FEMS Microbiology Letters 236 (2004) 163173

www.fems-microbiology.org

MiniReview

What drives bacteria to produce a biolm?

Kimberly K. Jeerson

The Channing Laboratory, Brigham and Womens Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA
Received 26 March 2004; received in revised form 1 June 2004; accepted 3 June 2004
First published online 15 June 2004

Nearly 40 years ago, Dr. R.J. Gibbons made the rst reports of the clinical relevance of what we now know as bacterial biolms
when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans [Sci. Am.
238 (1978) 86]. As the clinical relevance of bacterial biolm formation became increasingly apparent, interest in the phenomenon
exploded. Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fundamental questions remain. The intent of this review is to consider the reasons why bacteria switch from a free-oating to a biolm
mode of growth. The currently available wealth of data pertaining to the molecular genetics of biolm formation in commonly
studied, clinically relevant, single-species biolms will be discussed in an eort to decipher the motivation behind the transition from
planktonic to sessile growth in the human body. Four potential incentives behind the formation of biolms by bacteria during
infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area
(colonization), (3) utilization of cooperative benets (community), (4) biolms normally grow as biolms and planktonic cultures
are an in vitro artifact (biolms as the default mode of growth).
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Biolm; Genetics; Microbial communities

1. Introduction
Genetic adaptation is the cornerstone of tness and
survival and can ensue from mutations and recombination within genes, acquisition of new genetic material,
or from the regulated expression of existing genetic
material. Flexibility in bacterial gene expression permits
survival in environments with rapidly changing conditions, and bacteria, being particularly adaptable, have
ourished in nearly every environmental niche on our
planet. Bacterial species that are able to colonize humans are especially creative in their regulatory processes. Many pathogenic and commensal bacteria are
capable of transitioning between life in the environment
and in the human host, and all must be able to adapt to
sudden shifts in nutrient availability as well as to primary and secondary host immune defenses. One par*

Tel.: +1-617-525-2680; fax: +1-617-731-1541.

E-mail address: kjeerson@rics.bwh.harvard.edu.

ticularly important and clinically relevant example of

bacterial adaptation through systematized gene expression is the ability to grow as part of a sessile, exopolymer-enshrouded community referred to as a biolm.
Scientic interest in the process of bacterial biolm
formation has erupted in recent years and studies of the
molecular genetics of biolm formation have begun to
shed light on the driving forces behind the transition to
the biolm mode of existence.
It is now recognized that biolm formation is an
important aspect of many, if not most bacterial diseases,
including native valve endocarditis, osteomyelitis, dental
caries, middle ear infections, medical device-related infections, ocular implant infections, and chronic lung
infections in cystic brosis patients [2]. Established
biolms can tolerate antimicrobial agents at concentrations of 101000-times that needed to kill genetically
equivalent planktonic bacteria, and are also extraordinarily resistant to phagocytosis, making biolms
extremely dicult to eradicate from living hosts [3].

0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.06.005

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Abstract

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K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

quired for biolm formation. The formation of a biolm

in turn alters the microenvironment of its own inhabitants which then leads to additional alterations in gene
expression and further maturation of the biolm, and so
on. Complicating the study of gene expression in biolms even further, biolm inhabitants are heterogeneous. Disease-related biolms can be multi-species or
even multi-kingdom, such as the biolms involved in
tooth decay, or single-species such as those involved in
endocarditis, but even bacteria within single-species
biolms are heterogeneous with respect to gene expression. This is due to diusion limitations imparted by the
biolm, which result in local variations in pH, nutrient
and oxygen availability, and concentrations of bacterial
metabolites. On top of all of these complications, biolm studies are confounded by the intrinsic limitations
of the in vitro biolm models and the techniques available to study the roles of these genes.

Table 1
Genes required for biolm formation
Gene

Protein/function

Species

References

Adhesion
abpA
sspA/B
gbpA
tarC
icaADBC
hla
clfA
dltA
atlE
aap
bopABCD
esp
agn43

Amylase binding
Human salivary protein and collagen binding
Polysaccharide formation
Regulator of glucosyltransferase S and glucan binding protein
Intercellular adhesin synthesis
Hemolytic toxin
Clumping factor A, brinogen binding protein
D -alanine esterication of teichoic acids
Autolysin/adhesin
Accumulation associated protein
Biolm on plastic surfaces operon
Enterococcal surface protein
Antigen protein involved aggregation

S. gordonii
S. gordonii
S. mutans
S. mutans
S. aureus, S. epidermidis
S. aureus
S. aureus
S. aureus
S. epidermidis
S. epidermidis
Enterococcus faecalis
E. faecalis
E. coli

[7]
[7]
[7]
[50]
[8]
[51]
[52]
[53]
[8]
[54]
[55]
[56]
[57]

