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Quorum Sensing

Ibrahim Vazirabad
Dr. Eryn Hassemer
Winter 2010-11
Submitted: February 3rd, 2011
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In classical microbiology, bacteria have been considered as independently functioning
organisms. It was believed that bacteria found food, reproduced, and performed cellular
functions with little involvement from other bacteria. Any action taken by a bacterium was
assumed to be the result of its own decision-making machinery with no stimulus from other
bacteria. However, there is now evidence that bacteria are not the asocial organisms that they
were once thought to be. They actually love to talk and are constantly communicating with each
other, planning their next collective action, be it bioluminescence, pathogenicity, or a plethora of
other processes. With this new information, novel ways of producing antibiotics can be invented,
and the very framework that bacteria are viewed in will change dramatically.


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In the early 1960¶s, a Harvard professor named Woody Hastings found a peculiar species of
squid living off the coast of Hawaii. It was named the Hawaiian Bobtail Squid, and a colony of
light-producing, Gram negative bacteria was found to live in an organ in the squid¶s body. They
were named •
    è1). The bacteria live inside this light organ at a concentration
roughly 1011-1012 cells per milliliter è1). At this high concentration and at only this high
concentration, •
   produces light. The reason for this behavior is that •
   produces
a hormone-like molecule, called an auto-inducer or ³AI´ for short, and when the cell density
increases, so does the auto-inducer concentration. At a certain threshold of this molecule, the
molecule is bound by a regulatory protein, causing light to be produced by a cascade causing a
change in gene expression è1). This regulation of gene expression is termed ³quorum sensing´.
Because quorum sensing allows bacteria to coordinate the behavior of the group, it enables them
to take on some of the characteristics of multicellular organisms è2). Hundreds of bacteria have
conserved these same proteins that activate the gene expression è1). All that differs between the
bacteria are the actual genes that are expressed when the specific auto-inducer is bound, since
every bacterial species has their own specific molecular ³language´. Gram negative and Gram
positive bacteria have chemically dissimilar auto-inducers. Therefore, the quorum sensing
system used by Gram negative bacteria is only casually similar to the methods of Gram positive
bacteria. To complicate things further, there is a second auto-inducer that all bacteria use to
count the total bacterial population è1). A more in-depth portrait of quorum sensing will be
elucidated as the paper progresses.

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Quorum sensing is the technique in which bacteria regulate their gene expression to better serve
the needs of the group rather than the individual. Three major quorum-sensing circuits have been
described: one used primarily by Gram-negative bacteria, one used primarily by Gram-positive
bacteria, and one that has been proposed to be universal è3). Most Gram negative bacteria use a
certain molecule called an ³acyl homoserine lactone´ or ³AHL´ that is used as the signaling
molecule. Gram positive bacteria use oligopeptides as their signaling molecule, which have been
cut from a large polypeptide synthesized in the cytoplasm. These two systems are species-
specific, and each Gram negative or Gram positive bacteria produce their own specific auto-
inducer. Thus, these molecules enable intra-species communication. These are private, secret
conversations è1). The third system is proposed to be a ³Bacterial Esperanto´ that is used for
inter-species communication and is called ³AI-2´ è1). It is a novel type of auto-inducing
molecule which prominently uses Boron.
 º

 

 
 

The Gram negative quorum sensing system has been heavily studied in the past fifty years due to
the discovery of •
     by marine biologist Woody Hastings è1). As mentioned
beforehand, •
   lives in the light organ of the Hawaiian Bobtail Squid at very high cell
density and uses quorum sensing to perform bioluminescence. •
 also uses quorum
sensing to bio-illuminate, but it is free-living rather than a symbiont. The quorum sensing
circuitry of •
   is a model of the collective processes that take place in many Gram
negatives è2). The quorum sensing circuitry of •
 is more complex and is nearly identical
to the circuitry of a more notorious Gram negative, •
   . The basis of the •
  
quorum sensing system will be described below.

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Most Gram negative bacteria produce acyl homoserine lactones èAHLs for
short) as the signaling molecule of choice. Each AHL has a standard ester
moiety which is shown in Figure 1. However, the R-group confers species-
specificity to the communication. Each of these molecules interacts with its
own specific binding protein, but with no other è1). These molecules are
small and can easily enter and exit the cellular membrane. At a certain
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threshold value of auto-inducer, the molecule binds to the binding protein
and group behavior becomes expressed.


