Biofilms, Flagella
Biofilms, Flagella
Biofilms, Flagella
of Pages 11
Review
It is generally agreed that motility and biofilm development are mutually exclusive events, and a transition from
motility to sessility occurs during the earliest steps in
biofilm development [1,4]. The phase of the switch is
determined by a sensory transduction mechanism termed
surface sensing that often involves the bacterial flagellum
[5]. Thus, flagella are not only required for propulsion but
also have a critical mechanosensory role in surface sensing
and the initial stages of surface adhesion that leads to the
formation of a biofilm [6]. Thus, a long-range goal of
research on biofilm formation is a thorough understanding
of the surface-sensing mechanism, which will allow the
discovery of drugs that inhibit the ensuing response and
thereby prevent biofilm formation.
The goal of this article is to review the state of our
knowledge on the role that flagella have as mechanosensors (see Glossary) of surfaces, and to highlight what we
Glossary
Bis-(30 50 )-cyclic dimeric guanosine monophosphate (C-di-GMP): a secondary
messenger used by many bacteria to regulate biofilm formation. High levels of
c-di-GMP promote biofilms, whereas low levels of c-di-GMP promote motility.
Flagellin: protein subunit that comprises the flagellar filament.
Lateral flagella: flagella that are used for swarming, and that are distributed
around the surface of the cell in some bacteria that also possess polar flagella.
Lateral and polar flagella are encoded by different sets of genes.
Mechanosensor: an organelle, a biological complex, or individual protein that
detects and responds to physical forces exerted by the local environment and
transduces this signal to control the transcriptional machinery.
Membrane potential (DC): the potential (electrical charge) across a bacterial
membrane relative to the fluid outside of the concentration of potassium,
sodium, chloride, and other diffusible ions. DC is one component of proton
motive force. (The second is DpH.)
Polar flagella: flagella used for swimming, and localized to one or both ends of
a rod-shaped bacterium.
Proton motive force (PMF): energy that is generated by the transfer of protons
across a membrane that can be used for a variety of purposes, including
synthesizing ATP. It is composed of the difference in proton concentration
(DpH) and the electrical charge (DC) across a membrane.
Swarming: is a bacterial flagella-dependent motile behavior that allows cells to
move over surfaces in a coordinated manner and expand the population to
new locations. The process of swarming is distinct from swimming in that
swarming is a multicellular process that occurs on solid surfaces or in viscous
liquids, and requires differentiation of a vegetative swimmer cell into a
specialized cell type called a swarmer cell.
Swarmer cell differentiation: morphological change of some bacteria from a
planktonic or vegetative form that moves by swimming in liquid medium to a
form in which the cells move across solid surfaces. Swarmer cell differentiation
results in an increased number of flagella per cell and, in some bacteria, an
alteration in how the flagella are distributed around the cell surface, as well as
a significant elongation of the swarmer cells due to an inhibition of septation.
Two-component regulatory system: in its simplest form, composed of a
membrane-bound sensor histidine kinase protein that senses specific environmental stimuli and its cognate response regulator protein that mediates the
response, frequently through direct binding to DNA and subsequent differential expression of target gene transcription.
Review
produce a tumble. Swimming cells can perform chemotaxis by moving up or down chemical gradients, using an
elaborate signaling system that modulates the counterclockwise/clockwise bias of the motors [13].
The flagellum can be subdivided into three substructures that are assembled in a temporal sequence [14]
(Figure 1). The first component to be assembled is the
basal body, which anchors the flagellum to the cell membrane, provides the power for rotation, and secretes the
more distal components. The next component is the hook,
which is connected to the basal body and serves as a flexible
universal joint changing the angle of flagellar rotation. The
third structure is the helical filament, which is composed
primarily of the protein flagellin, one of the most abundant
proteins made by the cell. Hook and filament proteins are
secreted through the basal body, and a substrate specificity
switch ensures that hook proteins are secreted first and
flagellin proteins are secreted thereafter [14]. Synthesis of
the flagellum is an ordered process that is controlled by a
set of hierarchical regulatory checkpoints that ensure
proper synthesis and assembly of the flagellar components
(Box 1).
