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Published in final edited form as:

Nature. 2017 November 15; 551(7680): 313–320. doi:10.1038/nature24624.

Bacterial quorum sensing: the progress and promise of an

emerging research area
Marvin Whiteley1, Stephen P. Diggle2, and E. Peter Greenberg3,*
1Departmentof Molecular Biosciences, Institute of Cellular and Molecular Biology, John Ring
LaMontagne Center for Infectious Disease, The University of Texas at Austin, Austin, TX USA
2School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA USA
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3Department of Microbiology, University of Washington School of Medicine, Seattle, WA USA,

Guangdong Innovative and Entrepreneurial Research Team of Sociomicrobiology Basic Science
and Frontier Technology, South China Agricultural University, Guangzhou, China

Preface
This review highlights how we can build upon the relatively new and rapidly developing field of
bacterial communication or quorum sensing (QS). We now have a depth of knowledge about how
bacteria use QS signals to communicate with each other and coordinate activities. There have been
extraordinary advances in QS genetics, genomics, biochemistry, and diversity of signaling
systems. We are beginning to understand the connections between QS and bacterial sociality. This
foundation places us at the precipice of a new era where researchers can advance towards
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development of new medicines to treat devastating infectious diseases, and in parallel use bacteria
to understand the biology of sociality.

Keywords
Sociomicrobiology; bacterial communication; Vibrio; Pseudomonas aeruginosa; orphan receptors;
solo receptors; biogeography; quorum quenching; anti-virulence strategies

‘Competing is intense among humans, and within a group, selfish individuals

always win. But in contests between groups, groups of altruistsa always beat groups
of selfish individuals.’
E.O. Wilson
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Main text
Humans have provided descriptions of the natural history of animals for millennia. Apart
from basic anatomy and physiology, it was also noted that a number of animal species
showed signs of “social intelligence”. Aristotle provided a description of animal social

*
Corresponding Author: E. Peter Greenberg, Department of Microbiology, The University of Washington, Phone: 206-616-2881,
epgreen@uw.edu.
aThe ethological definition of altruism is a behavior by an individual that increases fitness of another individual while decreasing
fitness of the actor1.
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behavior in his 4th century BC book ‘History of Animals’, noting how ants march one after
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the other when putting away food, while bonitos swarm together when they catch sight of a
dangerous creature. The application of evolutionary principles to social behaviors in the 19th
century brought forth the field of sociobiology that was further explored and popularized in
the 1970’s2. While the last 40 years have been a time of great advancement and debate in
sociobiology, these ideas were largely unexplored in the field of microbiology until the 21st
century. We now know that microbes are highly gregarious communicating organisms and
bacterial communication can modulate a range of behaviors important for fitness
(reproductive success).

Background and a Brief History of Quorum Sensing

Bacterial QS involves self-produced extracellular chemical signals, which can accumulate in
a local environment to levels that are required to activate transcription of specific genes3–5.
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The first hints about QS came in the late 1960s and early 1970s, when investigators showed
that genetic competence in Streptococcus pneumoniae6 and luminescence in two species of
marine bacteria7,8 required production of extracellular molecules. Cell-cell signaling via
these molecules was proposed as a form of chemical communication, but these early
publications were met with skepticism and generally ignored for the next 10–20 years. The
1980s brought two landmark discoveries: (i) the luminescence (lux) genes from the marine
bacterium Vibrio fischeri were identified, and the genes required for what is now called
quorum control of luminescence, luxI and luxR, were show to control lux gene
transcription9,10; and (ii) the QS signal from V. fischeri was determined to be N-3-
oxohexanoy-L-homoserine lactone (3OC6-HSL)11 (Fig. 1A). The luxI gene codes for the
autoinducer synthase required for 3OC6-HSL production, and luxR codes for a 3OC6-HSL-
responsive transcriptional activator of the lux genes (Fig. 1A).
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General interest remained muted for another decade. In the 1990s DNA sequencing and
comparative sequence analysis became everyday laboratory procedures, and gene pairs with
homology to luxR and luxI began to attract the curiosity of some investigators. This led to an
explosion of findings that other bacterial species controlled genes for conjugation,
exoenzyme production and antibiotic synthesis with LuxI-LuxR-like systems3. A common
theme emerged; the LuxI homologs catalyzed synthesis of an acylated homoserine lactone
(AHL) and the LuxR homologs all showed specificity for their cognate AHL. This
convergence of discoveries led to the QS concept (Fig. 1A); that the diffusible AHLs served
as a proxy for cell density and allowed a bacterial species to produce costly extracellular
public goods only when there was a sufficient biomass to benefit from the public goods4.
Shortly thereafter the QS signal from S. pneumoniae (often referred to as a pheromone) was
shown to be a small peptide12, and Staphylococcus aureus was shown to use small cyclic
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peptide pheromones to activate genes for production of extracellular toxins13. Quorum

