1. Quorum sensing is a mechanism of communication between microorganisms where bacteria produce and detect signal molecules called autoinducers to coordinate gene expression based on cell population density. (2) Gram-negative bacteria use acyl-homoserine lactones and gram-positive bacteria use peptide signals. (3) Vibrio fischeri uses the LuxI/LuxR system where LuxI produces the autoinducer that binds LuxR to activate bioluminescence only at high cell density.

1. Sharavanakkumar SK
I MSc Biotechnology
University of Mysore

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Introduction:
• Communication is the key role in social behavior.
• We know that animals use sound and gesture as a tool for
communication.
• Plants also emit odours, that animals and humans are attracted to,
they can sense that, their neighbour has been cut, it has been eaten by
a bug.

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• The paper published in 1983 with ground-breaking results, about
communication between plants.
• Rapid Changes in Tree Leaf Chemistry Induced by Damage:
Evidence for Communication Between Plants. Ian T.
Baldwin, Jack C. Schultz Science 15 Jul 1983: Vol. 221, Issue
4607, pp. 277-279.
• Like all these complex organisms, microorganisms – the most basic
life forms also communicate with their own species, also with other
kind and act as a whole population to gain some useful
characteristics.
• This mechanism of communication between microorganisms is
called quorum sensing.

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History:
• First these kind of microbial communications was observed in gram-
negative, marine – luminous bacteria vibrio fischeri by Kenneth
Nealson, Terry Platt, Woodland Hastings in 1970.
• This bacteria is known to be in symbiosis with Hawaiian bobtail
squid.
• Vibrio fischeri, produce luminescence when they are present in high
cell density (1010−1011 CFU/ml), which helps the squid to adjust its
shadow and escape itself from predators in the night.

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 When the cell density is low (102 CFU/ml) during day time the
bacteria is not producing luciferase, so no luminescence.
 They observed when there is high cell density, some kind of
signaling molecule is released when these chemicals reach the
critical concentration, they get sensed by the receptors then
luciferase is expressed leads to bioluminescence. Then this
phenomenon is referred as ‘Autoinduction’.
 In1994, Dr. Steven Winans, who first reviewed the articles about
autoinduction used the term quorum sensing to refer this microbial
communication phenomenon.

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Fig: Euprymna scolopes and vibrio fischeri symbiosis
(Source: https://www.youtube.com/watch?v=saWSxLU0ME8&t=654s)

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Quorum Sensing (QS) – Definition:
 By using these communication, they can express some common
characteristics, most of these characteristics were due to specific gene
regulation.
 These characteristics are helpful for the microbe, e.g. some bacteria
use this mechanism for gaining virulence, sporulation, competence,
bioluminescence.
 Generally quorum sensing is defined as the regulation of gene
expression in response to fluctuations in cell population density.
 How this is taking place, basically the microbial cells have receptor
which sense the signalling molecule (autoinducer) as a result, all the
microbial cells in this certain community express some common
characteristics as a whole population.

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Quorum Sensing in Bacteria:
 Even though the basic principle of signalling mechanism is same,
both gram positive and negative bacteria have different working
systems.
 Gram – negative bacteria use Homoserine Lactone (HSL)
compounds as signalling molecule. [e.g. Vibrio fischeri,
Pseudomonad aeruginosa, Agrobacterium tumifaciencs, Erwinia
carotovora ]
 Gram – positive bacteria use peptide molecules, Auto-Inducing
peptide (AIP) as signaling molecule. [e.g. Staphylococcus aureus,
Bacillus subtilis, Streptococcus pneumoniae]

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Quorum Sensing [Gram – Negative Bacteria] :
 Most of the gram negative bacteria uses the LuxI/LuxR type quorum
sensing circuits or they use canonical quorum sensing circuit.
 Homoserine lactone molecules are small molecules (signaling) and
they easily diffuse through the cell wall of gram – negative bacteria.
Fig: Acyl Homoserine Lactone, signaling molecule used in Vibrio fischeri -
bioluminescence system