Quorum sensing
comX
comABCDE
luxS?
lasI

Competence
Competence
Quorum sensing
Synthesis of 3OC12-HSL quorum-sensing signal

S. gordonii
S. mutans
S. mutans
P. aeruginosa

[58]
[12]
[33]
[35]

Cell wall
PBP2B
PBP5
glmM
bacA
brpA

Peptidoglycan synthesis
Peptidoglycan synthesis
Peptidoglycan synthesis
Peptidoglycan synthesis
Possible regulator of autolysis

S.
S.
S.
S.
S.

gordonii
gordonii
gordonii
gordonii
mutans

[7]
[7]
[7]
[7]
[34]

Metabolism
ccpA
crc

Carbon catabolite control protein

Global carbon metabolism regulator

S. mutans
P. aeruginosa

[34]
[59]

Stress response
dgk
rB ?
purR
rpoS?
mutT

Stress response regulator, lantibiotic regulator

Alternate sigma factor-stress response
Regulator of purine synthesis, metabolism
Regulator involved in slow growth
DNA mismatch repair

S. mutans
S. aureus, S. epidermidis
S. epidermidis
E. coli
S. gordonii

[12]
[13,14]
[60]
[15,18]
[7]

Plasmids
tra

Conjugative pilus of F plasmid

E. coli

[41]

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Consequently, biolm-related infections that appear to

respond to a therapeutic course of antibiotics may relapse weeks or even months later, making surgical removal and replacement of the infected tissue or medical
device a frequent and unfortunate necessity.
The serious and pervasive clinical impact of bacterial
biolms has inspired many researchers to investigate the
regulatory mechanisms behind their formation and dissolution, with the ultimate goal of pinpointing specic
targets for chemotherapeutic agents. A number of target
gene-directed as well as global proteomics- and genomics-based studies have lead to the identication of a
plethora of genes associated with biolm development.
Sorting out the roles of all of these genes is however, an
exceedingly complex task. For one thing, the regulatory
processes of biolm elaboration are cyclical and dynamic. In other words, external conditions trigger
alterations in the expression of a subset of genes re-

K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

Despite the complications associated with the study

of biolms, comparative and comprehensive analysis of
all of the existing data can enable one to elucidate
congruencies and subsequently, globally signicant

165

truths become unveiled. Tables 1 and 2 present a compilation of genes from various clinically relevant bacteria that have been implicated in the biolm mode of
growth. Table 1 lists genes that appear to be required for

Table 2
Gene expression UP or DOWN in biolms with respect to planktonic cells
Protein/function

Regulation

Species

References

Adhesion
algC
wcaB
csgA
clfA
scaA
abpA
rggD
int/CoA
pA
has

Alginate synthesis
Colanic acid synthesis
Curli
Clumping factor/brinogen binding
Co-aggregation
Amylase binding
Glucosyltransferase inducer
Intrageneric co-aggregation-relevant adhesin
Fibronectin-binding
Streptococcal hemagglutinin

UP
UP
UP
DOWN
DOWN
DOWN
UP
DOWN
DOWN
DOWN

P. aeruginosa
E. coli
E. coli
S. aureus
S. gordonii
S. gordonii
S. gordonii
S. gordonii
S. gordonii
S. gordonii

[6]
[5]
[61]
[62]
[63]
[63]
[63]
[63]
[63]
[63]

Quorum sensing
pA4296
Probable two-component response regulator
comD,E
Competence factors

DOWN
UP

P. aeruginosa
S. mutans

[19]
[64]

Cell wall
mreC
dltA
ddl

UP
DOWN
DOWN

P. aeruginosa
S. gordonii
S. gordonii

[19]
[63]
[63]

Stress response
rpoH
Stress/stationary phase s factor
rpoS
Heat shock s factor
proU
Transport-osmotic adaptation
DnaK: Protein folding(heat shock)
Grpe: folding(heat shock)
60 kDa chaperonin: heat shock
htgX
Putative heat shock protein

UP
DOWN
UP
UP
UP
DOWN
DOWN

P. aeruginosa
P. aeruginosa
E. coli
S. mutans
S. mutans
S. mutans
S. gordonii

[19]
[19]
[5]
[65]
[65]
[65]
[63]

Carbohydrate metabolism
Fructose bisphosphate aldolase
Pyruvate kinase
6-Phospho-B-galactosidase
pbg
Phospho-b-glucosidase
D -ribose-binding periplasmic protein
P02925
D -galactose-binding protein
P02927
Q6150
Malate dehydrogenase

DOWN
DOWN
DOWN
UP
UP
UP
UP

S. mutans
S. mutans
S. mutans
S. gordonii
E. coli
E. coli
E. coli

[65]
[65]
[65]
[63]
[66]
[66]
[66]

minicell associated protein Div IVA: cell division

FTSZ: septum formation
ATP-dependent DNA helicase RECG: DNA
replication
H-NS: DNA binding

