Quorum Sensing- Communication Between Bacteria

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 Mrs. Lakshmi Sivasubramaniam

Mrs. Lakshmi Sivasubramaniam

The latest discoveries in the field of microbiology have proved that bacteria
communicate between each other. It is common knowledge that bacterial diseases
such as cholera, anthrax, meningitis, and many others are among the deadliest
in the world. It may be the case, however, that bacteria cannot cause an illness
in small quantities. Only when there are a sufficient number of them can they
act. Some, Vibrio Fischeri or Vibrio Harveyi, can glow in the dark. Others,
like Pseudomonas Aeruginosa, form biofilms on the surface of human organs, and
attack virulently those organs multiplying with tremendous speed, making it
practically impossible for antibiotics to interfere.

What is quorum sensing? Bacteria produce and release chemical signals - autoinducers
- in search of similar cells in their close surroundings. This is also called
"cell-cell communication." Other bacteria release the same autoinducers in response.
One-cell organisms in effect become multi-cellular organisms and can act together.
People or animals may have bacteria that cause some serious infectious diseases
and yet not be infected unless the number of bacteria is large enough; that
is to say, unless the quorum has been reached. Even though this phenomenon has
been known to scientists since the 1960s, only now they are able to study it
in detail. This new branch of microbiology, quorum sensing, discovered by Bonny
Bassler, professor of microbiology from Princeton, is dedicated to studying
this phenomenon.



Professor Bassler discovered that bacteria could send signals not only to their
own kind, but to other bacteria as well. She describes this phenomenon as "bacterial
ESPERANTO." This very important discovery allows scientists to work on new drugs
that can fight bacterial diseases. Bacteria develop immunity to antibiotics,
and new bacteria reproduce this quality with greater strength.


Developing new antibiotics that do not kill bacteria, but only neutralize them,
may be the hope for future medicine. Today, that hope drives new research behind
the doors of academic and pharmaceutical labs. This pathfinder addresses a broad
audience, from undergraduate students to scholars who are interested in investigating
quorum sensing either for their class projects or choosing the topic for their
scientific research.


The discovery that bacteria are able to communicate with each other changed our general perception of many single, simple organisms inhabiting our world. Instead of language, bacteria use signalling molecules, which are released into the environment. As well as releasing the signalling molecules, bacteria are also able to measure the number (concentration) of the molecules within a population. Nowadays we use the term 'Quorum Sensing' (QS) to describe the phenomenon whereby the accumulation of signalling molecules enable a single cell to sense the number of bacteria (cell density). In the natural environment, there are many different bacteria living together, which use various classes of signalling molecules. As they employ different languages they cannot necessarily talk to all other bacteria. Today, several quorum-sensing systems are intensively studied in various organisms such as marine bacteria and several pathogenic bacteria.


Bacteria that use quorum sensing produce and secrete certain signalling compound,
(called as autoinducers or pheromones) which are normally N-Acyl Homoserine
lactone
(AHL). The bacteria also have a receptor that can specifically detect
the inducer. When the inducer binds the receptor, it activates trnascription
of certain genes,  including those for inducer synthesis.


When only a few other bacteria of the same kind are in the vicinity, diffusion
reduces the concentration of the inducer in the surrounding medium to almost
zero, so the bacteria produce little inducer. With many bacteria of the same
kind, the concentration of the inducer passes a threshold, whereupon more inducer
is synthesised. This forms a positive feedback loop, and the receptor becomes
fully activated. This induces the up regulation of other specific genes, such
as lucifrase.


The purpose of quorum sensing is to coordinate certain behaviour or actions
between bacteria of the same kind, depending on their number. For example, opportunistic
bacteria, such as Psuedomonas aeruginosa can grow within a host without harming
it, until they reach a certain concentration. Then they become aggressive, their
numbers sufficient to overcome the host's immune system and form a a biofilm,
leading to to disease. It is hoped that the enzymatic degradation of the signalling
molecules will prevent the formation of such biofilms and possibly weaken established
biofilms. Distrupting the signalling process in this way is called quorum
quenching
.


The first organisms in which quorum sensing was observed were Myxobacteria
and Streptomyces species. However, the most popular example is the regulation
of light production in Vibrio fisheri, a bioluminiscent bacterium that
lives as a symbiote in the light-producing organ of deep-sea bobtail squid.
When the bacteria are free-living, they are at low concentration and do not
luminesce. In the light-producing organ (photophore) they are highly concentrated
(about 1011 cells/ml) and transcription of luciferase is induced,
leading to bioluminescence.


