254
Is there a role for quorum sensing signals in bacterial biofilms?
Staffan Kjelleberg* and Soeren Molin†
Bacteria form multicellular biofilm communities on most
surfaces. Genetic analysis of biofilm formation has led to the
proposal that extracellular signals and quorum-sensing
regulatory systems are essential for differentiated biofilms.
Although such a model fits the concept of density-driven
cell–cell communication and appear to describe biofilm
development in several bacterial species and conditions,
biofilm formation is multifactorial and complex. Hydrodynamics,
nutrient load and intracellular carbon flux have major impacts,
presumably by altering the expression of cellular traits essential
for bacterial adaptation during the different stages of biofilm
formation. Hence, differentiated biofilms may also be the net
result of many independent interactions, rather than being
determined by a particular global quorum sensing system.
Addresses
*Department of Microbiology and Immunology, School of
Biotechnology and Biomolecular Sciences, Centre for Marine
Biofouling and Bio-Innovation, The University of New South Wales,
Sydney 2052, Australia; e-mail: S.Kjelleberg@unsw.edu.au
† Section of Molecular Microbiology, BioCentrum-DTU, The Technical
University of Denmark, Building 301, DK-2800 Kongens Lyngby,
Denmark; e-mail: imsm@pop.dtu.dk
Current Opinion in Microbiology 2002, 5:254–258
1369-5274/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 13 May 2002
Abbreviations
AHL acylhomoserine lactone
QS quorum sensing
Introduction — is biofilm development
a genetically controlled pathway?
The realisation that, to a large extent, bacterial life in most
natural environments involves surface attachment and
development of biofilms has stimulated considerable
interest among many groups of microbiologists, initially
in connection with the engineering of bioprocesses in
reactors of different types and in the field of microbial
ecology, and lately as a new research field for microbiologists
with special interest in microbial physiology, differentiation
and global gene control. Much interest and rapid progress
in biofilm research followed the publication of a paper by
Davies et al. [1], in which a correlation between biofilm
development and cell–cell signalling in Pseudomonas
aeruginosa was demonstrated. Subsequently, an increasing
number of reports on the dissection of genomic functions
affecting biofilm performance and properties (see, for
example, [2–5]) have led a majority of scientists studying
bacterial biofilms to adopt a consensus model that proposes
that: first, microbial community development on surfaces is
a step-wise process involving adhesion, growth, motility and
EPS formation; and second, quorum sensing (QS)-mediated
controlling signals are directly involved as biofilm
development regulators in a stringently controlled
differentiation process.
The aim of this review is to assess the merit of the recently
proposed model for QS-mediated biofilm formation. We
compare and evaluate observations and data that involve
not only the QS model but also the notion that biofilm
formation and differentiation are multifactorial processes and
reflect the activation of additional genetic control systems.
Moreover, we identify questions that, if experimentally
addressed, provide a guide for further exploring the impact
of QS-based regulation of bacterial biofilm development.
The AHL-based quorum sensing system and
biofilm formation
In bacteria, the regulation of many important changes in
gene expression is mediated by systems of signalling
between cells known as QS cells [6]. Cells in a population
will sense their density and number through the presence
of signals that diffuse freely across cell membranes and
between cells. Via an autoinduced positive feedback
mechanism, a population of cells can quickly induce the
appropriate phenotypes required for responding to a
particular environmental condition or for proceeding with
the differentiation process of the population. A limited
number of QS systems, mainly the acylhomoserine lactone
(AHL) system of several Gram-negative bacteria [7], the
autoinducer 2 (AI2) system of a range of both Gram-
negative and Gram-positive bacteria [8], and the peptide-
mediated QS signalling system in several Gram-positive
species [9], have been characterised in some detail.
In contrast to the other QS systems, the AHL-mediated
QS signalling system appears to control genes essential for
colonisation of eukaryotes across a large number of bacterial
species. This process is facilitated by a high density of
cells, such as in bacterial biofilms [10]. The notion that
signal-producing cells in close proximity to each other
would acquire a higher intracellular concentration of the
QS signal and, hence, allow for induction of signalling-
controlled genes led to the first detailed set of experiments
that showed that the formation of differentiated multicellular
microcolonies in bacterial biofilms can be mediated by AHLs
[1]. A functional Las QS regulatory switch in P. aeruginosa
was found to be essential for formation of a highly structured
biofilm community. Recently, the AHL-dependent QS system
in P. aeruginosa was also found to govern biofilm formation
on living surfaces, as evaluated in a murine model of
corneal infection ([11]; H Zhu, personal communication).
Concurrent with and apparently in support of such
findings, reports in the literature also provided evidence for
the presence of AHLs in both mixed- and monoculture
species biofilms on inanimate surfaces [12–14]. Also, claims
 Is there a role for quorum sensing signals in bacterial biofilms? Kjelleberg and Molin
Figure 1
(a)
(c)
Scanning confocal microscope images of a 72-hour flow cell biofilm
formed by (a) the Serratia liquefaciens wild-type strain and (b) the
AHL mutant strain. Diagrammatic representations are given below
for (c) the wild-type strain and (d) the AHL mutant strain (swrI–).