sensing was therefore shown to occur in Gram-positive and Gram-negative bacteria via
diverse chemical signals (Fig. 1B). An early study showed that a luminescent marine
bacterium called Vibrio harveyi could sense a self-produced signal and also a signal or
signals produced by other bacterial species to induce light production7. This phenomenon is
considered to be a type of QS, where cells of many species in a mixed microbial community
sense the general bacterial population density via a molecule termed Autoinducer-2

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(AI-2)14,15. The strengths and weaknesses of this concept are discussed later in this review.
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Further studies also described QS-like systems in eukaryotic microbes (the pathogenic fungi
Candida and Histoplasma)16,17 and very recently in viruses18, thus providing clear examples
of convergent evolution. Some QS signals are volatile, for example the DSF and PAME
signals shown in Figure 1, and there is some evidence that volatile signaling can occur in a
local atmosphere19. Functional studies followed the discovery of many of these systems and
revealed that for many plant and animal pathogens, QS mutants showed greatly reduced
virulence13,20,21. The early connection between QS and pathogen virulence brought forth
excitement about the idea of targeting QS as a novel approach to treat bacterial infections
(Box 1).

Box 1

The challenge to QS therapeutic development

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Early discovery that QS mutants of important plant and human pathogens are attenuated
for virulence20,21 led quickly to the concept of using QS inhibitors to control some
diseases110. In fact, a variety of small molecule inhibitors of QS signal receptors and
LuxI-type QS signal synthases have been discovered, as well as enzymes, which degrade
AHL signals90–92,99–104. Yet we face obstacles in moving from the bench to the clinic.
Many of the obstacles are inherent to drug discovery such as lack of potency in animal
models, delivery, toxicity, stability, and a narrow spectrum of activity. But what are other
obstacles? Fundamental questions remain unanswered. At what point in an infection will
QS inhibition be of value? Will QS inhibitors find general utility or will they function
best as prophylactic agents? Can biological interference approaches be contemplated?
Can we imagine introducing a benign bacterial species, which produces an AHL
lactonase to interfere with a QS pathogen into a human? We note that such an anti-QS
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approach has been developed to control membrane fouling by biofilms in water

purification plants (see image below)111. What pathogens should we target? What are the
regulatory hurdles for a QS inhibition therapeutic to even enter a clinical efficacy trial?
How quickly will resistant bacteria emerge? There is an idea that because QS inhibitors
are anti-virulence agents rather than antibacterial agents, resistance is less likely to
emerge. Laboratory experiments do not support this idea98 but in theory the route to the
spread of resistance may be slow and depending on the resistance mechanism it may be
self-limiting. The route to testing anti-QS approaches may be much less tortured in the
case of crop diseases where any number of approaches can be easily tested in the
laboratory or greenhouse. Ultimately, if QS inhibition is to gain therapeutic utility for
human diseases, this will likely occur in the context of combination therapies with
conventional antimicrobials.
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Box 1 image.
Quorum-quenching membrane filtration (QQ-MBR) pilot plant system at sewage
treatment facility in Seoul, South Korea. Professor Chung-Hak Lee of Seoul National
University (center) has led a decade-long program to develop this technology

Sociomicrobiology: An evolutionary perspective on quorum sensing

Early research assumed that QS is a social trait (a trait that impacts both the individual
performing a behavior and a recipient22), but this concept was not tested experimentally. In
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principle, QS could be non-social, for instance allowing individual bacteria to sense their
physical environment (this has been termed diffusion sensing)23,24. However, there is now a
large body of evidence showing that in certain environments, QS is social and that at the
population level, QS regulates the production of extracellular public goods22,23,25–29.
Importantly, whilst public goods directly benefit the producing cell, they also indirectly
socially benefit surrounding cells. Because the production of a public good is costly, these
behaviors are potentially exploitable by non-producing ‘cheats’, creating bacterial social
dilemmas (Fig. 2). QS has now been shown to be exploitable by cheats in laboratory
cultures26–29. One well-studied example is with the pathogenic bacterium Pseudomonas
aeruginosa. This bacterium requires QS to induce production of extracellular proteases
required for growth on proteins. QS mutants fail to grow by themselves on milk protein, but
in co-culture with QS competent protease producing cells the mutants have a fitness
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advantage when at relatively low relative abundance25,26,29. In fact mixed infections of