A first X-ray structure of a receptor (LuxP) was discovered in Vibrio harveyi in 2002, together with its inducer (AI-2), which is one of the few biomolecules containing containing boron.


Streptococcus pneumoniae uses quorum sensing to become competent.


Quorum sensing is a cell density dependent gene regulation that allows bacterial cells to express certain or specific genes only when they reach high cell density.


The study of Quorum sensing is important because:


1. It has provided some very important insights into the mechanism of parasitism


2. It provides a novel means to control infections of plants and animals. Gram-negative bacteria secretes acyl-homoserine lactone quorum sensing signals, which are also known as Autoinductors AIs.


3. Gram-positive bacteria used peptide-based signaling molecules


Why do bacteria talk to each other?



QS enables bacteria to co-ordinate their behaviour. As environmental conditions
often change rapidly, bacteria need to respond quickly in order to survive.
These responses include adaptation to availability of nutrients, defence against
other microorganisms, which may compete for the same nutrients and the avoidance
of toxic compounds potentially dangerous for the bacteria. It is very important
for pathogenic bacteria during infection of a host (e.g. humans, other animals
or plants) to co-ordinate their virulence in order to escape the immune response
of the host in order to be able to establish a successful infection.


Background


Quorum sensing refers to a bacterial cell-to-cell communication process. Signal molecules as acyl-homoserine lactones (HSL) or Autoinductors (AIs) are released by certain gram-negative bacteria and regulate various processes in a cell density dependent manner. When the bacteria are at low cell density, HSL signal molecules only accumulate to low concentrations. However, once bacteria reach a high density, a high concentration of HSL accumulates in the surrounding medium and this induces production of antibiotics, virulence factors and biofilms and infection of  plant or other Eukaryotic organisms. Gram-positive bacteria use peptides that mimic the signaling activity of acyl-homoserine lactones.


Studies of the Gram-negative Bacterium Vibrio fisheri led to the discovery
of Autoinduction or Quorum sensing. The marine symbiotic organism Vibrio
fisheri
produces bioluminescence in the light organs of marine fish and
cephalopods. The bacteria colonise the marine organisms by entering through
the pores of the light organs and then emptying them into the crypts where the
bacteria grow to high density. When the bacterial cells reach a high density
inside the squid’s light organ, a large accumulation of Autoinducer is formed.
The Autoinducers move through the membranes of the neighboring bacterial cells
by passive diffusion and then bind to LuxR protein forming a positive transcriptional
complex. The transcriptional complex induces transcription when the LuxR is
activated and binds to DNA. Such regulatory mechanisms involved in autoinduction
allow the production of light in certain marine organisms.


Image


Autoinduction: Production of light in marine organisms by regulatory mechanisms
in autoinduction


Bacilliuc cereus is a Gram-posiitve bacterium that is an aerobic spore-former and is widely distributed in nature and foods. When foods are contaminated with some strains of pathogenic  B. cereus. This leads to two distinct types of illnesses


1. A diarrhoes illness


2. An emetic illness


B. cereus is also used as biocontrol agent for plant diseases. It was recently shown that a biocontrol strain, UW85, inhibits quorum sensing. Such findings may lead to the possibility of quenching quorum sensing signals of Gram-negative acteria for disease control, thereby benefiting the fields of agriculture and medicine


Do all bacteria use the same signal molecules?


Different bacterial species use different molecules to communicate. There are several different classes of signalling molecule. Within each class there are also minor variations such as length of side chains etc. In some cases a single bacterial species can have more than one QS system and therefore use more than one signal molecule. The bacterium may respond to each molecule in a different way. In this sense the signal molecules can be thought of as words within a language, each having a different meaning.


Structures of AHL s


Image


Can bacteria from one species communicate with those from another species?

There is evidence that interspecies communication via QS can occur. This is
referred to as quorum sensing cross talk. Cross talk has implications in many
areas of microbiology as in nature bacteria almost always exist in mixed species
populations such as biofilms.


What are the benefits of quorum sensing research?