 
The LuxIR system was the first one to be described in •
   è3). It is operated by two
proteins, one called LuxI, and one called LuxR. A schematic of this circuit can be seen in figure
2. The LuxI-type protein catalyzes the formation of the specific AHL
auto-inducer, and the LuxR-type protein binds the auto-inducer
ligand when the concentration of auto-inducer reaches a threshold
level è2). The ligand bonding to LuxR induces a conformational
change in LuxR. The LuxR±AHL complex then is able to activate
transcription of target genes by recognizing and binding specific
DNA sequences at quorum-sensing-regulated promoters è2). In the
case of •
   , this induced transcription produces the required
proteins to bio-illuminate, which are called luciferases. Homologues of
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homologues produce and bind their own specific auto-inducer and the genes expressed are
specific to the bacteria. It seems that because of their high importance, the DNA sequences
coding for these two types of proteins were highly conserved in many Gram negative bacteria.

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The Gram positive quorum sensing system does not have the big names of •
    ,
    , or    , but the circuit has
been studied well enough to understand its mechanism. Gram-
positive quorum-sensing systems typically make use of small post-
translational processed peptide signal molecules è ). These
oligopeptides build up in the inter-cellular space in the same
fashion as the acyl homoserine lactones.

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When the concentration of auto-inducer reaches a threshold level, the peptides are recognized by
sensor kinases that initiate phospho-transfer to a response regulator, which then phosphorylates a
piece of RNA that encodes for genes useful in group behavior è3). Many Gram positive bacteria
use this two-component regulatory system, which consists of a sensor and response-regulator
protein. This circuit can be viewed in Figure 3.

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Both Gram positive and Gram negative bacteria have quorum sensing systems that provide
excellent intra-species communication. However, bacteria do not live in homogenous colonies.
Bacteria live in areas where there may be dozens or hundreds of other bacterial species. It would
be logical to propose that if bacteria are able to talk to their siblings, they should be able to talk
to their cousins, or aunts, or other bacterial relatives. With the knowledge of how many of ³us´
and how many of ³them´, bacteria could make wise decisions on when to perform cooperative
actions. As stated earlier, this language would be the bacterial ³Esperanto´. In the year 2002, the
identity of this theoretical auto-inducer was discovered è
). AI-2 was originally identified in the
bioluminescent marine bacterium •
  as one of two auto-inducers that regulate light
production in response to cell density è
), the first being an AHL. Figure shows the structure
of AI-2. The details of this discovery are beyond the scope of this introductory article, but here
are some interesting details of the AI-2 structure and molecular formation. ³The synthase
required for AI-2 production, LuxS, is widely conserved among Gram-
negative and Gram-positive bacteria´ è
). It can then be surmised that
because the method of AI-2 synthesis is conserved by both Gram
negative and Gram positive bacteria and the methods of intra-species
communication èdeemed AI-1 communication) were not conserved, AI-2
communication developed first. This makes sense with the assertion that
more general modes of communication developed first and then became
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prominently featured in the AI-2 molecule. ³The proposed AI-2 structure
contains two fused five-membered rings stabilized within the LuxP binding site by numerous
polar interactions. Several candidates were considered for the atom bridging the diester. We
suggest that it is boron, on the basis of several considerations. Although boron and carbon are not
reliably distinguishable on the basis of electron density at this resolutionèThey had crystallized
the LuxP binding protein with AI-2 in it), the presence of carbon at this position would result in a
highly unstable orthocarbonate moiety, whereas the borate diester is relatively stable´ è
). The
above statement is a lot of jargon, but it is clear that boron figures prominently in AI-2, which
has a conformation which is much unlike any other previously found quorum sensing molecule.

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Îp to this point, the applications of quorum sensing have not been extrapolated. The reason why
quorum sensing is conducted is to perform certain collective actions simultaneously so that these
actions will have a high chance of success. There are many examples of both AI-1 and AI-2
being used in unison to further the collective goals of bacteria. These examples include
bioluminescence, secretion of virulence factors, biofilm formation, sporulation, conjugation, and
pigment production. One of the most pertinent examples will be discussed below.