cooperating and cheating cells have been shown to be less virulent than single strain
cooperator infections30. This has led to the idea of using bacterial cheats to help treat
infections by reducing virulence or by introducing ‘Trojan Horse’ genes into virulent
populations31.

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Given that QS can be readily exploited by cheats, why are numerous functional QS systems
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maintained in natural populations? This was a dilemma first discussed by Darwin: if cheats
in a population have a fitness advantage over cooperators they should survive, take over the
population and cooperation should be inherently unstable. Kin selection theory has been
invoked to help explain this dilemma32,33. Put simply, by helping a relative reproduce, an
individual indirectly passes its genes into the next generation. Kin selection has been
proposed to be important in microbial social behaviors such as QS because of clonal
reproduction and relatively local interactions26,34. Recently the importance of kin selection
in maintaining cooperative behaviors in single and multicellular organisms has been
theoretically challenged, and microbial systems provide powerful experimental platforms to
help resolve this debate35–37. The concept of kin selection is impacted by spatial structure,
which can maintain cooperation and public goods because it keeps cooperators and relatives
close together. Surface associated microbial communities (termed biofilms, described in
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detail later) are a good example of spatially structured populations The biofilm lifestyle
helps restrict the invasion of cheats and affects population dynamics28,38. In addition, recent
studies of QS have revealed molecular mechanisms for stabilizing cooperator populations.
We have learned that metabolic prudence can constrain cheaters. Here an expensive public
good is co-regulated by QS and nutrient availability such that the public good is produced
when two conditions are met; there is a quorum and an ample nutrient supply such that the
cost of public goods production is not critical39. We have also learned that there are
metabolic constraints on cheating where QS co-regulates private goods with public goods.
Experimentally, transcriptomic studies revealed that although P. aeruginosa QS controls
production of a battery of extracellular products, which may be considered public goods, it
also controls some cell-associated products, one of which is a cellular enzyme required for
growth on adenosine. As discussed above, when P. aeruginosa is grown on milk protein QS
is required for expression of genes for extracellular proteases, and there is a fitness
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advantage for QS mutants. When P. aeruginosa is grown on milk protein plus adenosine
cheats are unable to utilize the adenosine and their fitness advantage is nullified; a penalty
has been placed on cheating40. Finally, we are beginning to understand policing, defined as
an ability of bacterial cooperators or a host organism (host sanctioning), to hinder the fitness
of cheats41–43. A recent report describes how QS regulation of pairs of genes coding for
toxins and toxin immunity can serve as a policing mechanism. QS competent individuals
deliver toxins to other individuals. QS competent cells are immune but QS mutants are not.

Although we now have a wealth of mechanistic data showing how QS systems function at
the molecular level, the true biological function of many QS systems remains a research area
of great opportunity. Evolution and ecological approaches can help address this knowledge
gap. For example, are the chemically diverse QS molecules described in the literature always
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acting as signals? In an evolutionary context, a true signal evolved because it alters the
behavior of a receiver and the receiver’s response must have co-evolved. This is distinct
from a ‘cue’ where the production of a substance has not evolved because of its effect on a
recipient. If the production of a substance forces a costly response from a receiver we can
differentiate this from signaling and term it coercion or chemical manipulation44–46. The
current literature sometimes conflates signaling, cueing and coercion, and whether bacteria
are interacting via a signal, a cue or coercion can lead to different biological outcomes. This

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is becoming increasingly important as we begin to study microbiota interspecies interactions

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and as we seek to develop anti-QS therapeutics. It has been exciting for us to see these
concepts introduced into the field of microbiology and to see the power of microbiology and
microbial genetics brought to bear on questions about the mechanisms and consequences of
communication and social interactions in ways that are immensely more difficult with higher
organisms. Although there are certainly limitations in studies of social behavior and social
evolution in bacteria there are important advantages. One can execute an experiment with
hundreds of millions of individuals on the bench top. Experiments with ten generations of
offspring can be done in a day. Correlating genes with social activity is routine. In this
article, we focus a considerable amount of discussion on the pathogenic bacterium
Pseudomonas aeruginosa. We do so in part because it is a particularly well-studied model in
the QS field, and it is one on which the laboratories of all three co-authors have worked.
There are mutant libraries of this bacterium. As a first analysis of gene-social function
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relationships one needs only to order a mutant and study it in the context of sociality.

