Quorum sensing allows bacteria to communicate and coordinate behaviors based on cell population density. It involves the release and detection of signaling molecules that increase in concentration as more bacteria are present. This system regulates behaviors like biofilm formation, virulence factor production, and bioluminescence. Many human pathogens use quorum sensing to regulate virulence and form biofilms, which contribute to infection persistence. Understanding quorum sensing mechanisms in different bacteria could help develop new anti-virulence therapies that disrupt communication without killing cells, reducing resistance development.

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QUORUM SENSING AND BIOFILMS
Quorum sensing, a recent and vital discovery in the world of bacteria, is a
system of inter and intra communication between bacteria that allow
bacterial populations to collectively carry out gene expression based on
signaling and response from each other and their environment. The system is
coined “quorum sensing” because the bacteria are able to sense the
accumulation of released molecules from neighboring organisms and
determine how many cells are present around them. Afterwards they can
simultaneously determine if the cell density is substantial to carry out the
release of the wanted protein. The vibrio fischeri bacteria that are found in
the body system of squids are gram negative and use this basic signaling
system.
A single bacterium has a genetic sequence that codes for an autoinducer, or a
signaling molecule that will be released into the environment. This molecule
can vary for different types of bacteria. Gram negative bacteria, which are
typically cocci and have a thick cell wall use acyl-homoserine lactones as a
signaling molecule (AHL). They diffuse easily in and out of a bacterial cells
where they then bind to matching AHL receptors on the surfaces of other
bacteria. Once the molecule reaches a certain threshold concentration it
binds to and activates a regulatory protein. The regulatory protein then binds
to a specific site on the DNA where it initiates the sequence to create a
quorum sensing specific protein as well as more enzymes to make the AHL
molecules necessary to continue the process.

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In gram positive bacteria the autoinducer are precursor oligopeptides. The
oligopeptides are cleaved into functional signaling molecules of 10-20
amino acids. Unlike AHLs, these autoinducers cannot easily diffuse across
the membrane of the bacteria and must be exported via active transport
proteins. When the amount of oligopeptides autoinducers reach a certain
threshold amount outside of the cell, they are detected by sensors on the
outer membrane of the cell. When the oligopeptide reacts with the sensor
protein, the intracellular part of the sensor becomes phosphorylated. The
phosphate is then transferred to a response regulator protein. The response
regulator protein then allows to bind to a specific site on the DNA. It
becomes a transcription factor as it alters the transcription of target genes. In
this way quorum dependent proteins such as virulence factors are produced.
This is the intra communication system that is used by like bacteria to
establish presence of their own kind. These molecules are released and
reciprocated continuously. Each bacteria is able to send out a signals and
receive one as well. This allows each individual bacteria to count how many
other bacteria there are. Once the bacterium can assess that there is a proper
amount of other bacteria present, it can simultaneously, along with the other
bacteria emit a response such as virulence or bioluminescence
However, there is also the intra species communication system. The
universal molecule used in interspecies communication is called
autoinducer-2 or AI-2.The enzyme LuxS is responsible for creating the Al-2
molecule. The gene encoding the LuxS enzyme has been detected in 35 of