The rotor in the hook-basal body (HBB) consists of an
axial rod, the FliF (MS) ring (Figure 1), which is embedded
in the cytoplasmic membrane, and the C ring, which is
composed of FliG, FliM, and FliN [14]. Rotation of the HBB
structure is achieved via the motor force generators, MotA
Flagellum
Hook
FliF
Fli
Fl
liF
OM
L ring
P ring
PG
MotB
Stator
H+
Basal body
FliL
MotA
FliF
IM
YcgR
MS ring
FliG
Rotor
FliM
EpsE
H+
C ring
FliN
TRENDS in Microbiology
Figure 1. Flagellar structure. Simplified diagram showing the main components of the flagellum: the basal body, hook, and flagellar filament. The motor is composed of the
stator (MotA and MotB proteins) and the rotor (C ring, composed of FliG, FliM, and FliN). Ion (H+ or Na+) flow through the MotAB channel provides the power to rotate the
flagellum. On the left side of the diagram is a schematic of the Gram-negative structure, together with YcgR brake, whereas the right side depicts a Gram-positive envelope
and the EpsE clutch protein. Both YcgR and EpsE are functional inhibitors of motor rotation. Abbreviations: IM, inner (or cytoplasmic) membrane; OM, outer membrane; PG,
peptidoglycan.
Review
Box 1. Regulation of flagellar genes in enteric bacteria
Enteric bacteria, such as Escherichia coli and Salmonella enterica
serovar Typhimurium, serve as useful examples of the hierarchical
control system regulating bacterial flagella. The flagellar regulon of E.
coli and Salmonella is organized into a transcriptional hierarchy that
is based on three promoter classes temporally regulated in response
to assembly [86]. The flagellar master operon flhDC is at the top of this
hierarchy and controls the fundamental decision of whether to
produce flagella. The flhDC operon is expressed from the sole class
1 promoter. The FlhDC proteins form a heteromultimeric complex
(FlhD4C2) that functions as a transcriptional activator to promote s70dependent transcription from the class 2 flagellar promoters [87]. The
class 2 promoters direct transcription of the genes that encode
components of the flagellar C-ring (also known as the motor switch),
export apparatus, basal body, and hook, and are needed for the
structure and assembly of the hook-basal body (HBB). They also
include the gene for the flagellum-specific sigma factor FliA (s28). On
HBB completion, class 3 promoters are transcribed by s28 RNA
polymerase [86], which is specific for flagellar class 3 promoters [88]
encoding later-assembled components, including flagellin. A s28specific anti-sigma factor, FlgM, provides feedback to s28 regarding
the state of flagellar assembly [14]. On HBB completion, FlgM is
secreted from the cell, presumably through the completed HBB
structure, and s28-dependent transcription ensues. In this way, genes
such as the flagellin filament genes, the products of which are needed
after HBB formation, are only transcribed when there is a functional
motor onto which they can be assembled [14].
and MotB, which are anchored around the basal body and
act as stators against the C-ring part of the rotor. MotB is a
membrane protein with a peptidoglycan binding domain,
whereas MotA interacts with FliG. MotA, MotB, and FliG
are specifically involved in torque generation. The MotAB
(or PomAB homolog) complex creates an ion channel (H+ or
Na+ ions), such that ion flow induces a conformational
change in MotA that interacts with the C-terminal domain
of FliG, resulting in torque generation [15]. Thus, rotation
of the flagellar filament is powered through the proton/
sodium motive force (PMF/SMF), and not ATP [12]. The
filament-generated torque powers the cell to swim through
liquid or flagellum-dependent swarming over solid surfaces [8]. Although slight increases in viscosity (by the
introduction of crowding polymers, e.g., Ficoll and dextran)
enhance swimming speed, high viscosity generally
impedes flagellar rotation and performance [16].
