Download Regulation cascade of flagellar expression in Gram‐negative bacteria

FEMS Microbiology Reviews 27 (2003) 505^523
www.fems-microbiology.org
Regulation cascade of £agellar expression in Gram-negative bacteria
Olga A. Soutourina a , Philippe N. Bertin
b
b;
a
Laboratoire de Biochimie, UMR 7654, CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, France
Dynamique, Evolution et Expression de Ge¤nomes, Universite¤ Louis Pasteur, 28 rue Goethe, 67000 Strasbourg, France
Received 25 September 2002 ; received in revised form 11 February 2003 ; accepted 14 March 2003
First published online 16 June 2003
Abstract
Flagellar motility helps bacteria to reach the most favourable environments and to successfully compete with other micro-organisms.
These complex organelles also play an important role in adhesion to substrates, biofilm formation and virulence process. In addition,
because their synthesis and functioning are very expensive for the cell (about 2% of biosynthetic energy expenditure in Escherichia coli)
and may induce a strong immune response in the host organism, the expression of flagellar genes is highly regulated by environmental
conditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellated
bacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largely
unknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of the
cascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatory
regions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similar
hierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests an
evolutionary conservation of regulatory mechanisms in Gram-negative bacteria.
7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Polar and lateral £agellar systems ; Master regulator; Environmental control ; Hierarchical organisation
Contents
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chromosomal organisation of £agellar genes . . . . .
Hierarchical organisation of £agellar systems . . . . .
Master regulators of class I . . . . . . . . . . . . . . . . . .
4.1. FlhDC in lateral £agellar systems . . . . . . . . . .
4.2. Regulators of polar £agellar systems . . . . . . . .
Motility control by environmental factors . . . . . . .
Comparison of £agellar cascades . . . . . . . . . . . . . .
6.1. Functional similarities of £agellar systems . . . .
6.2. Important di¡erences between £agellar systems
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Bacterial motility and chemotaxis is one of the complex
* Corresponding author. Tel. : +33 (3) 90 24 20 08;
Fax : +33 (3) 90 24 20 28.
E-mail address : [email protected] (P.N. Bertin).
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processes allowing £agellated micro-organisms to survive
under a wide variety of environmental conditions by a coordinated control of their gene expression in response to
external stimuli [1].
Flagellar motility represents an important advantage for
bacteria in moving towards favourable conditions or in
avoiding detrimental environments and it allows £agel-
0168-6445 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6445(03)00064-0
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Fig. 1. The role of motility in the interactions of bacteria with their natural environment.
lated bacteria to successfully compete with other microorganisms [2]. For example, it has been recently demonstrated that a Fe(III) oxide-reducing bacterium, Geobacter
metallireducens, speci¢cally expresses £agella and pili to
move towards the insoluble electron acceptor, which
may explain the predominance of Geobacter species in a
wide variety of sedimentary environments [3]. In addition
to having locomotive properties, bacterial £agella play a
crucial role in adhesion, bio¢lm formation and colonisation of micro-organisms, such as Pseudomonas aeruginosa
[4], Escherichia coli [5], Vibrio cholerae [6], Salmonella
typhimurium [7] and Helicobacter pylori [8]. Motility in
pathogenic micro-organisms is usually considered a virulence factor, essential for colonisation of host organism or
target organ [9,10]. However, the £agellar ¢lament bears
strong antigenic properties in contact with animal and
plant hosts [11^13]. Furthermore, motility by means of
£agella is very expensive for cellular economy in terms
of the number of genes and the energy required for £agellar biosynthesis and functioning [1]. Consequently, it is
not surprising that the synthesis of £agella is highly regulated by external factors, including the interaction of bacterial cells with their host (Fig. 1). With respect to this
dual pathogenicity/antigenicity e¡ect of £agella, it must
be mentioned that several highly pathogenic bacteria,
such as Bordetella pertussis [14,15], Shigella sp. [16], and
Yersinia pestis [17], in contrast to their close relatives Bordetella bronchiseptica, E. coli and Yersinia enterocolitica,
respectively, have lost the capacity to synthesise £agella,
but yet possess the £agellar genes. In Y. pestis the loss of
motility has been associated with a frameshift mutation in
£hD master regulatory gene-coding sequence [18], while
nucleotide sequence comparisons of £agellin cryptic genes
suggest that loss of motility in Shigella is a recent evolutionary event [16]. The absence of motility in these species
may re£ect di¡erences in pathogenesis or life cycles and/or
the existence of particular adaptations in response to sim-
ilar conditions that necessitate motility in related strains
[14].