UP
UP
DOWN

S. mutans
S. mutans
S. mutans

[65]
[65]
[65]

UP

E. coli

[66]

Probable mbrial protein

Pilin protein
Flagellar basal-body rod modication protein
Flagellin type B
Flagellar capping protein
Flagellar hook protein
Flagellar synthesis

DOWN
DOWN
DOWN
DOWN
DOWN
DOWN
DOWN

P.
P.
P.
P.
P.
P.
E.

aeruginosa
aeruginosa
aeruginosa
aeruginosa
aeruginosa
aeruginosa
coli

[19]
[19]
[19]
[19]
[19]
[19]
[5]

Coat protein of bacteriophage Pf1

Helix destabilizing protein of bacteriophage Pf1
Probable coat protein of bacteriophage Pf1

UP
UP
UP

P. aeruginosa
P. aeruginosa
P. aeruginosa

[19]
[19]
[19]

Cell morphology
D -Alanine-D -Alanyl

carrier
D -Alanine: D -Alanine ligase

Division

Motility
PA2128
pilA
gD
PA1092
iD
gE
iC
Phage

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Gene

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K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

biolm formation (the cause genes) and Table 2 lists the

genes that are dierentially regulated in an established
biolm (the eect genes). Answers from many questions
can be extricated from trends apparent in these tables.
For example, the conspicuous presence of surface adhesions in Table 1 addresses the question How do
biolms adhere to solid surfaces?. Answering such
mechanistic questions is, inarguably, critical to our understanding of the biolm mode of growth. However,
excellent reviews dedicated to understanding the
How? of biolm formation have already been published [2,4], and I would like to venture instead, to
address the fundamental, albeit somewhat philosophical
question Why do bacteria form biolms in the human
host?.

According to Darwins theory of evolution, the only

true driving force behind the course of action of any
organism is reproductive tness. Any action that increases proliferation will endure within a species.
Therefore, when we talk about the driving force behind
biolm formation we are asking the question How does
the biolm mode of growth promote survival and
propagation of the cell? It almost seems counter-intuitive that the biolm mode of growth could confer a
reproductive tness advantage when one considers that
biolm bacteria have a reduced rate of growth relative
to bacteria growing planktonically in broth culture.
Outside of the laboratory, however, bacteria rarely, if
ever, nd themselves in an environment as nutrient rich
as culture media, and in these less-than-ideal conditions,
there are a number of tness advantages imparted by the
biolm mode of growth. This review proposes four advantages which are illustrated in Fig. 1. The more we
learn about the genetic regulation of biolm formation,
the more we understand about the relative roles of these
benets and about the forces that drive the switch to the
biolm mode of growth.
2.1. Defense: biolm formation as a stress response
Biolms are resistant to physical forces such as the
shear forces produced by blood ow and the washing
action of saliva. Organisms within biolms can withstand nutrient deprivation, pH changes, oxygen radicals,
disinfectants, and antibiotics better than planktonic organisms. Biolms are also resistant to phagocytosis, and
the phagocytes that attempt an assault on the biolm
may actually do more harm to surrounding tissues than
to the biolm itself. The chronic nature of certain infections is inarguably due to the development of a resilient biolm. The invulnerability of biolms is not
completely understood but is likely dependent upon a

Fig. 1. Dr. Sean D. Tavernas artistic interpretation of the four driving

forces behind bacterial biolm formation that are discussed in this
review.

number of biolm-specic characteristics including slow

growth and physiologic heterogeneity of the inhabitants.
Another important trait that forties biolm resistance
is the sticky matrix which may contain DNA and other
polymers but in general, is predominantly composed of
exopolysaccharides.
The important role of exopolysaccharide (EPS) in
both the early and late stages of biolm formation is
exemplied by the conspicuous presence of genes involved in polysaccharide synthesis in Tables 1 and 2. In
Escherichia coli, csgA, which encodes a protein involved
in the synthesis of colanic acid, is involved in aggregation and algC, the gene required for alginate synthesis
plays a role in Pseudomonas aeruginosa biolms [46].
EPS synthesis is important in the development of grampositive biolms as well. Glucan binding protein GbpA
is a glucosyltransferase that has been implicated in sucrose-dependent polysaccharide production and biolm
formation in S. mutans [7]. In addition, the intercellular
adhesin locus (icaADBC) in Staphylococcus aureus and
Staphylococcus epidermidis encodes the gene products
responsible for the synthesis of a b-1-6-linked poly-Nacetylglucosamine polymer called PNAG or PIA
(polysaccharide intercellular adhesin) [8]. Fig. 2 illustrates the important role for the exopolysaccharide
PNAG in the morphology of S. aureus biolms. A weak
PNAG-producing strain retains the simple morphology
of a young biolm whereas a strong PNAG-producing
strain forms tight mushroom-like microcolonies separated by wide channels. In addition to its roles in aggregation and biolm structure, EPS plays a part in
defense, enabling biolms to resist shear forces and

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2. Why do bacteria form biolms?