How and why the distinction between signals, cues and coercion is important is exemplified
by the AI-2 molecule described earlier (Fig. 1B). AI-2 is a furanosyl borate diester produced
by V. harveyi14. The identification of the luxS gene, which is required for AI-2 production47,
sparked a huge interest into AI-2 because this gene is found in both Gram-positive and
Gram-negative bacteria15. This led to the hypothesis that AI-2 allows widespread
communication between bacterial genera, a type of ‘bacterial Esperanto’48. However,
evolution and signaling theory question whether AI-2 can be defined as a true interspecies
signal. For this to be the case AI-2 must (1) diffuse from the producing cell; (2) interact with
a receiver cell; (3) elicit a response from the receiver cell that has co-evolved with signal
production by the producer; and (4) benefit both producer and receiver. Points 1 and 2 are
met with respect to AI-2, but points 3 and 4 are often not met between two or more species.
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Despite AI-2 being produced by many genera there are few instances, including V. harveyi
luminescence47 and the Lsr ABC transporter in Salmonella typhimurium49,50, linking it with
direct activation of specific genes. While a number of studies have reported that AI-2
‘signaling’ impacts specific bacterial phenotypes, many of these studies have relied on the
use of luxS mutant strains. As LuxS is involved in recycling of S-adenosyl-L-methionine, of
which AI-2 is a non-toxic metabolic byproduct, luxS mutant phenotypes may simply be due
to metabolic perturbations51,52. In these cases, AI-2 cannot be considered a signal at either
an intra or interspecies level. By understanding that in some cases AI-2 might serve as a
signal where in other cases it is a cue, or even a waste product, studies of AI-2 should lead to
evolutionary perspectives about how metabolic waste products can evolve to become signals,
and about the evolution of chemical communication itself.
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A next step: QS in natural habitats

The bulk of studies driving our current understanding of the molecular biology and evolution
of QS utilize well-mixed laboratory cultures and growth environments not intended to mimic
the natural environment. These in vitro systems provide reproducible conditions for
biochemical and evolution studies, as well as the ability to grow large culture volumes often
necessary for signal purification and identification. These systems have also provided a

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wealth of solid fundamental knowledge on which we can build to study the role QS plays in
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modulating the composition and function of natural microbial communities.

Working in natural microbial habitats is challenging and requires QS scientists to embrace

the complexity of these environments while leveraging state of the art multi-disciplinary
approaches. One elegant system that has been developed to begin to study QS within a
natural bacterial community in an animal host is the mutualistic symbiosis between the squid
Euprymna scolopes and its light organ symbiont V. fischeri. The squid, which inhabits
coastal waters of the Hawaiian Islands is born with a sterile light organ. The light organ is
colonized specifically by V. fischeri, which occurs in low abundance in the surrounding
seawater. V. fischeri uses its QS system to activate genes for luminescence in the high-
density light organ environment. The mutualism is simple in that there are but two partners,
and it is amenable to laboratory manipulation. This model system has provided insight not
only about the role of QS in an animal host-bacterial interaction, but also about how a
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microbiome, albeit a simple one, can influence host development (for recent reviews
see53,54). Furthermore, juvenile squid are smaller than a pencil eraser and translucent, and
bacteria tagged with GFP can be easily visualized in whole light organs. This has provided a
means to study a difficult question discussed later in this review; can aggregates of bacteria
employ QS signals to communicate with other aggregates, and if so is there a discrete
distance over which this kind of communication can occur in a given condition (a calling
distance)?

An ultimate goal is to integrate our comprehensive understanding of QS derived from

elegant laboratory culture studies, with ecological principles to illuminate the role of
bacterial communication in natural habitats. Researchers are now beginning to study more
habitats with more complex microbiota than the squid symbiosis. Here, we focus on three
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areas of emerging interest that have important and relatively unknown functions in natural
ecosystems: (1) orphan LuxR homologs; (2) the link between QS and microbial
biogeography, and (3) quorum quenching.