Motility by means of £agella is widespread in the microbial world, and more than 80% of known bacterial species
possess these organelles, including various £agellated Archaea [19,20]. The structure and arrangement of £agella
on the cell di¡er from species to species and both seem to
be related to the speci¢c environments in which the cells
reside [21,22]. Flagella can be arranged on the cell body in
a variety of con¢gurations, including single polar, multiple
polar, and many peritrichous (or lateral) con¢gurations.
Some species, e.g. Vibrio parahaemolyticus [23] and Azospirillum brasilense [24] display a mixed £agellation and
form two structurally unrelated £agellar types on the
same cell.
Bacterial £agellum synthesis genes form an ordered cascade in which the expression of one gene at a given level
requires the transcription of another gene at a higher level
[1]. At the top of the hierarchy is the £hDC master operon
in enterobacteria [25^27], and £eQ or £rA master genes in
P. aeruginosa [28,29] and V. cholerae [30,31], respectively.
The organisation of the £agellar system has been extensively studied in enterobacteria and multiple levels of
£hDC regulation were observed, such as transcriptional
and posttranscriptional control in E. coli (Fig. 2) and protein stability control in Proteus mirabilis [32]. The expression of the c70 -dependent £hDC operon is controlled by
numerous environmental signals, e.g. temperature, osmolarity and pH [33^35] and by global regulatory proteins,
such as H-NS and the cAMP^CAP (catabolite gene activator protein) complex [36,37]. Moreover, the stability of
its mRNA is controlled by the RNA binding regulator
CsrA [38,39] (Fig. 2). In contrast, the regulation of master
genes governing the synthesis of polar £agella remains
largely unknown. Based on the recent advances in this
¢eld, the present review will focus on a comparative analysis of £agellar regulatory cascades in di¡erent bacterial
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Fig. 2. Multiple regulation of the £hDC £agellar master operon in E. coli. The main regulatory levels and e¡ectors that are involved in the control of
FlhDC synthesis are indicated : +, positive e¡ect ; 3, negative e¡ect; no indication, positive or negative e¡ect.
systems, especially in terms of motility gene regulation by
environmental factors and regulatory proteins.
2. Chromosomal organisation of £agellar genes
Flagellar genes in many systems are grouped in several
clusters on bacterial chromosomes. In E. coli and in
S. typhimurium nearly 50 genes, required for £agellum biosynthesis and functioning, are organised in 15 and 17 operons, respectively [40,41], clustered in several regions [1].
Cluster I (minute 24 and 23 on the E. coli and S. typhimurium chromosomes, respectively) includes the genes encoding £agellar structural proteins. Cluster II (minute 41
and 40) includes the genes encoding the proteins participating in the regulation of £agellar assembly, the £agellar
motor genes, motA and motB, and the chemotactic genes.
Cluster III contains three regions on minute 43 and 40 on
the E. coli and S. typhimurium chromosomes, respectively,
and includes the genes encoding £agellar structural proteins, export apparatus proteins and £agellar-speci¢c
c factor.
The complete analysis of polar £agellar biosynthesis system in V. parahaemolyticus has been recently published
[42]. The authors have identi¢ed 57 potential £agellar
genes showing sequence homology with the components
of bacterial motility and chemotaxis systems. These genes
are grouped into operons in ¢ve regions on the bacterial
chromosome, most of the genes being located in two of
them. Chromosomal organisation of homologous genes is
conserved in V. cholerae [31], Pseudomonas putida and
P. aeruginosa [42]. In V. cholerae in particular [31], the
organisation of polar £agellar genes is almost identical
to that previously described in V. parahaemolyticus, with
some exceptions. First, three main chromosomal loci for
£agellar genes were identi¢ed in V. cholerae instead of two
regions in V. parahaemolyticus: regions II and III are contiguous in this organism, but separated by about 48 kb in
V. cholerae. Second, an additional £agellin gene was found
in V. parahaemolyticus, thus having six £agellins as an
alternative to the ¢ve found in V. cholerae.