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Fig: Table showing the different HSL signaling molecules and their expression
characteristics in gram – negative bacteria
(Applications of Molecular Docking and Molecular Dynamics on the Inhibition of Quorum Sensing Systems
Santiago Medina, et.al. 2015)

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Lux I/Lux R – Bioluminescence system of Vibrio fischeri:
Fig: Vibrio fischeri - LuxI/LuxR quorum sensing circuit
Fig: LuxI/LuxR quorum sensing circuit of Vibrio fischeri
(Quorum sensing in bacteria, Melissa B. Miller and Bonnie L. Bassler, Annu.Rev.Microbiol. 2001, 55:165-99)

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Lux Operon:
Fig: Lux operon (Lux ICDABE) of Vibrio fischeri
• This system comprises of two regulatory protein called Lux I and Lux R

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• This system consists of two regulator proteins, LuxI and LuxR.
• Lux I – Autoinducer synthase enzyme. (Synthesize AHL)
• Lux R – It functions to bind the autoinducer and also activate
transcription of the Lux operon.
• Lux R protein conjugated with AHL will binds to the Lux box, then
RNA-polymerase will bind to promoter and leads to transcription.
• Then Lux A & Lux B genes will encode subunits of A and B of
luciferase.
• Later the both subunits binds and become activated and occurrence of
bioluminescence.

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Fig: Enzymatic roles of proteins produced in Lux operon
(Shedding light on bioluminescence regulation in Vibrio fischeri, Tim Miyashiro and Edward G. Ruby, 2012, Mol Microbiol)

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 Luciferase, which is a heterodimer composed of α and β subunits is the core
of light production.
 Luciferase releases light during the oxidation of a long-chain aldehyde and
reduction of Flavin mononucleotide (FMN).
 LuxD, diverts fatty acyl groups from the fatty acid biosynthesis pathway to
yield fatty acids for luminescence.
 LuxC, activates the acyl group with AMP, which is then reduced to the long-
chain aldehyde by LuxE.
 In this manner, LuxC and LuxE are also able to recycle the long-chain fatty
acid resulting from the luciferase reaction by reducing it back to its aldehyde
form.
 LuxG was shown to reduce FMN produced by the luciferase reaction.

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 LasI/LasR – Rhl I/Rhl R virulence system [Pseudomonas
aeruginosa]
Fig: LasI/LasR – Rhl I/Rhl R virulence system
(Quorum sensing in bacteria, Melissa B. Miller and Bonnie L. Bassler, Annu.Rev.Microbiol. 2001, 55:165-99)

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Operon system in Pseudomonas aeruginosa:
Fig: Two pairs of LuxI/LuxR homologues called LasI/LasR & Rhl I/Rhl R system

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 It has two pairs of LuxI/LuxR homologues, LasI/LasR & Rhl I/Rhl R
system.
 Las I synthesizes – N-(3-oxododecanoyl)-HSL binds to – Las R
 Rhl I synthesizes – N-(butryl)-HSL binds to – Rhl R
 Las R complex influences the seconds quorum sensing circuit by
activating Rhl I.
 Rhl I then releases its own HSL and binds to Rhl R, which activates
other set of virulence genes.
 This mechanism is autoregulatory loop, LasR-HSL also prevents Rhl
I-HSL from binding to Rhl R. So they can initiate their cascades
sequentially in appropriate order.

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PQS Signaling Circuit:
• PQS (Pseudomonas quinolone signal), a unique signaling mechanism
in Pseudomonas species.
• PQS – 2-heptyl-3-hydroxy-4-quinolone.
• Expression of PQS requires LasR, inturn PQS induces transcription
of Rhl I, it also partly controls expression of Las B gene.
• PQS initiates Rhl cascade by allowing production of Rhl I-HSL only
after establishment of LasI/LasR signaling cascade. This helps in
formation of bio-film.