As one considers how flagella function to transduce
surface signals into a bacterial cell, FlhDC and other
master regulators of flagellar gene expression [8,14] stand
out as putative checkpoints in the swim-or-stick switch, as
do the major genetic regulators of biofilm formation, such
as RpoS, CsgD, and CpxR [17]. For example, rpoS plays a
key role during biofilm formation because it encodes the
stationary phase sigma factor (sS), which regulates a
number of stress-related genes, including CpxR and CsgD,
that exert strong negative regulation on flagellar class 3
genes [18], including ycgR of E. coli, which encodes a
protein that acts as a monkey wrench that interacts with
the rotor protein FliG to impair rotation [19]. Other proteins that may impair flagellum function in response to the
surface-sensing signal include the Bacillus subtilis homolog of YcgR, YpfA, and B. subtilis EpsE, which acts as a
clutch to decouple the motor stator [20], and CheY of
Rhodobacter sphaeroides, which binds to the motor, acting
as a brake [21]. Adding to the complexity, several of these
functional regulators of the swim-or-stick switch, for example, YcgR, are controlled or influenced by the secondary
messenger bis-(30 50 )-cyclic dimeric guanosine monophosphate (c-di-GMP) [22]. High intracellular cyclic di-GMP
levels favor settlement, surface attachment, and biofilm
formation, whereas low levels correlate with motility and
planktonic behavior [23].
Making sense of the staggering array of controls on the
swim-or-stick switch requires a reductionist approach. As
such, I have chosen to focus on six models as examples of
flagellar mechanosensing: (in order) Pseudomonas aeruginosa, Vibrio cholerae, B. subtilis, Caulobacter crescentus,
Vibrio parahaemolyticus, and Proteus mirabilis. It should
be emphasized that bacteria have evolved other mechanisms to sense surfaces that do not directly involve flagella:
for example, the Wsp control circuit of P. aeruginosa
[24,25]. These nonflagellar surface-sensing systems are
only briefly discussed here.
Model systems of flagellar mechanosensing: P.
aeruginosa
P. aeruginosa, a Gram-negative opportunistic pathogen, is
a biofilm-forming bacterium that uses a single polar flagellum to swim in liquids and swarm over surfaces [26].
Flagellar motility is required to form a biofilm, is controlled
at multiple levels, is regulated by c-di-GMP, and is recognized as a major step leading to lung infections in patients
with cystic fibrosis [27]. P. aeruginosa responds to growth
on agar surfaces by producing c-di-GMP, which stimulates
biofilm formation. C-di-GMP affects the activity of the
master regulator of flagellar gene expression, FleQ, which
inhibits the expression of the pel genes required for biofilm
exopolysaccharide synthesis. FleQ binds to c-di-GMP, and
elevated levels of c-di-GMP in vivo relieve the inhibition of
pel gene expression by FleQ [28]. Transcriptome and proteome measurements of cells in biofilms suggest that
expression of virulence genes is upregulated and swarming
cells are more pathogenic [29].
Viscosity-dependent regulation of flagellar reversal frequency plays a critical part in the swim-or-stick switch of P.
aeruginosa [30,31]. Evidence for this role comes from the
discovery of surface attachment defective (Sad) mutants
that are defective in biofilm formation and have increased
swarming motility [32]. In this category are mutants defective in SadC, a diguanylate cyclase that elevates c-diGMP levels [32]. Strains with defects in SadC show increased flagellar reversal rates in high-viscosity media,
similar to those encountered during either biofilm formation or swarming, but not when swimming in low viscosity
liquids: for example, nutrient broths [30,32]. SadC receives
an unknown environmental signal, perhaps contact with a
surface, and transmits this information to the cytoplasm by
modulating production of c-di-GMP, which affects flagellar
reversals via chemotaxis cluster IV (CheIV cluster) in a
viscosity-dependent fashion, which in turn influences the
production of the Pel biofilm exopolysaccharide [30,32].
BifA, a c-di-GMP phosphodiesterase, counters SadC activity by decreasing c-di-GMP levels, suppressing swarming
motility, and decreasing flagellar reversals [33]. It is worth
noting that flagellar reversals are also important in bacterial swimming through semisolid agar: greater reversal
3
Review
Surface signal
?