3. Hierarchical organisation of £agellar systems
In Gram-negative bacteria, the hierarchy of the £agellar
regulatory system was ¢rst well characterised in micro-organisms with peritrichous £agella, such as E. coli and
S. typhimurium [25^27]. This regulatory cascade possesses
three classes of genes. Class I genes form the £hDC master
operon at the top of the hierarchy, which encodes a
FlhD2 C2 transcriptional activator of the second class
gene expression. The majority of class II genes encode
components of the £agellar export system and the basal
body. The £iA gene at this second level encodes a sigma
factor, c28 (or RpoF), speci¢c for £agellar genes [43]. The
operons of class III are positively regulated by c28 and
negatively controlled by an anti-sigma factor, FlgM
[44,45]. The anti-sigma factor is retained inside the cell
until the £agellar basal body and hook are completed
[46]. At that time, the FlgM protein is exported to allow
activation by c28 of the transcription of class III genes,
which encode the components of £agellar ¢lament (e.g.
£agellin, FliC), hook-associated, motor, and chemotaxis
proteins (Fig. 3A).
Another £agellar system has also been well documented.
In Caulobacter crescentus, which is a polarly £agellated
micro-organism (see Table 1), £agellar gene expression is
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Fig. 3. Flagellation cascades. Lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae, Vibrionaceae families) £agellar cascades are compared
and the factors controlling master regulator expression and the connections with other cellular processes are shown. A: The lateral £agellation cascade
discovered in Enterobacteriaceae with the £hDC master operon at the top (class I) encoding the FlhD2 C2 transcriptional activator of class II genes including £agellar-speci¢c c28 factor £iA gene. FliA (c28 ) is necessary for the transcription of class III genes including the £iC £agellin gene. B: The polar
£agellation cascade identi¢ed in Pseudomonadaceae and Vibrionaceae with £eQ and £rA genes at the top. These genes encode c54 -associated NtrC-type
transcriptional activators, FleQ and FlrA, respectively (see Table 1), which activate the transcription of class II genes, such as £eSR and £rBC. Both
are two-component regulatory systems for class III genes that include £agellar ¢lament genes £iC and £aA, in Pseudomonadaceae and Vibrionaceae, respectively. Another class II gene, £iA, encodes the c28 £agellar-speci¢c factor participating in the transcription of class IV genes, such as non-essential
£agellin genes £aB, C, D, E in Vibrionaceae. Other factors such as regulatory proteins, environmental signals and growth phase-controlling master regulatory gene expression are shown in ovals at the top. Other cellular processes controlled by £agellar regulators are indicated in boxes at the left and the
right sides.
strongly linked to the control of the cell cycle, which is
distinguished by an asymmetric cell division [47]. The regulatory cascade includes four classes of genes. The unique
class I gene, ctrA, is situated under the control of cell cycle
signals. CtrA regulates the c70 -dependent transcription of
the second class of genes encoding the subunits of membrane/supramembrane (MS) ring complex and export system. The gene encoding the regulator FlbD of the NtrC
family of c54 -associated transcriptional activators (see Section 4.2) [48,49] belongs to this class. In concert with c54 ,
this regulatory protein is essential for the expression of
class III genes encoding both the basal body and hook
structure and of the class IV genes encoding the three £agellin subunits of £agellar ¢lament.
Recently, the regulatory cascade components of bacteria
with one polar £agellum, such as V. cholerae and P. aeruginosa, have been characterised [28^31] (Fig. 3B, Table 1).
In V. cholerae [31], the class I gene at the top of the
hierarchy encodes the FlrA protein, a c54 -associated transcription activator of the NtrC family. This regulator, together with c54 , activates the expression of the class II
genes encoding structural components of the MS ring,
switch and export apparatus, the c28 £agellar-speci¢c sigma factor, FliA, and FlrB and FlrC, the sensor kinase and
transcriptional regulator, respectively, of a two-component
signal-transducing system. The FlrC regulator along with
c54 activates the expression of the class III genes encoding
the basal body, the hook and the £agellin essential for
motility, FlaA. The sigma factor c28 is required for the
transcription of the class IV genes, including the motor
genes and non-essential £agellin genes £aB, £aC, £aD,
£aE. The absence of these last four genes does not a¡ect
£agellar function, but they may help to maintain the antigenic and environmental variations in £agellar ¢lament
composition [50,51]. Surprisingly, in V. parahaemolyticus,
entirely distinct lateral and polar £agellation systems have
been identi¢ed, the lateral £agellar components being homologous to enterobacterial systems [52] and the polar
£agellar system components being homologous to Vibrionaceae systems [42,51].