K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

167

phagocytosis by inammatory cells [2]. Some evidence

suggests that EPS may also be involved in tolerance of
biolms to antimicrobial agents but this is still a matter
of debate [3]. However, if the sole force driving EPS
production and biolm formation is resistance to dangers encountered in the body, then what are the environmental cues that warn bacteria that they need to
bolster their defenses? Certain bacterial species may
have evolved to switch on transcription of genes required for EPS synthesis in response to certain environmental stimuli that are encountered upon host entry,
before the immune system mounts a specic attack [9].
For example, certain stimuli that mirror the physiology
of the human body, such as iron deprivation and
osmotic stress, induce the expression of genes encoding
proteins that synthesize EPS in staphylococci and
enterococci [10,11].
The involvement of stress response regulators in
biolm formation would support the hypothesis that the
motivation behind biolm formation is defense, but the
contribution of these regulators is somewhat unclear. As
indicated in Table 1, studies have implicated the stress
response regulators Dgk, rB , and RpoS, of S. mutans,
S. aureus, and E. coli (respectively) in biolm formation
[1215]. Other studies however, dispute the roles of rB
and RpoS in biolm formation [1618]. In P. aeruginosa
RpoS expression is down in biolms but expression of
RpoH, a sigma factor that has been linked to the stationary phase and stress, is elevated [19]. The role of the
stress response regulators may depend upon the conditions under which biolm formation is triggered or upon
the genetic background of the bacterial cell, again suggesting that what we categorize as a biolm actually
represents a collection of dierent growth states. Bio-

lms are inarguably resilient, but if defense is the major

driving force behind the bacterial mode of growth in the
human body, then why would the bacteria form a sessile
community in such an inhospitable place? In sum, it is
apparent that the stress is not the only trigger for this
mode of growth.
2.2. Colonization: biolm formation as a mechanism to
remain in a favorable niche
Humans and other animals have developed intricate
immune systems for one critical reason: microorganisms are continually trying to inhabit their bodies. The
body, or at least parts of it, is nutrient rich and relatively stable with respect to water content, oxygen
availability, and temperature. Consequently, there is a
never-ending race between the development of the host
immune system and the progression of bacterial strategies to evade it. In some cases, a compromise has been
made, and as such, the body is inhabited by a large
number of commensals, many of which exist as biolms. As the body is obviously an appealing place for
bacteria to live, it may be that the primary motivation
for switching to the biolm mode of growth is to remain xed.
Bacteria have a number of strategies to ensure that
they remain xed in the human body. Bacterial surface
proteins that bind to host extracellular matrix proteins
such as bronectin, brinogen, vitronectin, and elastin
are referred to as MSCRAMMs (microbial surface
component recognizing adhesive matrix molecules) and
often play a key role in initial adherence of bacteria to
solid surfaces within the host [20]. S. aureus is particularly notable for the abundance of MSCRAMMs that

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Fig. 2. Confocal microscopic imaging demonstrates the inuence of exopolysaccharide elaboration on the structure of S. aureus biolms. S. aureus
clinical isolate strain MN8, and the isogenic, constitutive PNAG over-producing derivative strain MN8m were allowed to form biolms on collagencoated glass coverslips for 48 h under ow conditions. The biolms were stained using the BacLight Live/Dead kit which stains live bacteria green
and dead bacteria red. These confocal images demonstrate that the level of PNAG synthesis plays a critical role in biolm structure. MN8 formed a
somewhat unstructured biolm, whereas the PNAG-overproducing strain MN8m formed a highly structured biolm with dense mushroom-shaped
microcolonies separated by large channels.

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K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

phase so that when the organism nds itself in an environment rich in nutrients it can occupy that niche.
With all of the complex mechanisms that pathogenic
and commensal bacteria have evolved to survive in the
human body, it is clear that the benets that we aord
them outweigh the hurdles imparted by our immune
systems.