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Quorum Sensing [Gram – Positive Bacteria]
 Gram – Positive bacteria uses peptides secreted via dedicated ATP-
binding cassette (ABC) transporter.
 These bacteria uses two-component adaptive response system of
proteins for detection of auto-inducers.
 Two component sensor kinases are the detectors for the secreted
peptide signals.
 Phosphorylation of the response regulator activates it, allowing it to
bind DNA and alter the transcription of the quorum sensing
controlled target genes.

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Fig: Table containing different AIP’s specific for different species with its
characteristics
(Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained Levels In vitro and In vivo,
Frederick Verbeke, et.al. 2017)

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Staphylococcus aureus – AgrC/AgrA virulence system:
Fig: Schematic representation of
AgrC/AgrA virulence system
(Staphylococcus aureus)

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Operon System in Staphylococcus aureus
Fig: Agr operon [agr BDCA]of Staphylococcus aureus

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 An RNA molecule called RNA III, regulates density-dependent
pathogenicity in S.aureus.
 AgrC – sensor kinase
 AgrA – cognate response regulator
 AgrD(46 aa) – precursor of AIP, which is processed to final
octapeptide to form active AIP, which contains thiol-lactone ring,
required for signaling activity.
 AgrB – putative ATP-processing or Transporter protein.
 This system activates the Agr operon and responsible for regulating
the levels of RNA III.
 RNA III functions as an effector to activate the expression of an array
of secreted virulence factors.

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Multi channel Lux circuit in Vibrio harveyi:
Fig: The hybrid HSL – two component quorum sensing circuit of Vibrio harveyi
(Quorum sensing in bacteria, Melissa B. Miller and Bonnie L. Bassler, Annu.Rev.Microbiol. 2001, 55:165-99)

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Hybrid-HSL/Two-component signaling circuit:
• Vibrio harveyi, gram-negative bacteria uses both gram-negative &
positive quorum sensing systems.
• V.harveyi uses two autoinducers to regulate density-dependent light
bioluminescence.
• AI-1 – synthesized by LuxLM and binds to LuxN
• AI-2 – synthesized by LuxS and binds to LuxQ
• AI-1 binds to LuxN and activates LuxU and LuxO and it will
activates LuxR and then transcription of Lux operon.

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Feedback mechanism:
• At low cell density, Lux N is auto-phosphorylated and as a result
LuxU and LuxO gets phosphorylated and repress the transcription.
AI – 2, a component for interspecies communication:
• V.harveyi, which is most commonly found wit multiple organisms.
So it had developed a mechanism to react with other species too.
• It helps V.harveyi to respond and monitor to its own species
population and other species cell density.
• Lot of species with LuxS gene has discovered, Yersinia pestis,
Escherichia coli, Salmonella typhimurium, Bacillus subtilis, etc.

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Inter – Kingdom Communication:
 In seaweed Delisea pulchra (seaweed) possess natural agents which
counteract quorum sensing in Serratia liquefaciens
 They coexist, Serratia liquefaciens colonizes Delisea pulchra, may
the sea weed can develop this mechanism to stop the virulence of
S.liquefaciens
Quorum Sensing in Archaea:
 Methanosaeta harundinacea 6AC, a methanogenic archaeon,
produces carboxylated acyl homoserine lactone compounds.
 These compounds facilitate the transition from growth as short cells
to growth as filaments.

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Quorum sensing in virus :
• Phages employ chemical communication to regulate collective activities
• QS like systems in phage helps it to optimize the timing of the lysis – lysogeny
switch
Fig: QS mediated communication in virus

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 A small-molecule QS-like phage communication process was termed
as “arbitrium system”.
 Following phage phi3T infection of Bacillus species, a phage-
encoded precursor peptide called AimP is produced and secreted.
 AimP is processed by extracellular proteases into the final arbitrium
signaling peptide.
 The mature peptide is internalized by bacteria, and if they are phage
infected, the peptide is detected by the phage AimR receptor, which is
a transcription factor.