The implication of c54 in bacterial motility regulation is
widespread in Gram-negative bacteria. Indeed, the ele-
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509
Table 1
Polar £agellar genes and their function
Species
Gene function
Master c54
regulatory gene
Two-component c54
regulatory systema
Speci¢c c28
factor
Anti-c28 factor
Filament £agellinb
Pseudomonas aeruginosa
Vibrio cholerae
£eQ
£rA
£eSR (FleS: S, FleR: R)
£rBC (FlrB: S, FlrC : R)
£iA
£iA
£gM
£gM
Vibrio parahaemolyticus
£aK
£aLM (FlaL : S, FlaM : R)
£iA
£gM
Pseudomonas £uorescens
adnA
£eSR (FleS: S, FleR: R)
£iA
Helicobacter pylori
Campylobacter jejuni
Caulobacter crescentus
Pseudomonas putida
KT2440
^
£iA
^
82% £eQ of
P. aeruginosa
£gR (HP703) : R, HP244: S
£gR (Cj1024c) : R, Cj073: S
£bD, £bE (FlbE: S, FlbD: R)
74% £eS and £eR of
P. aeruginosa
Contig 458 50% PA3351
of P. aeruginosa,
28% £gM of V. cholerae
£gM
£iC
£aA (c54 ); £aB, £aC, £aD,
£aE (c28 )
£aA (c28 ); £aB (c28 ), £aC,
£aD (c28 ), £aE (c28 )
£iC
Pseudomonas syringae
81% £eQ of
P. aeruginosa
75% £eS and 76% £eR of
P. aeruginosa
81% £iA of
P. aeruginosa
Desulfovibrio vulgaris
38% £rA of
V. cholerae
25^28% £rB and 38% £rC of
V. cholerae
34% £iA of
V. cholerae
^
82% £iA of
P. aeruginosa
^
53% hypothetical £gM
of P. aeruginosa ; 35%
£gM of V. cholerae
53% hypothetical £gM
of P. aeruginosa ; 32%
£gM of V. cholerae
30% £gM of V. cholerae
£aA (c28 ), £aB (c54 )
£jJ, £jK, £jL
40% £iC of P. aeruginosa
24% £iC of P. aeruginosa
38% £aA of V. cholerae
The % numbers mean the percentage identity of homologous sequence in a given organism with the indicated gene sequence.
a
For two-component regulatory systems in parentheses, S is the sensor kinase protein component, R is the regulatory protein component.
b
For ¢lament £agellin in parentheses is indicated a c factor participating in £agellin gene transcription.
ments of £agellar regulatory cascades including c54 -associated regulators have also been recently identi¢ed in micro-organisms, such as Rhodobacter sphaeroides [53^55],
H. pylori [56] and Campylobacter jejuni [57], whose genomes have been recently deciphered (http://igweb.integratedgenomics.com/GOLD) (Table 1). In R. sphaeroides, the
£agellar regulatory system is supposed to be a combination of the elements present in enterobacteria and in
C. crescentus systems including the c54 -associated regulators of class I, the c54 -dependent class II genes and the
c28 -dependent class III genes (e.g. £agellin-encoding £iC
gene) [55]. Similarly as in C. crescentus, the c54 and the
master transcriptional activator of the NtrC family, FlgR,
are required for the transcription of the genes encoding the
basal body, the hook structures, and the £agellin FlaB in
H. pylori. In contrast, the expression of the major £agellin,
FlaA in this bacterium, is controlled by the c28 , as in
enterobacteria, and repressed by FlgR.
A particular £agellar hierarchy, sharing some similarities with that of enterobacteria systems, has been recently
identi¢ed in the K-proteobacterium Sinorhizobium (Rhizobium) meliloti [58]. At the top of the hierarchy is the
master operon, visNR, encoding the global regulators
VisNR that control motility and chemotaxis genes in
this organism. The VisN and VisR proteins, belonging
to the LuxR family with characteristic DNA and ligand
binding domains, form a heterodimer. For the activation
of the transcription by these regulators, the binding of a
yet unidenti¢ed e¡ector is necessary. The main di¡erences
with the system of enterobacteria are the existence of novel
master activators and the position of motor genes in the
second class rather than in the third class of genes.