Orphan or solo LuxR homologs and interspecies interactions—LuxR-type

transcription factors consist of two domains, an N-terminal signal-binding domain and a C-
terminal DNA-binding domain, and simple homology searches can be used to identify LuxR
homologs in genomic sequences. Just such a search of the Salmonella typhimurium genome
revealed the first example of what we now call orphan55 or solo56 LuxR homologs (Fig. 3A).
The genome of S. typhimurium possesses a luxR homolog called sdiA but it does not
possess a luxI-type gene, and S. typhimurium does not produce AHLs55,57,58. In fact, many
Gram-negative bacteria possess luxR-type genes and do not possess luxI-type genes, and
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many more Gram-negative bacteria possess greater numbers of luxR-type genes than luxI-
type genes. In the case of S. typhimurium, we have learned that SdiA responds to AHLs
produced by other bacteria, leading to activation of specific genes58. There is also an sdiA
gene in E. coli. The E. coli SdiA has been reported to respond to AHLs and mammalian
host-produced small molecules59–61. These latter studies provided the first hints that some
orphan LuxR homologs might be involved in sensing the host environment, directly, rather
than serving as QS signal receptors (Fig. 3A).

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Some orphan LuxR proteins, such as QscR in the opportunistic pathogen Pseudomonas
aeruginosa, can respond to self-produced AHLs62–64, while others respond to non-AHL,
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self-produced signals. Two examples of the latter case involve members of the genus
Photorhabdus. One species uses an orphan LuxR protein to detect self-produced α–
pyrones65 and the other detects self-produced dialkylresorcinols and cyclohexanediones
(Fig. 1B)66. Finally, there is a group of orphan LuxR homologs in some plant-associated
bacteria that activate transcription of specific genes in response to small molecule(s)
produced by the plant67–69. The identity of these small molecules remains elusive and they
may in fact be produced by the plant microbiota. The orphan LuxR homologs and what we
now know about them bring this research area to an interface with advances in microbiome
research, and studies of this group of LuxR homologs represent a rapidly emerging area.

Advances in mammalian gut microbiome research are beginning to reveal how QS can
influence microbiome species composition, how QS research might lead to ways to control
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infectious diseases like cholera, and how the host itself may have evolved mechanisms to
affect bacterial QS to shape its microbiome. For example, the AI-2 molecule produced by
many bacterial species, and discussed earlier, was recently shown to promote Firmicutes
over Bacteroidetes gut colonization70 and production of AI-2 by a gut commensal bacterium
can limit Vibrio cholerae infections71. Although the interactions of bacteria in the
mammalian gut are much more complex than the two-partner squid symbiosis, it is
becoming clear that the involvement of QS in these microbiomes can provide an opportunity
to intervene in gut dysbiosis.

QS, biofilms and microbial biogeography—Biofilms are defined as high-density

bacterial clusters frequently attached to surfaces and encased in an extracellular polymeric
matrix72. Biofilm cells have several unique properties compared to their planktonic (free-
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living) counterparts, notably an enhanced tolerance to antimicrobials. Throughout the 1980s

and 1990s, the prominence of biofilms in nature stimulated development of experimental
laboratory systems. Several groups leveraged these systems for studying QS in biofilm
communities. One common laboratory biofilm system is the flow-cell73, in which biofilms
growing on glass coverslips covering small channels are imaged using a confocal scanning
laser microscope. A nutrient medium is continuously pumped through the channels to feed
the biofilms. Using flow-cell systems, several groups showed that QS can impact biofilm
construction as well as the ability of the biofilm to tolerate antimicrobial treatment74. In
some cases, QS is critical for building a biofilm, whereas in other cases it is important for
biofilm disassembly. The link between QS and biofilm formation led to a flurry of studies to
assess how microbial social behaviors impact this important mode of growth, but it was
quickly discovered that this link was dependent on environmental conditions75. Not
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surprisingly, there is interplay between environmental cues and intercellular communication.

This leads to the question: Is the link between QS and biofilms specific to flow-cell
laboratory conditions or is it relevant in natural ecosystems, and if so, which ecosystems?

