QS signaling molecules.
• General mechanism of QS
• QS in Gram positive bacteria
• QS in Gram negative bacteria
• Applications of QS
• Case studies
• Conclusion

3. WHAT IS QUORUM SENSING ?
• ‘Quorum’ is a Latin word.
• It means the number of members of a group required to be present to transact business or carry
out an activity legally.
• In this process bacteria communicate via secreted signalling molecules called “autoinducers”,
which contribute to the regulation of the expression of particular genes.

4. Occurrence
• Within a single bacterial species as well as
between diverse species.
• In some local insects use quorum
sensing to determine where to nest.

5. Why do bacteria talk to each other?
• As environmental conditions often change rapidly, bacteria need to respond
quickly in order to survive.
1) Quorum sensing enables bacteria to coordinate their behaviour.
2) It is very important for pathogenic bacteria during infection of a host to
coordinate their virulence in order to be able to establish a successful infection.

6. COLLECTIVE
BEHAVIOUR
BIOLUMINISCENCE
BIOFILM
FORMATION
CONJUGATION
VIRULENCE FACTORS
(such as toxin production, antibiotic
production etc)

7. History of Quorum Sensing
• The first such system was described in
Vibrio fischeri (Nealson &Hastings, 1979), a
symbiotic species that provides its marine
eukaryotic hosts i.e., Squid with light.
Euprymna scolopes

8. KEY PLAYERS IN A QUORUM-SENSING NETWORK
• Autoinducers: Autoinducers are usually small signalling molecules that either diffuse freely
across the cell membranes or are actively transported out of the cell.
• Common classes of signalling molecules are
• N-acylhomoserine lactones (AHLs) in Gram-negative bacteria,
• Autoinducing peptides(AIPs)/oligopeptides in Gram-positive bacteria and
• Furanosyl borate diesters (AI-2), in both Gram-negative and Gram-positive bacteria.

9. Other Examples of AIs in Bacteria
Gram Negative Bacteria Gram Positive Bacteria
• Non-AHL unknown signals in Xyllela,
Xanthomonas (water-, air-, soil- borne pathogens of
citrus and grapes)
• Volatile esters of fatty acids[3-hydroxy palmitic
acid methyl ester (3OH-PAME)] in Ralstonia, a
pathogen that causes wilt in tomato.
• Gamma-butyrolactone in Streptomyces controls
production of aerial hyphae and antibiotics.
• Myxococcus uses a mixture of aminoacids to
initiate sporulation (more later).
• Peptide signals in Bacillus, control competence,
virulence.
Bodman et al.,2003

10. General Mechanism of Quorum Sensing in Bacteria
Podbielski et al., 2003

11. QUORUM SENSING IN GRAM
POSITIVE & GRAM NEGATIVE
BACTERIA

12. Quorum sensing in Gram positive bacteria
• Peptide mediated quorum sensing.
Quorum sensing in Gram negative bacteria
• The language of LuxI/R genes or their homologs.

13. Quorum Sensing in Gram Positive Bacteria
At high concentration of AIP in the environment, AIP bind to a receptor
to activate kinase. The kinase phosphorylates a transcription factor, which
regulated gene transcription. This is called a two-component system.
(figure A)
(Rutherford and Bassler, 2014)
Another mechanism is that AIP is transported into the
cytosol, and binds directly to a transcription factor to
initiate or inhibit transcription.(figure B)

14. Quorum Sensing in Gram Negative Bacteria
• Gram-negative bacteria produce N-acyl homoserine lactones (AHL) as their
signaling molecule.
• AHLs do not need additional processing.
• When the concentration of AIs is sufficiently high, which occurs at HCD, they
bind cytoplasmic receptors that are transcription factors. The AI-bound receptors
regulate expression of the genes in the QS regulon(Fig. C).
• In some cases of Gram-negative bacterial QS, AIs are detected by two-
component histidine kinase receptors(Fig. D).

15. Quorum Sensing Signaling System
• A major bacterial intercellular signaling system in Gram negative bacteria is LuxI/R QS system
or their homologs. These regulatory systems help the organisms to adapt to different
conditions and to colonize specific ecological niches in response to environmental signals.
• Some plant pathogenic bacteria have a single QS system while others have more than one QS
system.
• Not all plant pathogenic bacteria encode the LuxI and in these situations the LuxR modulates
cell behavior in a cell density manner by utilizing signal molecules that are produced by their
plant hosts.