Finally, in Gram-positive bacteria, such as Bacillus subtilis, some gene families have been characterised that
roughly correspond in their structure to the enteric class
II and class III genes, including cD factor, homologous to
c28 in enterobacteria with nearly identical promoter speci¢city and function, and FlgM anti-sigma factor, which
antagonises cD activity. The regulatory genes corresponding to the class I master £agellar regulators have not yet
been identi¢ed in this organism and some data indicate
that cD may occupy a central position in the £agellar
regulatory system [59^61].
4. Master regulators of class I
4.1. FlhDC in lateral £agellar systems
In the well-characterised systems of E. coli and S. typhimurium, the £hDC operon is at the top of the cascade and
encodes FlhD2 C2 activator required for the expression of
all other genes of the £agellar regulon, FlhD alone having
larger regulatory functions (see below) (for a recent review
see [62,63]). The FlhD (13.3 kDa) and FlhC (21.5 kDa)
proteins form a heterotetrameric complex for activation of
transcription of £agellar class II genes [64,65], the protein
FlhD alone being not capable of binding to DNA and
activating transcription [64]. The sequence TT(T/A)GCCGATAACG in their promoter regions has been
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suggested to serve as a binding site for the complex
FlhD2 C2 [66]. However, footprinting analysis did not allow identi¢cation of any consensus sequence in E. coli [64].
Nevertheless, a recent study on FlhD2 C2 binding to the
P. mirabilis class II £agellar promoters combined with
analysis of several sequences of the class II £agellar promoter regions of E. coli and S. typhimurium [67] identi¢ed
an imperfect palindrome with two 17^18-bp inverted repeats (FlhD2 C2 boxes: TNAA(C/T)G(C/G)N2=3 AAATA(A/G)CG) separated by a 10^11-bp spacer showing
no consensus. Such a symmetric structure of binding sites
is in accordance with the proposed model of heterotetrameric FlhD2 C2 /DNA complex formation. The di¡erences
between class II promoter DNA targets may explain the
di¡erential a⁄nity of FlhD2 C2 contributing to the selection of promoters to be activated in a temporal sequence
[67]. FlhD and FlhC proteins belong to the transcriptional
factors of class I, the C-terminal domain of the K-subunit
of RNA polymerase being essential for the activation of
transcription [68]. Recently, the crystal structure of FlhD
O resolution [69] showing that this
was obtained at 1.8 A
protein is present as a homodimer with a disul¢de bridge
between Cys65 residues. The presence of such a disul¢de
bridge in the cytoplasmic protein is surprising, considering
the reducing environment of cytoplasm in E. coli. The
replacement of Cys65 by alanine a¡ects neither the capacity of FlhD protein to control transcription of £agellar
genes, nor the stability of FlhD dimer. As supposed by the
authors [69], this disul¢de bridge may be necessary for
another intra- or extracellular yet unknown function of
FlhD. The conservation of this residue Cys65 in di¡erent
enterobacteria favours this hypothesis [69]. The C-terminal
part (residues 83^116) of the protein is £exible and carries
a putative helix-turn-helix (H-T-H) motif responsible for
DNA binding. The conformation of this motif is stabilised
only when FlhD is complexed with other proteins, such as
FlhC. This may explain the multiple speci¢city of FlhD,
which participates in various cellular processes. Indeed,
each protein interacting with FlhD may in£uence the conformation and the speci¢city of binding via the H-T-H
motif. The extensive alanine scanning analysis allowed
the identi¢cation of residues implicated in the interactions
between FlhD and FlhC, as well as in the interactions with
DNA [70]. In contrast, Claret and Hughes [71] suggest
that the protein FlhC, and not FlhD, is the DNA binding
component of the FlhD2 C2 complex. This suggestion is
based on the fact that, unlike FlhD, the FlhC protein
alone is capable of binding DNA but with a 10-fold lower
a⁄nity than in complex with FlhD. These authors also
identi¢ed a potential H-T-H motif in the FlhC protein
sequence. However, the results of Campos et al. [69,70]
do not rule out the possibility that FlhC also participates
in DNA recognition and/or binding. More recent data of
Claret and Hughes [67] suggest that FlhC and FlhD subunits contact DNA and contribute together to stability
and speci¢city of the interaction. Determination of the
FlhD/FlhC crystal structure, the initial steps of which
have been recently reported [72], will contribute to a better
understanding of these protein^protein and protein^DNA
interactions.