We have learned that 3D biofilm architecture in flow-cells often does not mimic biofilm
structure in natural ecosystems76,77. Indeed, some naturally occurring microbial biofilms are
composed of high-density, micron-scale aggregates containing hundreds to thousands of
cells. These aggregates exhibit remarkable micron-scale spatial organization, and this

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biogeography is critical for fitness of the microbial community78,79. The fact that natural
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aggregates share traits with laboratory biofilms, including enhanced antimicrobial tolerance,
led to the hypothesis that QS is involved in aggregate formation. This hypothesis is
supported by studies of several bacteria including Pantoea ananatis, Rhodobacter
sphaeroides, Burkholderia thailandensis, and E. coli. In P. ananatis and R. sphaeroides, QS
inhibits formation of large aggregates via unknown mechanism(s), while aggregate
formation is promoted by QS in Burkholderia thailandensis and E. coli80,81. In the case of E.
coli, aggregate formation is promoted via active movement of individual cells towards
aggregates excreting AI-281.

Although QS appears to play some role in aggregate formation, is it also important for the
precise spatial organization of aggregates, and how does aggregate formation influence QS?
To answer these questions, it is not only necessary to understand the relationship between
aggregate size and QS-controlled behaviors but also the effective ‘calling distance’ of signals
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produced by an aggregate. Elucidating the number of cells required to reach a quorum has
been actively pursued over the past 10 years. As predicted by the QS hypothesis, confining
single bacterial cells in femto- to picoliter aqueous volumes has provided evidence that
single cells can QS, although it is not clear whether there are fitness benefits to doing so82,83
(for reference the volume of an E. coli cell is on the order of about a half to one or two
femtoliters). However, these closed systems do not allow exchange of solutes outside the
confinement volume, which ultimately results in lack of robust bacterial growth. As
microfluidics and laser printing technologies have continued to develop, it has become
possible to address these sorts of questions by confining small numbers of bacteria in
diffusive picoliter-scale hydrogel traps. One such study in which V. harveyi was trapped
showed that aggregates with diameters of about 25 microns demonstrated robust QS while
those ≤10 microns showed little QS-dependent gene expression84. Similar results were
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observed for P. aeruginosa, which had been confined in micro-3D printed bacterial ‘lobster
traps’. As few as 500 P. aeruginosa cells were shown to produce the QS-controlled
exoproduct pyocyanin when confined within 8 picoliter traps, indicating that aggregates of
this size are capable of initiating social behaviors in an open system85.

A next step in understanding bacterial communication revolves around whether aggregates

can communicate with each other: What is the ‘calling distance’ of QS signals, how far can
two aggregates be from each other and still interact? Several theoretical studies have focused
on this question, and the general consensus is that communication at distances greater than
10–100 microns is unlikely86–88. One empirical study provided a cursory set of experiments
aimed at assessing communication between spatially organized aggregates85. This study
employed state-of-the-art laser-printing technology to trap P. aeruginosa in aggregates
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separated by 8 microns. The ability of one aggregate to communicate with another via AHL
QS was assessed for aggregates of different sizes. Signaling across this 8-micron distance
required signal-producing aggregates to contain at least 2,000 cells. Although P. aeruginosa
aggregates greater than 2,000 cells have been observed in natural ecosystems including
chronic infections76,89, most aggregates in these communities are generally smaller and are
often spaced further than 8 microns apart (Fig. 3B). These data suggest that in some
environments, P. aeruginosa QS may primarily function as an intra-aggregate
communication system. The squid-V. fischeri symbiosis provides a unique opportunity to

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address questions of inter-aggregate signaling in a natural host-associate habitat (see Box 2).
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We believe that there is much yet to learn about how micron-scale biogeography influences
how bacteria interact. This will be critical as we begin to manipulate microbial communities
such as the human gut microbiome by influencing QS either chemically or using probiotic
approaches.

Box 2

Biogeography of V. fischeri in a squid light organ

As described in the text the squid-V. fischerilight organ mutualism has proven to be a
useful two-partner model for fundamental studies of quorum sensing in a real-world host-
microbe setting. It is also becoming an important model for biogeography studies
concerning the ability of spatially segregated aggregates to communicate with one
another. The light organ consists of two lobes, and each lobe possesses three crypts.
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Descending into a lobe the crypts become progressively smaller. If the experimenter
presents a new born squid with a mixture of V. fischerimutants or strains in the
surrounding seawater different crypts can be colonized with different mutants or strains.
We know this because V. fischeri can be tagged with genes coding for different
fluorescent proteins and whole light organs can be imaged by fluorescence microscopy
(see image below). AHL QS signals can diffuse through the crypt barriers. Can an AHL
signal produced by a wildtype V. fischeriin one crypt activate the luminescence genes in
an adjacent crypt? Can wildtype in a small crypt activate bacteria in a larger crypt or
perhaps maybe signaling is undirectional depending on crypt size? Can a signal-
producing luminescence mutant activate luminescence by a signal-negative mutant in an
adjacent crypt, and will the luminescent strain compensate for the luminescence mutant
by producing more light itself? The answers to these questions will be fascinating, and
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coupled with laboratory experiments on physically separated aggregates of bacteria will