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The Gram negative bacteria •
   is the pathogen that causes cholera. This bacterium
has captured the spotlight recently with the cholera epidemic in Haiti. Cholera kills by causing
vomiting and diarrhea, which causes rapid dehydration. •
  uses a novel quorum sensing
pathway to achieve its goals of infection and eventual escape from the host, which is not the
canonical LuxIR quorum sensing pathway. It is rather a pathway that is nearly identical to the
bioluminescent marine bacterium •
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There are two types of bacterial infections that we humans usually deal with, acute infections and
persistent infections. Acute infections are self-limiting infections that only last a certain period of
time. Persistent infections are not self-limiting and the bacteria that cause it will continue to
divide and fight to subdue the host. Most bacteria cause persistent infections, and their quorum
sensing strategy is similar to the strategy of •

   . They will perform binary fission until they
and their auto-inducers reach a very high level, and
then certain genes will be expressed at that period in
time. However, •
  has adopted an opposite
strategy. At low cell density, the auto-inducer
receptors on the cell surface are kinases and they
phosphorylate proteins down the pathway which
leads to the LuxR homologue called HapR, the
master regulator of •
  quorum sensing
behavior è6). One phospho-protein called LuxO
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activates the expression of four genes encoding small
RNAs which prevent expression of the LuxR homologue HapR by destabilizing its message è6).
This destabilization causes virulence factors and a toxin-coregulated pilus which allows biofilm
attachment to the intestinal wall è6). At high cell density, the auto-inducer receptors become
phosphotases and a de-phosphorylation cascade takes place è). LuxO is deactivated, and HapR
becomes stabilized, shutting off the virulence factors. HapR also activates the expression of a
protease which is thought to function as a detachase that cuts the pilus è6). The various binding
proteins, AI-1 and AI-2 can be seen in figure
. Hence, when •
  enters the body, it will
immediately attach to the intestinal wall and perform its virulent functions. When a sizable
portion of the intestine is colonized, it will detach itself from the intestines due to the
accumulation of auto-inducer, and leave to infect a new host.
The techniques used by V. Cholerae to induce pathogenicity are complex, but it seems that many
bacteria in the Genus •
 use this kinasephosphotase cascade duality to control quorum
sensing è).
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Since bacteria have developed such complex and highly conserved quorum sensing systems,
exploiting these systems to produce effective, non-lethal
antibiotics would be a step forward è). Since many
pathogenic bacteria use quorum sensing to initiate
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impede the ability of the bacteria recognizing the auto-inducer or stopping the bacterium from
producing auto-inducer, hence, making the bacterium ³deaf´ and ³mute´. Meet Dr. Bonnie
Bassler. She is a Princeton scientist who has been studying quorum sensing for over 20 years.
She conducted a series of experiments in which quorum sensing was used to cure animals of •

  è). The technique was as so: The specific auto-inducer for •
  was removed
from the supernatant in which the cells were reproducing. This auto-inducer is called ³CAI-1´. It
was purified and its chemical structure was elucidated. CAI-1 can be seen in figure 6 above. As
can be immediately seen, it is not the canonical acyl homoserine lactone seen in figure 2. The
synthetic CAI-1 was injected into wild-type •
  , and through Western blotting, •

  ¶s virulence factors were shut off after exposure to CAI-1 è). A mutant lacking the
CAI-1 receptor was exposed to CAI-1, and as expected, virulence factors were continuously
expressed no matter the CAI-1 concentration è). Therefore, the idea that this synthetic CAI-1
was indeed de-activating the virulence factors was supported. Finally, the synthetic CAI-1 was
injected into a mouse which was infected with wild-type •
  è). The mouse survived
and became healthy. This technique can help develop better therapeutics and be a step forward
on reducing the sickness time of cholera and many other bacteria which use quorum sensing è).

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Quorum sensing is proof that bacteria are not the single-celled, single-minded organisms of
classical microbiology. They are   . Working in unison, they are able to accomplish
many wonderful, and at times, terrible things. However, new discoveries are occurring rapidly.
Now that their bacterial language has been cracked, we will be able to exploit it to make the life
of humans better.

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1. Why is the use of quorum sensing a benefit to bacteria?
2. Explain how quorum sensing can be exploited to create new antibiotics.
3. Why do you think that Auto-Inducer-2 is used by both Gram negative and Gram positive
bacteria when the Auto-Inducer 1 molecules are so dissimilar in these two groups?
 References

1.     
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2010, Proceedings of the American
Philsophical Society, pp. 30-312.

2.       
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2003,
National Academy of Sciences, pp. 1
9±1

.

3. <     
  

6    
   
 


Dallas, TX : Federation of European Microbiological Societies, 200
.

.   <        

Ú  ë
 
 
£  
9, Rochester, New York : American Society for Microbiology, 2000, Vol. 68.

.     
       


 

Princeton NJ; Strasbourg France : Nature, 2002, Vol. 1
.

6. ë     !    "        
   •
   
 # $•   
  
r        
  
, Hanover NH :
Molecular Microbiolog, 200, Vol. 6 .

. £

£ 
•
   <  % 
 
>Video] Princeton
NJ : s.n., 2010.