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 AimX represses expression of the arbitrium Cl repressor gene, and
subsequently, the lytic cascade is deployed.
 At sufficient concentration, the AimP peptide binds and inactivates
AimR.
 Consequently, AimX is not expressed, Cl is made and represses lytic
development, and lysogeny is established.
 Thus, newly infecting phages can avoid triggering the lytic cascade
when there is low availability of uninfected hosts in the vicinity.
 In the un-liganded state, AimR binds DNA and activates transcription
of the gene encoding the AimX small RNA.

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Quorum Sensing in Fungi:
• Pheromones (or) autoinducers are signaling molecules embroiled in
cell-cell communication in fungi are considered as quorum-sensing
molecules (QSM), e.g. Isoprenoid farnesol and Farnesoic acids.
• In Cryptococcus neoformans, quorum sensing circuit is determining
the type of reproduction, unisexual or bisexual.
• QSP-1 is the autoinducer used, it binds and activated CSQ-2, which
plays an important role in the QSP-1 signaling cascade during
unisexual and bisexual reproduction of C.neoformans.

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Fig: Quorum sensing in Cryptococcus neoformans
(Fungal Quorum-Sensing Molecules and Inhibitors with Potential Antifungal Activity: A Review Arshad
Mehmood, et.al. 2019)

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Quorum Quenching (QQ):
• Quorum quenching is the process of preventing quorum sensing by
disrupting the signaling.
Strategies in quorum Quenching:
1. Inhibition of signaling molecules
Closantel: block Enoyl-acyl carrier protein reductase
2. Mimicking of signaling molecules
Halogenated furanones – mimic AHL, Synthetic AIP
3. Degradation of signaling molecules
Development of signaling molecules, degradation protein and target
to degrade AHL’s and AIP’s

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4. Modification of signaling molecules
• In Hydra vulgaris, the main colonizer of it’s epithelial cell surfaces,
Curvibacterspp produce 3-oxo-HSL quorum sensing molecules.
• Oxidoreductase activity of Hydra vulgaris is able to modify the 3-
oxo-HSL into their 3-hydroxy-HSL counterparts, so the quorum
sensing is inactivated.

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Applications of understanding the QS & QQ systems:
Strategies for QS inhibition to develop new anti-virulence
therapies:
• Various classes of natural compounds and chemical molecules could
interrupt with the cascade of the QS system.
• The multi-drug resistant bacteria, is a major problem and life
threating diseases, most of the bacteria gain their virulence after
forming bio-film.
• Development of drug molecules to inhibit the synthesis of signaling
molecule, competitive inhibition of the receptors, may be a different
approach to encounter bacterial infections .

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References:
 Quorum sensing in Bacteria, Melissar.B.Miller and Bonnie.L.Bassler, Annual
Review Microbiology, 2001, 55:165-199.
 Acyl-homoserine lactone-based quorum sensing in methanogenic archaeon, Zhang
Guishan, Zhang, Fan, Ding, et.al. The ISME journal, 2012, 6(7):1336-1344.
 Bacterial quorum sensing: It’s role in virulence and possibilities for its control, Cold
Spring Harbor perspectives in Medicine, 2012, 2(11): a012427.
 Quorum Sensing in Bacteria and a Glance on Pseudomonas aeruginosa, Mehrdad
Moghaddam, Samanesh Khodi, Ali Mirhossein, Clinical Microbiology, 2014, 3:4.
 Roles of Pseudomonas aeruginosa Las and Rhl Quorum-Sensing systems in control
of elastase and rhamnolipid biosynthesis genes, J.P.Pearson, E.C.Pesci,
B.H.Iglewski, Journal of Bacteriol, 1997(18): 5756-5767.

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 Quorum sensing across bacterial and viral domains, Olivia.P.Buddy,
Bonnie.L.Bassler, PLOS Pathogens, 2021.
 Quorum-Quenching microbial infections: Mechanisms and Implications, Yi-Hu
Dong, Lian-Huiwang anf Lian-Huizhang, Philosophical Transactions B, 2007,
362(1483): 1201-1211.