Recently, sequences encoding FlhDC homologous proteins have been identi¢ed in di¡erent enterobacteria
[70,73]. A sequence alignment of FlhD proteins suggests
a high degree of structural conservation among di¡erent
species, in particular for several important residues identi¢ed by crystallographic analysis, for example Cys65 forming a disul¢de bridge or Gly93 in the H-T-H motif (see
above). This further supports the essential role that these
residues might play in the functioning of this type of protein, in particular in dimer formation and in DNA binding.
4.2. Regulators of polar £agellar systems
The regulators at the top of the polar £agellar hierarchy
belong to the NtrC family of c54 -associated transcription
activators. In addition to its role in polar £agellar gene
expression in P. aeruginosa, V. cholerae, C. crescentus,
Pseudomonas £uorescens [30,74^76], this alternative sigma
factor participates in the transcription of genes having
di¡erent physiological functions, such as nitrogen assimilation in E. coli and S. typhimurium [48,77] and pilin synthesis in P. aeruginosa and Neisseria gonorrhoeae [78]. The
RNA polymerase in complex with c54 (RpoN) requires an
activator protein for transcription initiation [49]. Such regulators usually bind to the promoter region and activate
transcription via the direct contact with RNA polymerase
in complex with c54 [48]. The activity of these activators is
modulated in response to environmental changes. Recent
studies on FleQ regulatory mechanism in P. aeruginosa
[79] revealed the absence of consensus sequences for activation of transcription and the atypical location of probably the majority of enhancer binding sites on FleQ-regulated promoters.
The complete sequence determination of many bacterial
genomes provides new insight into £agellar regulators
(http://igweb.integratedgenomics.com/GOLD). In addition
to the recently identi¢ed master £agellar regulators in
V. cholerae, V. parahaemolyticus, P. aeruginosa, P. £uorescens, genes showing high sequence homology with the
master £agellar regulatory gene £eQ also exist in P. putida
and P. syringae (Table 1). The homology search for other
c54 -associated £agellar regulators in some polarly £agellated bacteria, whose genome sequencing is in progress,
e.g. Desulfovibrio vulgaris, makes it possible to identify
proteins having only limited sequence homology with
known polar £agellar regulators (Table 1). Such an analysis may be complicated by high sequence homology within the NtrC family of c54 -associated regulators, which
control various physiological processes. This observation
may also suggest the existence of entirely new but yet
unidenti¢ed £agellar regulatory proteins.
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511
Fig. 4. Protein sequence alignment of regulators of the polar £agellar gene system. The multiple alignment was performed by the CLUSTALW method
and re¢ned manually [191]. Amino acid sequences are indicated as follows : FleQ_pseae, FleQ of P. aeruginosa (GenBank accession number L49378);
FleQ_psesp, FleQ of Pseudomonas strain Y1000 (EMBL nucleotide sequence database accession number AJ308470) ; AdnA_pse£, AdnA of P. £uorescens (GenBank accession number AF312695); FlrA_vibch, FlrA of V. cholerae (GenBank accession number AF014113); FlaK_vibpa, FlaK of V. parahaemolyticus (GenBank accession number AF069392); FlbD_caucr, FlbD of C. crescentus (GenBank accession number AE005767); FlgR_camje, FlgR
(Cj1024c) of C. jejuni (GenBank accession number AL139077); FlgR_helpy, FlgR (HP0703) of H. pylori (GenBank accession number AE000583). The
arrowheads indicate the position corresponding to conserved residues involved in the phosphorylation of the NtrC family transcriptional activators. The
conserved amino acids constituting the ATP-binding site and the DNA-binding site (H-T-H motif) are boxed.
Unlike FlhD protein, structural data on polar £agellar
regulators are limited. However, some information is
available on the structure of both the DNA binding and
the receiver domains of related NtrC proteins [80,81]. All
proteins identi¢ed until now share structural and functional domains conserved among the NtrC family of transcriptional activators that work in concert with RpoN [28].