provide critical information as we begin to understand the social interactions in more
complex ecosystems like the human gut.
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Box 2 Images.
Left, Juvenile squid with infected light organ (boxed). The mantle has beed dissected to
expose the light organ. Right, Fluorescence microscopy of light organ crypts colonized

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with a mix of YFP-labeled V. fischeri FQ-A001 and CFP labeled strain ES114. The three
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crypts in each lobe are outlined and labeled I, II and II. Images provided by T. Miyashiro
Penn State University. Details of this type of experiment have been published
previously112.

Quorum quenching in natural ecosystems and as a therapeutic modality—As

it is clear that many natural microbial communities are polymicrobial and spatially
structured, it is important to consider how ecological interactions between species shape the
evolution of signaling. Important considerations include abiotic and biotic factors that
interfere with QS through degradation of signals (Fig. 3C), a process termed quorum
quenching (QQ)90. Signal degradation can result from the chemical characteristics of an
environment such as pH, or due to the action of enzymes produced by microbes or animals.
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In the latter case, two main types of AHL-degrading enzymes, lactonases and acylases, have
been described. Lactonases hydrolyse the HSL ring of an AHL to produce corresponding
acyl homoserines90, whereas acylases cleave the AHL amide bond generating the
corresponding fatty acid and homoserine lactone91. Determining the impact of QQ on
natural microbial communities, given the complexities of spatial structure and signal calling
distance, remains a key challenge for the future. For example, are QQ enzymes produced for
competition, cooperation or for the private benefit of producing cells? It is notable that
humans also possess lactonases (the paraoxonase (PON) family of enzymes), which are
better known for their ability to hydrolyze organophosphate toxins and low density
lipoproteins92–95. PON lactonase activity is considered to be the ancestral activity92,94, and
Drosophila PONs appear to serve a host-defense function96. It is possible that such enzymes
provide the host a means of manipulating the microbiome through modulation of microbial
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social interactions.

Regardless of the true functions of QQ enzymes, QQ presents an attractive and progressive

route for treating the rising number of antimicrobial resistant infections. A recent report
commissioned by the Wellcome Trust estimated that by 2050, antimicrobial resistance
(AMR) could cause 10 million additional deaths annually, and a cumulative loss to the
worlds GDP of $100 trillion97. QQ enzymes and other QS-blocking approaches do not kill
pathogens but instead block virulence factor production, and such anti-virulence agents have
been proposed to impart less selective pressures that lead to the development of resistant
mutants98 (see Box 1). The basis for QS inhibition as a therapeutic approach dates back to
the 1990s, when a brominated furanone produced by the Australian macro-alga Delisea
pulchra was shown to antagonize AHL-controlled phenotypes in a number of bacterial
species99. Importantly, halogenated furanones have been shown to be effective in vivo,
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resulting in clearance of P. aeruginosa from the lungs of infected mice100. Subsequent

studies identified a number of small molecule inhibitors of AHL QS, many of which have
potent activity against several bacterial pathogens laboratory culture99–104.

What diseases might be treated by QS inhibition? Obviously, we would like to be able to

treat infections that are not resolved by current therapies. The chronic P. aeruginosa lung
infections, which plague people with the genetic disease cystic fibrosis (CF), have been an

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 Whiteley et al. Page 12

inviting target, and targeting chronic P. aeruginosa infections presents a clear example of
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why fundamental research and therapeutic development are co-dependent and must proceed
hand-in-hand. These chronic infections are not resolved by antibiotics, and the chronic
microbial communities residing within the CF lung are comprised of bacterial aggregates89.
Because QS activates a battery of P. aeruginosa virulence factors, and P. aeruginosa QS
mutants have reduced virulence in animal models, LasR, the master QS signal receptor
became a focus of small-molecule inhibitor screens103,105,106. Subsequent ecological studies
showed that many patients harbor P. aeruginosa LasR mutants107,108. These ecological
studies were of course discouraging, but further investigation showed most LasR mutant CF
isolates have co-opted a second QS system, the RhlR system, to replace LasR in ways,
which remain unclear109. Perhaps a RhlR inhibitor or an inhibitor that targets both RhlR and
LasR might be an appropriate CF therapeutic, and one has recently been reported94. The
lesson is that we need continued basic research about QS in natural human habitats to know
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how, when or what to target with a QS inhibitor.