16. Organism Major signal molecule/
Autoinducer
LuxI/LuxR
Homologue
Functions
Vibrio fischeri 3-Oxo-C6-HSL LuxI/LuxR Bioluminescence
Pseudomonas aeruginosa 3-Oxo-C12-HSL
C4-HSL
LasI/LasR
RhlI/RhlR
 Multiple extracellular
enzymes,RhlR, Xcp, biofilm
formation.
 Multiple extracellular
enzymes,rhamnolipid, RpoS,
secondary metabolites.
Agrobacterium tumefaciens 3-Oxo-C8-HSL TraI/TraR Ti plasmid conjugation
Erwinia carotovora 3-Oxo-C6-HSL ExpI/ExpR Exoenzymes
Erwinia chrysanthemi 3-Oxo-C6-HSL
C6-HSL
ExpI/ExpR Pectate lyases
Erwinia stewartii 3-Oxo-C6-HSL EsaI/EsaR Exopolysaccharide, virulence factors
Rhizobium leguminosarum C6-HSL, C8-HSL,
3-Hydroxy-7-cis-C14-
HSL
RhiI/RhiR RhiABC rhizosphere-expressed
genes, nodulation
(Miller et al., 2001)

17. Detection of Quorum Sensing Signals in Vibrio fischeri
• The AHL molecule made by the LuxI synthase moves freely across bacterial
membranes.
• Inside the bacterium, AHL interacts with its receptor, LuxR, to form an active complex.
• AHL-LuxR complex binds to specific palindromic sequence in the DNA(Lux box)
near the promoter regions of genes in the lux regulon .
LuxI = AHL synthase → makes AHLs,
LuxR= AHL receptor → detects AHLs
luxCDABE = gene encodes production
of light in V. fischeri
Red triangle = AHLs that diffuse in and
out
(Miller et al., 2001)

18. Functions of the LuxI and LuxR Family of Proteins
LuxI FUNCTION:
S-adenosylmethionine(SAM) and acyl-acyl carrier protein
(acyl-ACP) are the substrates for the LuxI-type enzymes.
1. The LuxI type proteins direct the formation of an amide linkage
between SAM and the acyl moiety of the acyl-ACP.
2. Subsequent lactonization of the ligated intermediate with the
concomitant release of methylthioadenosine occurs.
3. This step results in the formation of the acylated homoserine lactone.
The SAM/acyl-ACP biosynthetic pathway is common for all LuxI homologues. Homoserine
lactone autoinducers differ only in their respective acyl side chains.

19. LuxR FUNCTION:
• LuxR like proteins are responsible for binding a cognate HSL autoinducer, binding specific target gene promoters,
and activating transcription.
• The amino-terminal domain is involved in binding to the HSL autoinducer, and the carboxyl-terminal domain is
required for DNA binding and transcriptional activation.
• In the absence of auto inducer, the amino-terminal domain inhibits DNA binding by the carboxyl-terminal domain.
• The LuxRs are extremely sensitive to alterations in the acyl side chains of the autoinducers, thus maintain the
specificity.

20. Mechanism of trait regulation

21. Behaviours controlled by QS in Plant Pathogenic Bacteria
• Structuring of multicellular communities
• Stress survival
• Production of
• Antibiotics
• Pigments
• Host tissue degrading enzymes
• Toxins
• Regulating
• Conjugative gene expression

22. How Quorum Sensing helps bacteria to survive
• Large percentage of the cells of pathogens such as Pseudomonas syringae occur in aggregates
on leaves.
• Through the use of Gfp-marked cells and viability stains it is found that
• Aggregates are required for tolerance of environmental stresses.
• While individual cells often die upon exposure to stressful conditions such as periodic
desiccation on leaves.
• Furthermore, such bacterial aggregates facilitate the successful immigration of other cells to a
leaf.

23. Virulence factors controlled by QS in plant pathogenic bacteria
• Cell wall degrading enzymes and exopolysaccharides (Erwinia spp, Pantoea spp.
and Pseudomonas solanacearum)
• Conjugal transfer of Ti plasmid in Agrobacterium tumefaciens
• Foliar penetration by Pseudomonas syringe pv. syringe.
• Expression of phc genes (phenotype conversion genes) in Pseudomonas
solanacearum
• At low density motility related genes
• At high density phcA gene (EPS)

24. QS systems in Pectobacterium carotovorum
• Pectobacterium strains encode one LuxI homolog and two or more LuxR homologs.
• LuxR homologs include:
1. CarR:
-regulates synthesis of
antibiotics.
-gives competitive
advantage over other bacteria
co-existing during infection.
2. ExpR and 3. VirR:
Regulation of the production of PCWDE

25. Pectobacteria carotovora ssp. carotovora strain ATCC 39048
• Produces detectable levels of the b-lactam antibiotic, Carbapenem , during the transition between late log and
stationary phases of growth.
• The Car biosynthetic and auto resistance functions of this strain are encoded by the carABCDEFGH gene cluster.
• The enzymes encoded by carA, carB and carC are essential for the production of Car.
• CarB catalyses the first committed step in Car biosynthesis, the formation of carboxymethylproline,
• But the carD and carE genes are not essential; disruption of either genes results in the reduction, but not the
complete abolition of Car production (McGowan et al. 1999)

26. Models for quorum sensing-dependent control of Car production
• At low cell densities, carA–H are not expressed, this is because transcription of carA–H is not activated in
the absence of quorum sensing signal .
• At high cell densities in Erwinia, the 3-oxo-C6-HSL quorum sensing signal binds and activates CarR, which
promotes carA–H transcription and hence activates Car Production.