Despite a relatively low homology in the N-terminal region (Fig. 4), these proteins possess residues believed to be
involved in their phosphorylation [82], e.g. the acid pocket-forming residues Asp11 and Asp12 present in all sequences with the exception of FlbD of C. crescentus. In
contrast, two other sites corresponding to the phosphorylation site Asp54 and the salt bridge-forming residue
Lys104 in other NtrC family proteins are not conserved
in most of the master polar £agellar regulators except for
the FlgR regulators of H. pylori and C. jejuni, and the
FlbD regulator of C. crescentus. This is in agreement
with the presumed absence of a cognate kinase for the
regulators at the top of the hierarchy of polar £agellar
systems [28]. The activity of these regulators may be
changed by phosphorylation at a serine or a threonine
residue present at position 54 instead of Asp54 in other
NtrC family proteins or may be controlled by a novel
signal-transducing mechanism. But the absence of experimental evidence of such an e¡ect and of any cognate kinase favours the hypothesis that these regulators probably
do not require phosphorylation for their activation. Alternatively, external signals may in£uence motility at the level
of master regulatory gene expression, as recently demonstrated for the regulation of £eQ transcription by environmental factors in Pseudomonas sp. [73]. In contrast, FlgR
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proteins in H. pylori and C. jejuni contain all four conserved residues known to be involved in phosphorylation
by a speci¢c kinase suggesting a classical phosphorelay
mechanism. In fact, the cognate sensor kinase HP244 protein that phosphorylates FlgR regulator has been identi¢ed in H. pylori [83]. Similarly, the CjO793 sensor identi¢ed in C. jejuni shows sequence homology with HP244
[57]. Finally, a strong conservation is observed in the
ATP binding site and/or the H-T-H DNA binding element
of the various analysed proteins (Fig. 4). Nevertheless, the
absence in the FlgR sequence of H. pylori of the H-T-H
motif conserved in all analysed regulators must be emphasised. This protein has been proposed to be the speci¢c
master activator of transcription of c54 -regulated £agellar
genes in H. pylori [56]. This could be related to the existence in FlgR of a DNA-binding domain di¡erent from
that present in regulators playing a more general role in
various micro-organisms.
5. Motility control by environmental factors
Motility and the chemotaxis system play a crucial role
in the adaptation of micro-organisms to multiple environmental conditions. These external factors a¡ect not only
the physiology of motility via the chemotaxis system and
the functioning of £agella (for a recent review see [84^86]),
but also the process of £agellum biosynthesis via the control of expression of £agellar genes at the top of the regulatory cascade. In E. coli, £agellum biosynthesis is inhibited by catabolite repression in the presence of D-glucose,
under conditions of high temperature or high concentration of salts, in the presence of carbohydrates or of lowmolecular-mass alcohols, at extreme pH and in the presence of DNA gyrase inhibitors [33,35,87] (Fig. 2). Moreover, the £agellin expression in E. coli is known to be
regulated by various metal ions [88], while oxygen-limited
conditions were shown to induce £agellar gene transcription in E. coli [89]. In the enteric pathogen Salmonella
serotype enteritidis, pH and temperature a¡ect £agellum
production [90]. In Campylobacter coli, the expression of
£agellar genes is also known to be modulated by several
growth conditions such as pH, temperature, the composition of the growth atmosphere, the concentration of inorganic salts and divalent ions [91]. Similarly, motility in
V. cholerae is a¡ected by temperature, NaCl, pH and organic nutrients [92]. In addition, sodium salicylate inhibits
motility in E. coli, Proteus, Providencia and Pseudomonas
spp. in relation with osmoregulation [93]. A negative e¡ect
of sodium deoxycholate on £agellum production and motility has also been observed in P. mirabilis and E. coli,
which could result from an action of this detergent on
£agellum assembly [94]. Moreover, chloride was recently
revealed as a new environmental signal molecule involved
in £agellar gene regulation in Halobacillus halophilus [95]
while the role of inorganic phosphate was demonstrated in
the motility of several pathogenic bacteria, such as
P. aeruginosa, V. cholerae, S. enterica, E. coli and Klebsiella pneumoniae [96]. Finally, the control of motility by
temperature, in relation with the transition between
growth outside and inside the host, has been reported in
various pathogenic bacteria, such as Y. enterocolitica [97],
Listeria monocytogenes [98], B. bronchiseptica [14], Legionella pneumophila [99] and Actinobacillus pleuropneumoniae
[100].