3. Community: biolms and communal behavior

3.1. Are biolms multicellular organisms?
The idea that bacteria often exist within nature as
biolms rather than individual, free-oating cells was
brought into wide acceptance by the ideas and intuition
of Dr. J. William Costerton [1]. Following the excitement generated by his trailblazing revolution of longstanding scientic dogma, other revolutionary
hypotheses about the nature of bacterial behavior began to emerge. One such hypothesis is that biolms
should be regarded as multicellular organisms and that
biolm bacteria exhibit cooperative, unselsh behavior
[26]. The behavior of bacteria within biolms has even
sparked skepticism about the Darwinian theories of
evolution [27]. Hypothetical challenges to well-accepted
theories are entertaining but without scientic evidence
to back them up they remain purely philosophical, and
there has not yet been an eort to scientically validate
the challenge to our current ideas about evolution.
Recently however, mathematical modeling of biolm
systems and scientic experiments are being designed to
test whether cooperative, altruistic behavior in bacteria
is compatible with the mainstream theory of evolution
[28].
There are indeed similarities between biolm bacteria
and multicellular organisms. For instance, bacteria (including planktonic bacteria) can sense their surroundings, and this enables them to adjust their metabolic
processes to maximize the use of available substrates
and to protect themselves from detrimental conditions.
When bacteria are growing within a biolm, these
changes in gene expression result in phenotypic heterogeneity within the biolm which can be interpreted as
specialization or division of labor similar to cellular
dierentiation seen in multicellular organisms. In addition, bacteria secrete substances referred to as autoinducing signals, which inuence gene expression and may
be a means by which cells communicate with one another. There is even a growing body of evidence that
bacteria exhibit altruistic behavior and can undergo a
process similar to programmed cell death, again suggestive of multicellularity [29]. However, there are fundamental distinctions between bacteria and multicellular
organisms. For example, while bacterial cells can react
and adapt to their environmental surroundings, they do

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it can produce, including clumping factors A and B

(ClfA/B), bronectin binding factors A and B (FnBA/
B), and a collagen binding protein (Cna). S. epidermidis
produces at least two autolysinadhesins that bind to
bronectin and the brinogen binding protein Fbe [8].
Streptococcus pyogenes contains genes for bronectin
(prtF) and brinogen (emm) binding proteins [20]. The
oral streptococci have evolved to bind to the pellicle or
conditioning lm on tooth surfaces which is composed
of salivary glycoproteins and lipids. The salivary agglutinin glycoprotein binding proteins (SspA and SspB
in Streptococcus gordonii and SpaP in S. mutans), and
salivary amylase binding proteins of various oral
streptococci aid in binding to the pellicle on the surface
of teeth [7,21]. Flagella, pili, and mbriae have also
been implicated in adherence of Vibrio cholerae, E. coli,
P. aeruginosa, and Salmonella enterica [22]. Interestingly, Table 2 indicates that once the biolm is established, expression of a number of the adhesins and
motility factors is suppressed. This suggests that the
main role of adhesins, pili, and agella is in initial
attachment, and that once the development of the
biolm has passed this stage, the proteins are no longer
needed and their expression is inhibited. Overall, bacteria produce an impressive array of adhesins that
appear to have evolved as a means to inhabit the human body.
Also in support of the hypothesis that biolm formation is a mechanism for organisms to stay put in the
favorable environment of the human host, is the nding
that carbon catabolite induced gene regulation plays a
critical role in biolm formation (Table 1). Exopolysaccharide expression and biolm elaboration are
markedly enhanced in certain bacteria, including the
pseudomonads, V. cholerae, and E. coli, the staphylococci and the streptococci, when glucose or another
readily utilizable carbon source is abundant [4,2325].
When nutrient sources are depleted, the bacteria detach
and become planktonic, suggesting that nutrient deprivation is a trigger to move on, in search of a better
habitat. Glucose-induced exopolysaccharide production
may be multi-functional. It is possible that glucose
simply serves as a substrate in the EPS synthesis
pathway but studies with S. aureus in our laboratory
suggest that, at least for this organism, this is not the
case. Glucose appears to augment EPS elaboration at
the level of transcriptional regulation rather than at the
level of EPS synthesis [25]. A second possibility, which
supports the biolm as a mode of defense is that bacteria may have evolved to interpret elevated glucose
levels as a cue that it is in the bloodstream, and that it
needs to form a biolm to remove itself from circulation and protect itself from the immune system. Alternatively, polysaccharide production may function as a
mechanism of glucose storage during times of plenty,
and/or as a mechanism to augment the accumulation

K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

3.1.1. Division of the metabolic burden

Diusion limitations imparted by the biolm structure result in local variations in nutrient availability, pH,
and oxygen tension. Therefore, the bacteria within biolms are inevitably heterogeneous with respect to gene
expression. Many biolms are made up of a variety of
bacterial species and some even contain mixtures of
bacteria and fungi. The members of these mixed biolms
have dierent requirements and perform dierent metabolic functions making commensalism a widespread
phenomenon in biolms [26]. For example, whereas
early colonizers of the oral cavity are aerobic or facultatively anaerobic, limited oxygen diusion through the
biolm provides an environmental niche allowing for
later colonization by obligate anaerobes [7]. A study in
which promoter activity was monitored as a function of
the expression of a uorophore indicated that heterogeneity in the gene expression proles of the individual
cells exists even within single-species biolms [30]. It is
likely that this heterogeneity translates into specialized
functions of cells within a biolm [30]. Fruiting body
formation by Myxobacteria is frequently cited as an
example of cell specialization in bacteria and it is an