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89 sequenced bacterial genomes with little variation. The LuxS enzyme uses
the substrate dihydroxy pentanedione to create the AI-2 molecule.
In 1998, Greenberg and his colleagues first described quorum sensing in
Psuedomonas aeruginosa, a gram negative bacteria known for causing fatal
systematic disease under certain conditions. Lung infections with biofilms
including this pathogen are found in patients with cystic fibrosis. Subsequent
studies further show that the quorum-sensing circuits in P. aeruginosa or
chestrate a symphony of several virulence factors.
In Gram-positve bacteria, many virulence factors involved in
staphylococcal infections, including surface-associated adhesins, hemolysin,
toxins and autolysins, are regulated by quorum sensing via
the accessory gene regulator (agr) system.
Observed and Studied Quorum Sensing in Biofilms Examples
Campylobacter jejuni
spiral or spirochete, rod-shaped Gram-negative bacteria that can cause
gastroenteritis and is commonly associated with foodborne illness. The
production of AHLs has not been established in C. jejuni The enzyme LuxS
was found in C. jejuni, and an AI-2-like product has been demonstrated.
However, chemical characterization of these AI-2-like compounds has not
been done. Utilizing the M129 strain of C. jejuni, scientists demonstrated
that a mutation in the luxS gene led to a decrease in biofilm formation when
compared to the isogenic wild-type strain. Addition from the wild-type strain
M129 led to an increase in biofilm formation.
Aeromonas hydrophila
A. hydrophila is a Gram-negative rod and facultative anaerobe present in all
freshwater environments and in brackish water. Some strains of A.
hydrophila are capable of causing illness in fish and amphibians as well as in
humans, who could acquire infections through open wounds or by ingestion
of a sufficient number of the organisms in food or water. A. hydrophila also
causes infections.
The formation of mature biofilms on stainless steel coupons by A.
hydrophila required the production of C4-HSL since an ahyI (AHL
synthase) mutant lacking the ability to form C4-HSL did not produce a
mature biofilm. A mutation in the ahyR (AHL receptor) gene had no effect

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on biofilm formation. An unidentified LuxR-like receptor bound the AHL
to produce biofilm in the ahyR mutant. Thus, quorum sensing regulates
biofilm formation, and a number of researchers have shown that mutations
in flagella formation decrease biofilm formation in A. hydrophila. Although
studies have not been reported, it is probable that flagella formation in A.
hydrophila is regulated via quorum sensing.
Bacillus cereus
B. cereus is a rod-shaped facultative aerobic Gram-positive bacterium that
forms endospores and has been associated with foodborne illness. The
quorum sensing system of B. cereus consists of PlcR (the plcR gene
encodes a transcriptional regulator) and PapR (the papR gene encodes a cell-
to-cell signaling peptide) as well as the LuxS/AI-2 system A plcR-negative
mutant produces approximately 4-fold more biofilm than its isogenic wild-
type parent.
Listeria monocytogenes
L. monocytogenes is a facultative pathogenic Gram-positive coccoid rod-
shaped bacterium and the cause of listeriosis. It is associated with foods
such as milk, cheeses, ice cream, raw vegetables, fermented raw-meat
sausages, raw and ready-to-eat meat and poultry, and raw and smoked fish.
Resistant to the injurious effects of freezing, drying, and heating, it is able to
grow at temperatures as low as 3°C. Biofilm formation by L.
monocytogenes has been demonstrated on polyvinyl chloride plates, glass
slides, stainless steel, polyethylene, teflon coupons, conveyer belt materials
(such as polypropylene, acetal, stainless steel), and floor drains of food
processing facilities.
While L. monocytogenes has a LuxS/AI-2 system, the luxS gene was shown
to repress biofilm formation A mutation in the luxS gene resulted in a 4-fold
thicker biofilm than in the wild type, and the addition of in vitro synthesized
AI-2 to cultures of the mutant did not repress biofilm formation . Therefore,
there is no indication that there is a quorum sensing role for AI-2 in L.
monocytogenes biofilm formation.
Little is known about possible peptide quorum sensing compounds in L.
monocytogenes. However, recent data indicate that L. monocytogenes has an
accessory gene regulator (agr) system.
There are 4 genes, agrB, agrD, agrC, and agrA, in the agr operon. There is
an approximately 62% decrease in the number of cells attached to glass