27. Quorum sensing in Agrobacterium tumefaciens

28. Agrobacterium tumefaciens infection process

29. Conjugal transfer of Ti plasmid pTiC58 by A. tumefaciens
• LuxI homologue: TraI (synthesize auto inducer AHL)
• LuxR homologue: TraR
• includes two linked tra operons, a separate trb operon, and the rep operon for the replication of the Ti plasmid.
• Together, the tra and trb operons encode the structural components for Ti plasmid conjugal transfer. In each
system, traR is part of a separate transcription unit near the tra gene cluster, whereas traI is the first gene in the
trb operon.
• AccR : Agrocinopine catabolism repressor
• TraM: Antiactivator

30. Figure 1 Quorum sensing regulation of the Ti plasmid conjugal transfer genes in A. tumefaciens.
Opines (triangles) induce synthesis of the TraR response regulator (open ovals) via AccR (circle). In
the presence of high concentrations of AHL signals(pentagons), produced by the TraI synthase
(rectangle), TraR activates the tra regulation. (Miller and Bassler, 2001)

31. Discovery of two component system in Gram negative
Bacteria
• The sensor kinase GacS, initially called LemA, was first described in Pseudomonas syringae
pv. syringae strain B728a as an essential factor for lesion manifestation by this pathogenic
strain on bean leaves (Hrabak and Willis, 1992).
• Two main properties of GacS/GacA mutants:
• Partial or complete loss of biocontrol ability in a group of plant-beneficial Pseudomonads
• Virulence in plant- or animal-pathogenic bacteria

32. GacS/GacA Two-Component System
• Controls the production of secondary metabolites
and extracellular enzymes involved in pathogenicity.
• The sensor undergoes auto phosphorylation and then
activates the response regulator (GacA) by
phosphotransfer.
GacS: Sensor kinase ; GacA: response regulator

33. Shoot the Message, Not the Messenger —
exploiting Quorum Sensing in managing
bacterial disease

34. QUORUM SENSING INHIBITOR
• The continuing emergence of multiple-drug-resistant strains of bacteria has necessitated finding novel
strategies for treating bacterial infections.
• QS inhibitors are molecules that interrupt the pathway of communication bacteria used to regulate expression of
virulence factors - A novel target for antimicrobial therapy (Quorum Quenching).
• Through blocking this cell-to-cell signaling mechanism, pathogenic organisms that use quorum sensing to control
virulence could potentially be rendered avirulent. This can be achieved by
(i) inhibition of the biosynthesis of QS signalling molecules;
(ii) destruction of the QS signalling molecules in the medium and
(iii) inhibition of the activation of QS receptors
(Helman and Chernin, 2015)

35. QQ as a natural phenomenon or engineered procedures causing weakening of the expression of QS-regulated traits in
bacteria. QQ strategies are nonlethal to bacteria and govern only the expression of virulence factors in pathogenic
bacteria.

36. EXAMPLES
• N-Acyl homoserine lactonase, encoded by aiiA(isolated and purified from Bacillus sp. strain
240B1),attacks the lactone bond, causing ring opening of AHLs.
• Transgenic plants harbouring the aiiA gene from Bacillus thuringenesis were less prone to maceration
by Erwinia carotovora.
• Transgenic plants expressing bacterial AHL-degrading enzymes are more resistant to infection by
phytopathogens. Some plants naturally synthesize QQ enzymes with comparable activities to bacterial
AHL-degrading enzymes.
• The construction of bifunctional recombinant strains by the transformation of natural PGPR with an
AHL-degrading gene enhance the efficiency of QQ biocontrol agents to fight plant diseases.

37. Conclusion

38. • Based on these studies, novel antimicrobial strategies could be designed, which suggests that
research on quorum sensing could have enormous practical applications in regulation of
bacterial pathogenesis and sustainable crop health management.

39. References:
• Achari, G. A. and Ramesh. R. (2019). Recent advances in quorum quenching of plant pathogenic bacteria. In : Meena, S.N. and Naik, M.
(eds) Advances in biological science research: a practical approach, 1st edn. Academic press, pp. 233-245.
• Bodman, S.B., Bauer, W.D., and Coplin, D.L. (2003). Quorum sensing in plant-pathogenic bacteria. Annu. Rev. Phytopathol., 41: 455–482.
• Helman Y., and Chernin L. (2015). Silencing the mob: disrupting quorum sensing as a means to fight plant disease. Mol. Plant. Pathol.,
16(3): 316-329.
• Kievit T.R., and Iglewski B.H.(2000). Bacterial quorum sensing in pathogenic relationship. Infect Immun., 68(9): 4839-4849.
• Nakatsu, Y., Matsui, H., Yamamoto, M., Noutoshi, Y., Toyoda, K. and Ichinose, Y. (2019). Quorum-dependent expression of rsmX and
rsmY, small non-coding RNAs, in Pseudomonas syringae. Microbiol. Res. 225 :72-78.
• Uroz, S., Cathy, D., Carlier, A., Elasri, M., Sicot, C., Petit, A., Oger, P., Faure, D., and Dessaux, Y. (2003). Novel bacteria degrading N-
acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria. Microbiol., 149 :
1981–1989.

40. Thank you