attractive idea that this phenomenon occurs in other

bacteria as well [26]. Although it has not been denitively proven, the heterogeneity within biolms may
indeed result in a division of labor of sorts and certainly increases the metabolic eciency of the population as a whole.
A popular notion is that such division of labor is
coordinately regulated within biolms through intercellular communication. Autoinducing signals are small
molecules, generally homoserine lactones in gram-negatives and peptides in gram-positives, that are constitutively released by bacteria and which, when present at
a critical concentration, will induce the expression of
certain genes. Autoinducing signals are frequently referred to as quorum-sensing signals because when a
bacterial population reaches a high enough density, the
local concentrations reach threshold levels and alter
gene expression. However, it has not been shown conclusively that bacteria actually respond to the accumulation of a quorum, and it has been suggested that the
more biologically signicant role of the autoinducing
signals is to relay information to the bacterial cell about
local diusion rates [31]. In her provocative review,
Dr. Rosemary Redeld suggests that expression of secreted proteins are induced in the presence of elevated
levels of autoinducing signals not because the bacteria
have evolved to work cooperatively, but because the
benets of secreted proteins are realized by an individual
bacterial cell when local conditions limit diusion and
mixing [31]. One example used is the secretion of a
protease which is required to degrade exogenous proteins so that the bacteria can assimilate amino acids.
Under conditions of reduced diusion or mixing, secreted proteases and the proteins degraded by them
would remain in the vicinity of, and benet the cell. It is
therefore logical that bacteria would restrict expression
of secreted proteins under conditions of high mixing and
diusion. There are also examples of autoinducer eects
that do not readily t this model. The diusion sensing
model suggests that a bacterial cell responds to its own
secreted signals, but recognition of and response to
signals secreted by heterologous species has been welldocumented [32]. It is likely that both diusion-sensing
and quorum-sensing are aspects of autoinducing signals
but a more precise answer incorporating roles for both
of these phenomena awaits further investigation.
While the primary function of autoinducing signals
remains unclear, their role in biolm development is
even more ambiguous. As is indicated in Table 1, one
study found a role for the LuxS system in S. mutans but
two additional studies indicated that LuxS was not required for biolm formation [12,33,34]. Equally confounding results were obtained when dierent
investigators studied the role of the lasRlasI quorumsensing system in P. aeruginosa [35]. Furthermore, there
is evidence that the accessory gene regulator (Agr) which

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not permanently dierentiate. For instance, one can

isolate colonic epithelial cells from a human and grow
them in tissue culture medium, and even though the cells
are suddenly faced with radical changes in their surrounding milieu, they continue to grow as colonic epithelial cells and can even form a polarized monolayer
similar to colonic epithelium. Scientists have devoted
much eort to developing methods to undierentiate
dierentiated cells, but the process of cellular dierentiation, even in simple multicellular organisms, is not
easily reversed by any natural means. This is because
their genetic regulatory patterns have been permanently
altered. If however, you remove bacterial cells from a
biolm and radically change their environmental conditions then they will quickly adapt to their new environmental surroundings and exhibit phenotypic
changes. Depending on the conditions in which they are
grown, they will even convert back to the planktonic
mode of growth. Bacterial cells do not dierentiate, rather they respond to their environmental surroundings
by adapting their gene expression to suit their own needs
for survival. For this reason, it is more accurate to refer
to biolms as interactive communities rather than
comparing them to multicellular organisms. Nonetheless, the community lifestyle is likely an important motivation for biolm formation and provides its members
with a number of benets. In addition to the advantage
of resistance to environmental changes, which is discussed in the defense section, the biolm may benet
from a number of properties of a communal existence
including division of the metabolic burden, gene transfer, and seless behavior.

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K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

3.1.2. Gene transfer

Nucleic acid is the basis for evolution and consequently its one true purpose is to replicate and perpetuate its own specic code. A reproductive tness
advantage will perpetuate the genetic material of an
individual. The same rules apply to obligate infectious
agents such as viruses and plasmids, and as these agents
can not replicate their own genetic material, they would
cease to exist without a means to spread from one organism to another. Bacteriophage and plasmids have
therefore both evolved mechanisms to promote their
maintenance within a bacterium and their spread to
other bacteria. For example, bacteriophage slow down
their replicative machinery and integrate into the bacterial chromosome so that their genome is replicated as
the cell divides. Plasmid replication is an expensive
process for a bacterial cell and plasmids are quickly lost
unless there are required or benecial. Plasmids have
therefore evolved a rather elaborate method for survival.
They carry toxinantitoxin gene pairs which make their
maintenance necessary for bacterial survival [38]. The
plasmid-encoded toxin is a stable protein and the antitoxin is a labile protein. When a bacterial cell divides the
daughter cell inherits both toxin and antitoxin. If the cell
fails to replicate the plasmid as it divides or if the
daughter cell fails to inherit the plasmid, then the supply