WspA
Periplasm
+CH3
Cytoplasm
CH3
WspC
WspF
WspR
P
WspB/D
WspE
P
Biolm
TRENDS in Microbiology
Review
Box 2. B. subtilis biofilm regulation
Control of Bacillus subtilis biofilm matrix gene expression is under
the control of a complex regulatory network that includes three
major transcriptional proteins: Spo0A, ComA, and DegU [7]. Signals
relayed through this network ultimately trigger expression of the
extracellular biofilm matrix amyloid protein, TasA, and proteins
required for the synthesis of the biofilm matrix exopolysaccharides,
respectively [7]. A third matrix protein, BslA (formerly YuaB), is also
required for biofilm formation [89]. The expression of bslA gene is
activated by phosphorylated DegU (DegUP) [90,91].
The DegSDegU two-component regulatory circuit also acts to
regulate biofilm formation. DegS is a cytoplasmic bifunctional
protein that exhibits kinase and phosphatase activities. As a kinase,
DegS phosphorylates DegU, which acts to control a myriad of
processes involved in biofilm formation, including motility and
assembly of the flagellar basal body [92], as well as synthesis of gPGA [48]. DegU activity is controlled at the level of degU
transcription, through DegU phosphorylation, and by DegUP
activity, and the protein has regulatory activity in both its unphosphorylated and phosphorylated states [92] (Figure 3).
In the absence of appropriate signals, DegS is at a low level and
unphosphorylated, and the level of phosphorylated DegU is also
low. Low levels of DegUP induce flagella biosynthesis and
swarming motility by binding to the promoter region of the fla/che
operon and thereby inducing flagellar gene transcription, which also
requires the activity of two other proteins, SwrA and SwrB, as well
as completion of the flagellar basal body [52].
Elevated levels of DegUP induce synthesis of BslA, leading to
biofilm development, as well as an extracellular protease (AprE;
subtilisin) and g-PGA, all of which are regulated in part by a positive
feedback loop [93]. DegUP inhibits the fla-che promoter for class 2
genes [94], and also indirectly inhibits sD (encoding the flagellar
gene specific sigma factor) by activating the expression of the antisigma factor FlgM [52]. DegUP-dependent activation of FlgM is
essential to inhibit flagellin expression when assembly of the basal
body is disturbed, which results in a non-motile, cell chaining
phenotype [52]. This suggests that the DegSDegU two-component
system may either directly or indirectly senses cell motility, flagellar
assembly, or the status of flagellar motors operation.
trigger phosphorylation of DegU is unknown, but regulation of flgM by DegU-P suggests that the DegSDegU twocomponent system may either directly or indirectly sense
the completion state of the flagellum [52].
Further evidence comes from a recent report by the Kearns
laboratory [50]. In a screen for mutations that reduced
mucoidy in a motA mutant, they found a class of transposon
insertions that disrupted proteins involved in the assembly of
the flagellar filament (FliD, FliT, and FlgL), suggesting that
disruption of filament cap assembly (and hence flagellum
function) reduces g-PGA synthesis. Although the precise roles
of FliD, FlgL, and FliT remain unclear, the underlying message is that the B. subtilis flagellar stator regulates g-PGA
synthesis and biofilm formation [50].
How does DegS sense the lack of flagellar rotation or
incomplete flagellum assembly? Several scenarios are conceivable (Figure 3). First, similar to E. coli YcgR [19], DegS
could directly interact with cytoplasmic components of the
flagellar motor to act as a brake. Second, DegS may interact indirectly through a protein component of the basal
body, such as the stator-associated transmembrane protein FliL, which is part of the P. mirabilis surface-sensing
mechanism [53]. Third, inhibited flagellar rotation is likely
to cause changes in DC or cellular energy status due to
restricted proton (ion) flux through the MotAB stators [42],
and this change may be sensed by DegS. Restriction of
Surface signal
DegS
DegS
-PGA
DegU
PdegU DegU
BslA
AprE
Biolm
formaon
SwrB
PgM
yvyF
DegU
Pa/che
gM yvyG gK gL
FlgM
ED
sigD swrB
a/che operon
DegU
SwrA
Swarming molity
(Low DegUP)
TRENDS in Microbiology
Review
Surface signal
TRENDS in Microbiology
Figure 4. Flagellar mechanosensing in Caulobacter crescentus involves inhibition of flagellar rotation, resulting in just-in-time production of the holdfast polysaccharide
adhesin. From left to right: swimming swarmer cells possess a single flagellum and polar pili. As a cell nears a surface, surface contact results in the rapid pili-dependent
arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide (depicted here as a red cone) and ultimately the formation of a stalk cell.