A difference is observed with respect to morphology, biofilm structure
and differentiation under flow cell conditions. The wild-type strain coats
the surface and bacteria position themselves vertically above this
for the role of AHLs in affecting several aspects of biofilm
dynamics, such as heterogeneity, architecture, stress
resistance, maintenance and sloughing, have been made
[1,15–18]. Comparisons between wild-type and mutant
strains lacking AHL signals have demonstrated that
multicluster-containing biofilms on inanimate surfaces in
Aeromonas hydrophila [19•], Burkholderia cepacia [20•] and
Serratia liquefaciens [16] also require an intact AHL system.
Moreover, a detailed analysis of cell differentiation in
S. liquefaciens revealed dramatic differences in intercell
arrangements and differentiation into cellular morphotypes
for anchoring the biofilm and assembling cells at specific
sites (Figure 1). In S. liquefaciens, the requirement of the QS
system for the regulation of all key steps in surface coloni-
sation appears to be met. AHL-controlled genes essential
for adhesion, swarming and biofilm cluster formation were
found in this species ([16]; M Labbate, personal communi-
cation). Interestingly, expression of the cytotoxic lectins
PA-IL and PA-IIL in P. aeruginosa is also controlled by the
QS system [21]. In support of these findings, which portray
a key role for AHL regulation in colonisation events and
255
(b)
(d)
Current Opinion in Microbiology
coverage. Vertically positioned cells filament to approximately 12 µm
and aggregate bacteria at their tip. Filament cells ranging from
50–200 µm associate with the aggregates and septate to form cell
chains. The AHL mutant is unable to form aggregates at the vertically
positioned filaments and is stalled at this stage of development.
Addition of BHL to the flow cell medium rescues the AHL mutant
biofilm to that of the wild-type (Bar = 10 µm).
biofilm formation, halogenated furanones, which are
produced by a marine red alga to protect the surface against
colonisation by bacteria and higher sessile organisms and
specifically interfere with AHL systems in bacteria [22–24],
have been shown to penetrate biofilms, shut down the
signalling system and induce sloughing of biofilms both on
inanimate surfaces [25•] and in vivo in the lung tissue
(M Givskov, personal communication).
These observations suggest that, for several organisms
known to produce AHLs, it is often possible to identify
differences between biofilm development and performance,
when comparing the wild-type strain with isogenic variants
deficient in the synthesis of the QS signal molecules. The
suggested connection between QS regulation and biofilm
properties is appealing, considering that, in natural environ-
ments, biofilms would be the dominant setting in which
QS regulation plays a significant role.
It appears, however, that although the mechanism of
regulation exerted by QS systems is relatively conserved
256 Ecology and industrial microbiology
Figure 2
(a) 1.0
0.8
Roughness
0.6
0.4
0.2
0.0
2 4 6 8 10 12 14 16 18 20
Average thickness (µm)
Quantification of flow cell biofilm structures for four strains of
Pseudomonas aeruginosa using the COMSTAT programme [38].
Roughness versus average thickness of (a) 98-hour- and (b) 146-hour-old
biofilms of P. aeruginosa wild-type (black circle), P. aeruginosa rpoS
(white inverted triangle), P. aeruginosa ∆pilHIJK (type IV pili mutant,
black square) and P. aeruginosa las I (white diamond). Images were
acquired by CLSM in three independent biofilm experiments and
across the different QS-containing bacterial species,
different bacterial species employ the QS control system
as a global regulator of operons encoding different
functionalities, including a series of non-household traits
[26]. Hence, mutations in the QS control system will have
pleiotropic effects with potentially complex consequences
for the expression of many genes. Although these effects
will be different in different species, given the many
genes and pathways involved, it is possible that they will
be different also for the same strain grown under different
sets of conditions. Thus, since the first reports on
AHL-mediated formation of differentiated biofilms and
the presence of AHLs in biofilms, conflicting data on the
role of extracellular signals and QS in governing the
formation and function of biofilms have been published
[27•,28,29,30•]. Common to these studies is the fact that
significant differences between wild-type and isogenic
AHL mutants with respect to biofilm development could
not be detected within the chosen experimental framework.
The role of QS in biofilm development and maintenance,
which require a series of genes for behavioural responses
such as adhesion, motility, chemotaxis, exopolysaccharide
or capsule production and stress resistance, is therefore
difficult to ascertain. These experiments further lead to
the suggestion that a clear-cut case for the role of QS
systems in biofilms requires a high-level regulatory function
and that the QS circuit always controls the expression of
biofilm-essential genes.