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slides with agrA and agrD deletion mutants of L. monocytogenes as
compared to the wild type . In addition, these mutants showed a 33%
decrease in the amount of biofilm formed on polystyrene during the first 24
hours. However, at 48 and 72 h, the amount of biofilm formed by the
mutants and wild type was approximately the same.
Escherichia coli
E. coli is a facultative anaerobic Gram-negative rod-shaped bacterium that
causes gastrointestinal and extra-intestinal infections. E. coli can also cause
hemorrhagic colitis and hemolytic uremic syndrome, and outbreaks have
been linked to contaminated water and foods such as ground beef, raw milk,
and produce. E. coli strains produce biofilms on the surfaces of glass,
stainless steel, etc. The LuxS/AI-2 system is present in E. coli
Enzymatically synthesized AI-2 increased motility and stimulated the
formation of biofilms when added to wild-type E. coli K-12 strains
Vibrio cholerae
Members of the genus Vibrio are facultative anaerobic Gram-negative
curved rod-shaped bacteria that are associated with foodborne and
waterborne diseases.V. cholerae causes cholera whereas V. vulnificus is
associated with wound infections, enteritis, bacteremia, and death in
immunocompromised individuals. A number of studies have examined
quorum sensing in vibrios. As a wound pathogen, the organism may form
biofilms in human tissue.
Biofilm formation in V. cholerae is tightly regulated and controlled by
multiple quorum sensing systems operating simultaneously to regulate the
transcription of genes involved in the production of exopolysaccharide. The
organism forms biofilms at low (rather than high) cell densities when signal
molecules have accumulated .
Furthermore, exopolysaccharide-over producing variants readily arose
during the time course of the biofilm assay, trapping smooth parental cells
within the biofilm. Mutations in hapR, a transcriptional regulator, were
responsible for enhanced biofilm formation. This suggests that at low cell
densities or early in the infection, it may be advantageous for V. cholerae to
be able to form biofilms and express virulence genes.
Role of Quorum sensing:
Quorum sensing plays an important role in biofilms. Biofilms are groups of
microorganisms that stick to each other and are frequently embedded in

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extracellular DNA, proteins and polysaccharides. The films can appear on
living or nonliving surfaces, such as a table or someone’s gums. Formation
occurs due to a number of factors including cellular recognition of specific
or non-specific attachment sites on a surface or nutritional cues. When a cell
starts growing in a biofilm, many genes are regulated differently.
Industries:
Biofilms are a major concern for the food and health industry. Human
pathogens form biofilms of food and food contact surfaces thereby
enhancing their ability to survive harsh environments, resists
antimicrobacterial treatments, and persist in the food processing
environment.
Social Interations:
The increases cell density environment of the biofilms favors chemical
signals to communicate for social interactions. Quorum sensing is used to
regulate biofilm formation and its maintenance as well as symbiosis between
species of the biofilm.
Biotechnological Applications of Quorum Quenching
 Naturally occurring quorum-quenching processes are being tested as
novel antimicrobial the rapies. Over expression of aiiA in tobacco and
potato plants confers resistance to E.carotovora, which requires AHL-
controlled virulence factor expression to cause disease.
 Like wise, co culture of Bacillus thuringiensis decreased
E.carotovora–mediated plant disease in an aiiA-dependent manner.
 Mice treated with synthetic antagonists of S.aureus AIPs how
resistance to infection.
 Similarly, purified halogenated furanones appear to attenuate
virulence of bacteria in mouse models.
 These and other examples predict that inhibition of quorum sensing
which offers an attractive alternative to traditional antibiotics because
these strategies are not bactericidal and the occurrence of bacterial
resistance there fore could be reduced.
 Like wise, approaches aimed at promoting bene ficia lquorum sensing
associations may enhance industrial scale production of natural or
engineered bacterial products.

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INHIBITION OF QUORUM SENSING
 Inhibition of quorum sensing has been proved to be very potent
method for bacterial virulence inhibition.
 Several QS inhibitors molecules has been discovered.
 QS inhibitors have been synthesized and have been isolated from
several Natural extracts such as garlic extract.
 QS inhibitors have shown to be potent virulence inhibitor both in in-
vitro and in-vivo, using infection animal models.
Studying the varied mechanisms of quorum sensing in biofilms allows
scientists to develop systems for disrupting the mechanism and preventing
illness and infection.
Prepared by Amjad Khan Afridi Reg:5149
Submitted to Dr. Abdul Rehman
Date: 13th October, 2016