Green circular arrows indicate rotating flagellum; red circular arrows indicate rotation is inhibited. Adapted from Kirkpatrick and Viollier [62].
Review
thereby preventing their rotation, triggers differentiation
of V. parahaemolyticus swarmer cells; (iii) the sodiumchannel-blocking drug phenamil, which poisons the energy
source that drives polar (but not lateral) flagellar rotation,
induces swarmer cell differentiation [66]; (iv) mutations
that cause defects in swimming motility by the polar
flagellum induce transcription of lateral flagellar genes
in liquid; and (v) mutants with defects in the motor stator
proteins induce swarmer cell differentiation [67]. Together, these results suggest that when the rotation of the polar
flagellum of V. parahaemolyticus is reduced (for example,
as it comes into contact with a surface), the cell senses and
responds to this signal by inducing synthesis of swarmer
cells. The identity of the signal is currently not known. The
cue may be torque or external force on the motor, which is
then sensed by an unknown mechanism, but the results
from perturbing ion flow using phenamil or through construction of stator mutants argue that what is being sensed
is a reduction or change in sodium ion flux. The V. parahaemolyticus surface-signaling pathway is also not known,
but is likely to be mediated through the s54-dependent
master regulator, LafK, which controls transcription of the
lateral flagella (laf) genes [67].
P. mirabilis, a Gram-negative Enterobacteriaceae that is
often associated with urinary tract infections [68], synthesizes a single type of flagella, such that vegetative swimmer cells possess four to eight peritrichous flagella,
whereas differentiated swarmer cells are elongated and
hyperflagellated (in a similar way to V. parahaemolyticus).
Similar to V. parahaemolyticus, P. mirabilis swarmer cell
differentiation is triggered by physical conditions that
inhibit the rotation of the peritrichous flagella of the
swimmer cell [69]. Agar surfaces, viscous liquids, and
antibodies specific to flagellar proteins, such as flagellin,
all induce differentiation and are thought to increase
torque on the motor [69]. Correct flagellar assembly is also
required for differentiation, and certain flagellar mutations result in constitutive swarmer cell elongation
[69,70]. In general, mutations in P. mirabilis flagellar
genes result in cells that do not differentiate and do not
swarm, but there are exceptions: mutations in fliL, fliG,
and to a lesser extent fliF, result in the inappropriate
production of swarmer-like cells (referred to as pseudoswarmer cells) in noninducing conditions: for example,
broth [69]. The production of a pseudoswarmer cell suggests that these mutants are defective in their response to
a surface and behave as though they are always on a
surface [53]. Thus, the P. mirabilis flagellum functions
as a mechanosensor of the surface signal.
The P. mirabilis surface signal ultimately affects the
flagellar master regulator, FlhD4C2, encoded by flhDC, the
transcription of which is upregulated during swimmer-toswarmer cell differentiation and the onset of swarming
motility [71]. Regulation of flhDC is complex and involves a
myriad of controls; however, only a few of these regulatory
circuits have been shown to interact with the P. mirabilis
surface-sensing pathway and are discussed next.
Both lipopolysaccharide (LPS) and O-antigen play a
part in P. mirabilis surface sensing [70,72]. Evidence
includes the observation that, when placed on solid surfaces, cells with mutations in waaL (rfaL), encoding
Review
RcsF
OM
WaaL
UmoA
PG
FliL
stators
(PMF/torque?)