of labile antitoxin rapidly declines whereas the toxin

lingers and destroys the plasmid-free daughter cell.
These cunning maintenance methods ensure vertical
transfer of phages and plasmids but in order to be successful, a phage or infectious plasmid must also utilize
horizontal transfer. A biolm is the ideal environment
for horizontal exchange of genetic material [39]. The
close proximity fosters rapid spread of phage as well as
conjugation and uptake of plasmid DNA by competent
bacteria. Plasmids and phage have consequently developed methods to induce the transition to the biolm
mode of growth in their host so that they can spread to
uninfected bacteria and sometimes even to cross the
species barrier [40]. Dr. Jean-Marc Ghigo found that the
pili encoded in a number of conjugative plasmids adhere
nonspecically to solid surfaces and to other bacteria
leading to a dramatic increase in biolm formation of E.
coli and other Gram-negative bacteria [41]. By inducing
the transition to the biolm mode of growth, the plasmid is likely increasing its chances for horizontal
transmission. In addition, a number of phage genes,
including phage coat protein genes, are activated in
P. aeruginosa biolms, supporting the idea that an effective strategy for horizontal phage transmission is to
re-enter the infectious cycle during the biolm mode of
growth [19]. In eect, we may need to consider that
biolm formation benets not only bacterial tness but
the proliferative potential of bacteriophage and plasmids as well.
Horizontal gene transfer within biolms may also
directly benet the bacteria through the exchange of
antibiotic resistance determinants. In S. gordonii, the
expression of competence factors has been implicated as
both a cause and an eect of biolm formation supporting a role for exchange of genetic material in biolms [39,42]. Whether the primary function of
competence factors is to assimilate external DNA as a
means to increase their genetic diversity or simply to use
it as a nutrient source is controversial, but the end result
is that the biolm is an ideal environment for the exchange of genetic material. The motivation of bacteria
themselves as well as plasmids and bacteriophage to
exchange genetic material may all play an important
role in the process of biolm development.
3.1.3. Seless behavior
Overall, bacteria enjoy a number of benets due to
their community mode of growth but are bacteria truly
cooperative and capable of exhibiting unselsh or even
altruistic behavior? Indeed, experimental evidence from
mathematical modeling supports the concept that bacteria can exhibit altruistic behavior [28]. While this may
initially appear to defy the rules of survival of the ttest,
the models indicate that unselsh behavior in biolm
inhabitants can increase the overall growth yield.
Therefore, altruistic bacteria, despite a sacrice in

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is involved in quorum-sensing in the staphylococci actually inhibits biolm formation [36]. A more recent
study suggests that the Agr eect is dependent upon the
ow strength over the biolm and that under static
conditions, Agr reduces biolm formation, under low to
moderate ow it does not aect biolm formation, and
under very strong ow it increases biolm formation
[30]. These results may support the diusion-sensing
theory if one considers that under rapid ow, the autoinducing signals may rapidly diuse out of the biolm.
Overall, a biolm represents both a quorum and
imparts restrictions on diusion, so regardless of whether one accepts the diusion-sensing or the quorumsensing hypothesis, it would seem, at least supercially,
that auto-inducing signals inuence its development. In
parts of the human body however, especially in places
such as heart valves and teeth, biolms are subjected to
strong shear forces which may keep autoinducing signal
levels low. A recent report indicates that in the human
lung, the acyl-homoserine lactone quorum-sensing signal of P. aeruginosa is inactivated by some unidentied
host cell-associated factor [37]. Does this indicate the
dispensability of quorum-sensing and mean that
P. aeruginosa can establish a biolm in the lung despite
immune-mediated inhibition of quorum-sensing, or is
the nding that the immune system targets this signal
suggestive of its importance in biolm formation? The
answer is unclear and the role of quorum-sensing in
biolm formation remains elusive.