QS research has many potential applications, most of these involve controlling bacteria by interfering with their signalling systems. For example many bacteria rely on QS to control the expression of the genes, which cause disease. If we can block the QS systems we may be able to prevent these bacteria from being dangerous


Quorum sensing in Vibrio fischeri


Research into AHL based quorum sensing started in the late 1960s. The marine
bioluminescent bacteria Vibrio fischeri was being grown in liquid cultures
and it was observed that the cultures produced light only when large numbers
of bacteria were present. The initial explanation for this was that the culture
media contained an inhibitor of luminescence, which was removed by the bacteria
when large numbers were present. This was suggested because when grown in media
"conditioned" by preliminary exposure to the bacteria, luminescence could be
induced even at low cell densities. It was later shown that the luminescence
was initiated not by the removal of an inhibitor but by the accumulation of
an activator molecule or "autoinducer". This molecule is made by the bacteria
and activates luminescence when it has accumulated to a high enough concentration.
The bacteria are able to sense their cell density by monitoring the autoinducer
concentration. This mechanism of cell density sensing was termed quorum sensing
(QS). The molecule produced by V. fischeri was first isolated and characterised
in 1981 by Eberhard et al. and identified as N-(3-oxohexanoyl)-homoserine
lactone (3-oxo-C6-HSL). Analysis of the genes involved in QS in V. fischeri
was first carried out by Engebrecht et al. This led to the basic model
for quorum sensing in V. fischeri which is now a paradigm for other similar
quorum sensing systems.


For many years following this, it was thought that AHL-based QS was limited to marine bacteria such as V. fischeri and V. harveyi. Research into antibiotic synthesis caried out at Nottingham and Warwick led to the discovery that QS was far more widespread than previously thought.


QS relies on the synthesis, accumulation and subsequent sensing of a signal
molecule. In V. fischeri, the signal (3-oxo-C6-HSL, an N-Acyl Homoserine
lactone
or AHL) is synthesised by the protein LuxI and sensed by the protein
LuxR. At low cell densities (i.e. when only a small number of bacteria are present)
the signal is produced by the bacteria at a low level. The AHL diffuses out
of the bacterial cells and into the surrounding environment. As the cell density
increases (i.e. more bacteria are present) the signal accumulates in the area
surrounding the bacteria. When the signal reaches a critical threshold concentration,
it is able to interact with the LuxR protein. The LuxR/AHL complex binds to
a region of DNA called the lux box causing the luminescence genes to
switch on. In addition, the LuxR/AHL complex also causes the AHL (via LuxI)
to be produced at a higher level. Thus the AHL is said to autoinduce its own
synthesis. V. fischeri exist at low cell densities when free living and
at high cell densities when colonising the light organ, QS can therefore explain
why the bacteria are dark when free living and light when in the the light organ.


Image


The bacteria are effectively communicating, as a single bacterium is able to
detect and respond to signals produced by the surrounding bacteria. Bacteria
sense their cell density by measuring the amount of signal present. A large
number of Gram negative bacteria have been found to have AHL-based QS systems
similar to V. fischeri.


At low cell densities, the luxICDABE genes (luxCDABE genes are
responsible for bioluminescence) are transcribed at a low level and the small
amounts of 3-oxo-C6-HSL produced diffuse out of the cell.  high cell densities,
3-oxo-C6-HSL accumulates in the local environment and therefore also inside
the cell. Transcription of luxICDABE appears to be increased by a complex
of the LuxR protein and 3-oxo-C6-HSL binding to a region of DNA called the lux
box. In this way the 3-oxo-C6-HSL autoinduces its own synthesis and hence
amplifies the quorum sensing signal


Quorum sensing in Vibrio harveyi


Vibrio harveyi, like Vibrio fischeri is a bioluminescent marine
bacterium in which the luminescence genes are regulated by QS. As in all QS
systems, a signal molecule is produced and subsequently sensed by the bacteria.
V. harveyi has two quorum sensing systems, one employs an N-acyl-homoserine
lactone , 3-hydroxy-C4-HSL as the signal. This system is however distinct from
that of V. fischeri as the genes involved are not homologous to luxR
and luxI from V. fischeri. The second system in V. harveyi
relys on the accumulation of a molecule of unknown structure designated AI-2.
Both the AHL and AI-2 systems regulate the luminescence genes via two-component
regulatory systems. The AHL signal (3-hydroxy-C4-HSL,), which is generated by
the protein LuxM is received by the LuxN protein. AI-2l, generated via LuxS
is received by the LuxP and LuxQ proteins. LuxN and LuxQ contain both sensor
kinase and response regulator domains of two component systems. The two receptor
systems converge on a protein LuxO, which is homologous to the response regulator
domains of two-component signal transduction systems. At low cell densities
LuxO is phosphorylated by LuxQ and LuxN (via a phosphorelay protein, LuxU).
In its phosphorylated state, LuxO activates the transcription of a repressor
protein which blocks transcription of the luxCDABE genes. At high cell
densities when the two signal molecules are present, LuxN and LuxQ dephosphorylate
LuxO preventing from, actvating transcription of the repressor. This allows
a transcriptional activator LuxR, not homologous to LuxR from V. fischeri)
to activate lux gene expression.