UmoD
UmoC
IM
RcsC
UmoB
Surface signal
RcsD
RcsB
Swarming
aA
hDC
TRENDS in Microbiology
Figure 5. Surface-sensing in Proteus mirabilis. Conditions that inhibit flagellar rotation induce surface-dependent swarmer cell differentiation mediated by FliL, a flagellar
protein thought to be associated with the MotAB stator. The role played by FliL is unknown, but it may empower the motor, perhaps through modulating ion flow. The
signal mediated through FliL involves UmoA, which upregulates flhDC, encoding the flagellar master regulator, FlhD4C2, the activity of which is required for swarming and
swarmer cell differentiation. A second nonflagellar mechanosensing circuit has been proposed that senses cell wall stress or perturbations through lipopolysaccharide
(LPS) and O-antigen changes, mediated by WaaL, UmoD, and UmoB. Both mechanosensors are likely to control the activity of the Rcs regulatory circuit, which in turn
inhibits flhDC expression. Abbreviations: IM, inner (or cytoplasmic) membrane; OM, outer membrane; PG, peptidoglycan. Unbroken lines indicate interactions determined
by empirical tests, broken lines are hypothetical interactions, arrowheads indicate positive effects, whereas T-end lines indicate inhibitory effects.
Review
the P. mirabilis surface-sensing pathway that lead ultimately to FlhD4C2.
Concluding remarks and future directions
How does a bacterium know it is in contact with a
surface? Hopefully, the examples provided in this review
offer one answer, if not the answer: they use a flagellar
mechanosensor. These mechanosensors utilize the rotating flagellum and are able to detect subtle changes in
the function of their motors during surface contact.
However, although prevalent in many bacterial species,
flagellar mechanosensing is not the only means used to
detect and respond to surfaces. Obviously, not all surfacesensing pathways directly involve flagella or their
function, and nonmotile bacteria form biofilms without
the need for flagella. For this reason, I included at least
two examples of non-flagellum-mediated surface sensing
(the P. aeruginosa Wsp surface-sensing circuit and the
P. mirabilis LPS/O-antigen circuit). With that caveat
out of the way, I firmly believe that a better understanding of the surface-sensing function of the flagellar nanomachine is critical to our understanding of biofilm
formation.
In contemplating how flagellar mechanosensing function, two features stand out in the model systems described
here. The first is that surface sensing takes place at the
level of the flagellar stators, and second, surface contact
seems to alter the flow of ions through the stators and
perturb PMF or one of its components, DC and DpH. Such
changes could result in a hyperpolarized membrane and/or
acidification of the cytoplasm (in the case of increased
unabated proton flow into the cell). Understanding the
molecular mechanisms the cell uses to detect and respond
to a hyperpolarized membrane or to cytoplasmic acidification resulting from stator impairment is a high priority
that offers great potential for future research on flagellar
mechanosensory systems.
Many questions remain to be answered (Box 3). What
cellular components (proteins, regulatory RNAs, small
molecule effectors, and so on) constitute the surface-sensing pathway? What is the signal sensed by WspA or the P.
mirabilis LPS/O-antigen surface-sensing circuit? For species that have multiple means of sensing a surface (for
example, P. aeruginosa SadC and Wsp surface-sensing
circuits), how is the hierarchy of biofilm control orchestrated? Do the pathways converge or remain separate, controlling separate and unique components needed to initiate
biofilm formation?
I am of the opinion that study of the flagellums function
in sensing and response to surfaces is crucial for understanding biofilm development, for the development of
strategies to prevent biofilm formation, and in the fabrication of stealth surfaces that go undetected by the flagellar mechanosensor. Using mechanosensing flagella as a
model provides a tractable system to understand surface
sensing that will have implications beyond flagellated
bacteria, given the central role of the proton-driven motors
identified in this review. Moreover, although the flagellar
motor is the best-understood nanomachine, one big mystery is the mechanism by which ion flow generates rotational force. Gaining a better understanding of the
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