(b) 0.7
0.6
0.5
Roughness
0.4
0.3
0.2
0.1
0.0
0 5 10 15 20 25 30
Average thickness (µm)
quantified by the computer program COMSTAT. In each of the three
rounds, 18 image stacks were acquired from two flow-channels totalling
54 image stacks for each strain at each time-point. Each spot represents
a single stack of images. It is clearly seen from these quantifications that
the wild-type strain and the lasI mutant form biofilms with identical
structures, whereas the rpoS and type IV pili mutants show significantly
different biofilm development. Adapted, with permission, from [27•].
Biofilm development — the processes and
their regulation
The attention created by the report by Davies et al. [1] is in
striking contrast to the apparent lack of attention created by
a number of publications that arrived at the one general
conclusion that biofilm development (understood as formation
of heterogeneous communities and structures, as demon-
strated by Davies et al. [1]) is a predictable consequence of
the physicochemical conditions in the biofilm environment
[31,32]. The latter publications concluded that employment
of relatively simple mathematical models, such as the
“cellular automaton” [33], combined with changing nutrient
conditions would lead to predictions of biofilm behaviour
and development. In other words, biofilm differentiation
into mature biofilms of “organized communities with
functional heterogeneity” [10] does not necessarily require a
genetic programme, but may in fact constitute the sum of a
large number of cellular adaptations and growth cycles influ-
enced by the nutrient diffusion conditions in the community.
Moreover, by varying the nutritional and flow-dynamic
conditions, biofilm development should follow different
routes. In this model, the bacterial cells do not perceive
that they are within a biofilm — they simply respond to
local environmental conditions (such as stress and nutrient
gradients) as would planktonic cells.
From a physiological point of view, it should be recalled
that bacteria possess genetically determined response
 Is there a role for quorum sensing signals in bacterial biofilms? Kjelleberg and Molin
pathways enabling them to survive and even thrive under
conditions of nutrient limitation or deficiency and other
kinds of stress conditions (such as osmotic stress, anoxia
and pH changes). Many complex regulatory pathways,
such as those governing catabolite repression [34], stationary-
phase gene expression [27•,35], motility and chemotaxis
[36], have been shown to play important roles for biofilm-
associated bacteria and for biofilm performance and
development, even though these control systems are
probably not exclusively evolved for biofilm development
(Figure 2). In a recent report [37••], the CsrA regulator
responsible for controlling carbon flux and storage in
Escherichia coli was shown to be a major determinant of
biofilm formation and resolution under a variety of cultivation
conditions. Thus, the challenges of the diverse and
complex environmental conditions, most often associated
with biofilm life, require that the bacteria exploit their
entire repertoire of regulatory systems when adapting to
these changing conditions. It is likely, therefore, that more
than one control system is needed to develop stable and
physiologically optimal communities, which prevail
because of the regulatory flexibility.
Is quorum sensing regulation a niche-specific
biofilm control circuit?
Biofilm establishment and development is a complex
multifactorial process, which is governed by a combination
of the environmental conditions and the resulting cellular
household functions, as well as a range of specifically
controlled activities that are highly influenced by genetic
control systems operating under high-cell-density condi-
tions. In many cases, QS regulation has evolved as niche-
specific control circuits operating, in particular, in biofilm
scenarios in which cell densities are high. In addition to
controlling the expression of niche-related factors (for
example, those related to host–microbe interactions), QS
systems may also have direct impacts on the quality and
properties of the community that produces the signals, that
is, as a feedback loop operating at the community level
to ensure a stable and robust continuation of related
functionalities. However, it may be an oversimplification
to interpret this control as the dominant, let alone exclusive,
route for biofilm development. It is, therefore, pertinent to
address some of the important questions, outlined in
Box 1, that are still unresolved.
Conclusions
The title of this review poses the question: is there a role
for QS signals in bacterial biofilms? The current state-of-
the-art in biofilm research suggests that it is highly likely
that QS regulation is important for biofilm development
for several organisms under certain sets of conditions, but
that there is no reason to assume that this type of regulation
is the only important effector. In a given setting, the
biofilm-associated community will exploit all available
adaptive mechanisms and the corresponding network
of regulatory activities (including QS), and it is not
possible to unequivocally assign a specific determining
257
Box 1
Important questions to be resolved.
Is it the case that biofilms develop in discrete steps? If so, how are
these defined functionally and at the level of genetic control?
If QS is shown to have an impact on biofilm development, which
target genes are directly involved in discrete events in the
development of the biofilm?
Is QS-mediated control of biofilm performance for a given organism
unconditional, or are there experimental conditions under which no
apparent correlation exists?
What proportion of QS-regulated genes in specific organisms is
controlled directly (for example, indicated by the presence of a ‘lux’
box), and how many of the genes may be indirectly controlled
because of pleiotropic effects?
Are there any examples of biofilm development that resemble that
of P. aeruginosa and of others that are completely independent of
QS or any other global regulation?
regulatory factor for the structure/function relationship
of the community.
Acknowledgements
The constant inspiration from our colleagues at the University of
New South Wales and the Technical University of Denmark has been
instrumental in this writing and, in particular, we wish to thank Michael
Givskov and Peter Steinberg.
References and recommended reading
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• of special interest
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