Image


At low cell densities, the sensor kinases LuxQ and LuxN autophosphorylate,
passing the phosphate signal via LuxU, to LuxO. In its phosphorylated state,
LuxO activates the transcription of a repressor of the luxCDABE genes.
At high cell densities, the two autoinducer molecules accumulate and interact
with the sensors LuxP and LuxN. This causes them to dephosphorylate LuxO (via
LuxU) preventing it from activating the expression of the repressor protein.
This allows the protein LuxR to activate transcription of the luxCDABE
genes.


Although two component systems similar to those of V. harveyi have been
found in other Vibrio species including V. cholerae, V. parahaemolyticus
, V. anguillarum V. vulnificus and V. fischeri, most other
bacterial species do not appear to have proteins homologous to those of the
V. harveyi
QS system with the exception of LuxS.


LuxS is the protein responsible for the synthesis of the AI-2 molecule. Recently
homologues of LuxS have been found in Gram negative and Gram positive bacteria
including E. coli, Salmonella typhimurium, Haemophilus influenzae,
Helicobacter pylori, Bacillus subtilis, Neisseria meningitidis,
Yersinia pestis, Mycobacterium tuberculosis, Staphylococcus
aureus
and Streptococcus pneumoniae,.


Interestingly, none of the species listed above appear to have homologues of
any of the other V. harveyi QS proteins. The mechanisms by which they
detect and respond to the AI-2 signal are unknown. It is possible that LuxS
and AI-2 have functions other than QS in these bacteria. Vibrio species
maybe the only species which use AI-2 for QS and may use their QS system to
sense the presence of other bacterial species.

References


  1. Bluth, Brian J., Frew, Sarah E. and McNally, Brian. 1997. “Cell-cell
    communication of Lux operon in Vibiro fisheri
    ”,
  2. Cha, Chung, Gao, Ping, Chen Yu-Ching, Shaw, Paul D and Farrand, Stephen
    K., Production of Acyl-Homoserine Lactone Quorum-Sensing signals by Gram-negative
    Plant-Associated Bacteria, 1998, Mol. Plant-Microbe Interact 11: 1119-1129
  3. Dong, Yi-Hu, Lian-Hui, Xu, Jin-Ling, Zhang Hai-Bao, Zhang, Xi-Fen and Zhang
    Lian-Hui. Quenching quorum-sensing-dependent bacterial infection by an N-acyl
    homoserine lactonase. 2001. Nature. 411: 813-817.
  4. Summer, Susan s. and Albrecht, Julie A. January 1994. “Bacillus
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    ” . University of nebrask-Lincoln
  5. Teplistski, Max, Robinson, Jayne B., and Bauer, Wolfgang D. Plants secrete
    substances that mimis bacterial N- Acyl Homoserine Lactone signal activities
    nd affect population density-Dependent Behaviours in associated bacteria.
    2000. The Americam Phytopathological Society. 13: 637-648.
  6. The quorum-sensing site: The
    home of bacterial cell-cell communication on the Web
    .
  7. Segelken, R. (2002). Molecular
    structure is solved for key protein of quorum-sensing bacteria
    .
  8. Intercellular
    communication and quorum sensing in bacteria

  9. Small talk:A Cell-to-cell communication in bacteria. (A lecture at the meeting
    of the American Physical Society on Jan 31, 2004 given by Prof. Bassler)
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    The signals for bacterial communications differ in Gram-positive and Gram-negative
    bacteria, but as the studies of quorum sensing proceed, it is apparent that
    bacteria can communicate between each other successfully. Authors hypothesize
    that bacteria may have certain qualities of higher organisms.
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About Authors

Madhumathi Seshadrib and Mrs. Lakshmi Sivasubramaniam
a, *


 Mrs. Lakshmi Sivasubramaniam

Mrs. Lakshmi Sivasubramaniam


a Lecturer, Department of Pharmaceutical Analysis, College of Pharmacy,
SRM Institute of Science and Technology, Deemed University, Katangulathur, Chennai,
India.

* aAuthor for Correspondence: Lakshmi Sivasubramaniam, Lecturer,
Department of Pharmaceutical Analysis, College of Pharmacy, SRM Institute of
Science and Technology, Deemed University, Katangulathur, Chennai, India. E
mail: laxmisiva@rediffmail.com.


 


Madhumathi Seshadri

Madhumathi Seshadri


b Department of Chemistry, Pharmaceutical Chemistry unit, Vellore
Institute of Technology, Vellore - 632 014, India.


Madhumathi Seshadri successfully completed M. Tech. in Pharmaceutical Chemistry
at Vellore Institute of Technology, Vellore (a deemed university) in I Class
with Distinction.

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