K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

a biolm. And yet they are often grown planktonically

in the laboratory.
It is possible that the presence of a suitable substrate
for attachment is all that is required to trigger biolm
formation. There is mounting evidence that immediately
subsequent to the initial adherence of bacterial to a solid
surface, changes in gene regulation begin to occur [4,44].
This suggests that cells actually sense the solid surface to
which they are attached and that this sensing system
triggers a signaling cascade that may lead to some of the
early gene expression patterns necessary for biolm
formation. For example, in P. aeruginosa, expression of
algC, a gene required for alginate synthesis, is increased
within minutes of attachment, and when S. epidermidis
makes contact with a solid surface, the normally
spherical cell forms a leg-like appendage [45,46]. These
ndings suggest that, similar to eukaryotic cells, bacterial cells possess surface-sensing systems that induce
intracellular signals powerful enough to result in transcriptional and morphologic changes.
The sensing mechanisms utilized by bacteria to detect
adherence are not well understood. Changes in the
perceived osmolarity caused by charges on solid surfaces
may be an important cue for bacteria to recognize surfaces [22]. The EnvZOmpR two-component system
which is involved in sensing environmental osmolarity,
has also been shown to regulate expression of curli and
colanic acid [47,48]. The brillar surface structure curli
plays a role in adherence and colanic acid is an exopolysaccharide involved in aggregation. The role of osmolarity in biolm regulation has also been noted in
staphylococci [13] and Pseudomonas uorescens [49].
Overall, the biolm mode of growth may be the default
mode of growth for at least some bacterial species suggesting that we should be questioning what triggers the
planktonic mode of growth rather than what motivates
the biolm mode of growth.

5. Conclusions
4. The biolm as the default mode of growth
In the laboratory, bacteria are generally grown
planktonically, but the utopian microcosms created in
culture vessels are designed to maximize bacterial
growth rates, not to replicate natural growth conditions
of the bacteria. In fact, some bacterial species appear to
constitutively utilize the biolm mode outside of the lab.
The oral streptococci are very highly adapted to sessile
growth on the surface of teeth. Most of the oral bacterial
species lack an environmental niche and are found almost exclusively within the mouth [24]. For these bacteria, planktonic growth would cause them to be quickly
washed away by saliva, swallowed and destroyed within
the acidic juices of the stomach. These bacteria likely
spend the majority of their natural existence growing as

Scientic interest in biolms has exploded in the past

decade. This fascination with biolms is due in large
part to their presumed clinical relevance. But it is also,
undeniably, due to the appeal of projecting traits of
higher organisms on these life forms that were once
thought to be so simple and autonomous. The ability of
prokaryotes to adapt to their surroundings is indeed
remarkable, but whether they actually communicate,
coordinate, and specialize within biolms for the benet
of the community, as opposed to simply reacting to their
environments in order to selshly promote their own
survival, has not yet been suciently established. Unfortunately, we often make the erroneous conclusion
that certain questions are purely philosophical and can
not be tested scientically. In fact, such questions can

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growth rate are, under certain conditions, actually more

t. Results from these studies also suggest that altruism
is more ecient when the bacteria exist in small, clonal
clusters, perhaps explaining the microcolony formation
characteristic of biolms. Fitness need not entail aggression, benecial symbiotic relationships can increase
yield, and Darwins theories about evolution through
competition and tness can be aptly applied to unselsh
behavior.
There is mounting evidence that a process similar to
apoptosis may actually occur in bacteria. Homologues
of pro-apoptotic genes such as caspases are widespread
among bacteria [43]. Additionally, several examples of
toxinantitoxin cassettes, similar to those that ensure
plasmid maintenance, have been found within the
chromosomes of gram-negative and gram-positive bacteria, and it has been suggested that programmed cell
death may occur within the biolm community [38]. One
rationalization for altruistic behavior in bacteria is that
it reduces the metabolic load and increases nutrient
availability to the survivors. This sort of unselsh behavior could be explained in a clonal population of
bacteria where death of some still results in perpetuation
of the genetic code. This type of phenomenon is rationalized by the kin selection model [43]. However, bacterial biolms are rarely clonal, and surviving bacteria
would quickly out-compete sacricial bacteria. A more
plausible hypothesis regarding the chromosomal toxin
antitoxin cassettes, has recently been proposed by
Dr. Kim Lewis. He suggests that the cassettes induce, in
a fraction of the bacterial population, not death, but a
quiescent, persister state which enables survival in the
event of a sudden unfavorable environmental change
[3]. Overall, the idea that bacterial cells can undergo
programmed cell death, while appealing, does not
make immediate sense with respect to the laws of evolution, but only further experimentation will resolve this
question.

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K.K. Jeerson / FEMS Microbiology Letters 236 (2004) 163173

often, if not always, be objectively examined, and scientists have recently begun to develop mathematical
models that predict the relative impacts of altruistic vs.
selsh behavior on the survival and propagation of
bacteria [28].
In conclusion, it is evident that bacteria reap a
number of benets from the biolm mode of growth and
it is likely that dierent forces motivate bacteria to
transition to one of a variety of biolm states depending
on the genetic makeup of the organism and its environment. The alternative motives for biolm formation
presented in this review are by no means exhaustive or
mutually exclusive and because it is such a complex
process, they may all have a role.

I thank Dr. Sean Taverna (Rockefeller University)

for his artistic contribution. I would also like to acknowledge that there are a number of excellent publications on the molecular genetics of biolm formation
which could not be referenced here due to space
limitations.

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