In conclusion, there has been an explosion of activity on bacterial QS and the field continues
to expand rapidly. One area ripe for advancement involves complex adaptive microbial
communities. QS likely controls behaviors critical for development and success of these
communities in diverse environments like the human gut microbiome and chronic infections
of humans. Elucidating the roles and functions of QS in natural ecosystems requires a
continued willingness to embrace the complexity of these microbial communities and
incorporate systems level ecological principles. We will continue to see incorporation of the
fundamental biology of bacterial social dynamics into thinking about how to interfere with
certain infectious diseases, and there will be a continued use of bacteria to understand the
biology of communication and sociality. We have come to understand that there are many
different small molecule-dependent interactions between microbes and between microbes
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and their hosts. There are certainly more to be discovered and there is an opportunity to sort
out fundamental differences between these diverse systems. Understanding these issues will
be critical as we move towards translating basic studies of QS to meet future needs including
functional studies of the human microbiome.

Acknowledgments
We acknowledge support for of our research programs from US Public Health Service (USPHS) Grants GM59026
and P30DK089507 (to EPG), and R01GM116547 and NIH R01DE023193 (to MW).

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Fig. 1. Canonical QS and the chemical diversity of signals

(A) Quorum sensing in Vibrio fischeri. LuxI produces 3O-C6-HSL (AHL, yellow spheres),
which specifically interacts with the LuxR transcriptional regulator when it reaches
concentrations in the nM range. This leads to expression of the luxICDABE operon and
bioluminescence. (B) Examples of quorum sensing signals from Gram-negative and Gram-
positive bacteria. (i) AHL, N-acyl homoserine lactone; (ii) 3-Hydroxy-AHL, N-(3-
hydroxyacyl)homoserine lactone; (iii) 3-oxo-AHL, N-(3-oxoacyl)-L-homoserine lactone. R
can be a fatty acyl group of 4–18 carbons with or without one unsaturated carbon-carbon
bond, the terminal carbon can be branched and some R groups are aromatic acids (p-
coumaric acid or cinnamic acid); (iv) The V. harveyi AI-2, autoinducer-2, furanosyl borate
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ester form; (v) PQS, Pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4(1H)-quinolone;

(vi) DSF, diffusible factor, methyl dodecenoic acid; (vii) PAME, hydroxyl-palmitic acid
methyl ester; (viii) Autoinducing peptide 1 (AIP-1) from Staphylococcus aureus.
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Fig. 2. Social cheating in QS populations

Bacterial cells act as cooperators (green) when secreting QS-dependent public goods (e.g.
protease, red spheres) into the surrounding environment and this imposes a fitness cost on
individual cells. Cheater cells (yellow) do not secrete these enzymes and pay no fitness
costs. Cheats can benefit from the action of public goods and therefore gain a fitness
advantage in mixed populations with cooperators. Orange halos depict a nutrient source
liberated by a public good (e.g. protease) from which all cells (cooperators and cheats) can
benefit.
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Fig. 3. Factors that impact QS in natural environments

(A) LuxR orphans (solos) can detect and respond to signals produced by eukaryotic hosts,
self, or other microbial species. (B) Top: Aggregates of two P. aeruginosa strains (green,
strain PA14 and red, strain PAO1) in synthetic cystic fibrosis sputum 101. Bottom: Diagram
of P. aeruginosa aggregates (3 clusters of red rods) spatially separated and socially isolated
(yellow halos represent QS signals and QS-controlled exoproducts). (C) Top: Diagram of a
high-density aggregate of QS positive cells (green) secreting a diffusible QS signal into the
surrounding environment (orange halo). The signal can activate QS in a nearby cells (green)
but not in more distant cells (yellow). Bottom: quorum quenching (orange stars), either by
the action of enzymes or due to environmental conditions, degrades the QS signal and limits
the ability of an aggregate to induce QS in nearby cells.
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