A possible mechanism of environmental regulation of
motility may be proposed in some cases. In pathogenic
bacteria, two-component signal-transducing systems are
usually involved in the control of both motility and virulence. In these systems environmental signalling is performed via a phosphorylation cascade, i.e. the sensing of
external factors is mediated by a sensor kinase activity
that phosphorylates and, thus, activates the corresponding
transcriptional regulator. The polar £agellar regulatory
cascade contains such two-component regulatory systems.
One example is the co-ordinated control of the expression of virulence and motility genes in V. cholerae by
the ToxR transcriptional regulatory system in response
to environmental changes in pH, osmolarity and temperature [6,101^103]. In other pathogenic bacteria, such as
B. bronchiseptica, motility and virulence genes are coordinately regulated by the two-component system BvgAS
in response to environmental stimuli, including temperature. Inside the host, this system represses £agellar
master operon transcription and activates the expression
of virulence factors [14,15,104^106]. Another member of
two-component signal-transducing systems, SirA, participates in the control of motility and virulence in various
bacteria of genera Pseudomonas, Vibrio and Erwinia [107]
and in L. pneumophila [108]. Its orthologue UvrY has a
similar function in E. coli [109]. In V. parahaemolyticus, a
novel operon scrABC has been shown to inversely a¡ect
two gene systems that are pertinent for colonisation of
surfaces, i.e. swarming and capsular polysaccharides
[110].
On the other hand, the molecular mechanism by which
catabolite repression a¡ects £agellar biosynthesis in E. coli
has been recently elucidated. This phenomenon is observed in the presence of D-glucose, when the concentration of cAMP in the cell is low and CAP is present essentially in a form that is unable to activate the transcription
of speci¢c genes without its cAMP ligand. With respect to
motility, the catabolite repression of £agellar gene expression was observed many years ago [87,111], and implication of the cAMP^CAP complex in this process has been
proposed on the basis of the non-motile phenotypes of
E. coli and S. typhimurium strains mutated in cya and/or
crp genes encoding adenylate cyclase and CAP, respectively [111^114]. Recent experimental data provide evidence that the cAMP^CAP complex positively controls
the biosynthesis of £agella by activation of £hDC master
operon transcription via binding to its promoter and a
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513
Fig. 5. Nucleotide sequence alignment of regions encompassing the promoters and the +1 translational start site of £hDC-like genes (A) and £rA-like
genes (B). The multiple alignment was performed by the CLUSTALW method and re¢ned manually [191]. A: Enterobacterial £hDC nucleotide sequences are indicated as follows: £hD-ecoli, E. coli (GenBank accession number AE005411); £hD-salty, S. typhimurium (GenBank accession number
D43640) ; £hD-entsp, Enterobacter strain 22 (GenBank accession number AJ308469); £hD-erwca, Erwinia carotovora (GenBank accession number
AF130387); £hD-serma, S. marcescens (GenBank accession number AF077334); £hD-yeren, Y. enterocolitica (GenBank accession number AF081587);
£hD-xhene, X. nematophilus (GenBank accession number AJ012828) ; £hD-promi, P. mirabilis (GenBank accession number U96964). B: L-Proteobacterial sequences are indicated as follows : £rA-borbr, B. bronchiseptica (GenBank accession number U17998, http://www.sanger.ac.uk/Projects/B_bronchiseptica), £rA-borpe, B. pertussis (http://www.sanger.ac.uk/Projects/B_pertussis), £rA-borpa, B. parapertussis (http://www.sanger.ac.uk/Projects/B_parapertussis), £hD-ralso, R. solanacearum (http://sequence.toulouse.inra.fr/ralsto/public/doc/gb/rs_home.html), £hD-raleu, R. eutropha (http://www.jgi.doe.gov/
JGI_microbial/html/ralstonia/ralston_homepage.html). Black lines correspond to DNA regions of low homology in all sequences, the number indicates
the length in bp of missing region. The positions of the CRP (cAMP^CAP complex) binding site, the 310 and 335 promoter sequences and the transcriptional start sites (+1) are boxed. The transcriptional start sites have been identi¢ed experimentally in E. coli [37], S. typhimurium (one of six transcriptional start sites) [65], Enterobacter spp. [73], P. mirabilis [144], B. bronchiseptica [104]. RBS indicates a putative ribosome binding site, Start codon
indicates the translation initiation codon.
direct interaction with the K-subunit of RNA polymerase
[37].
Additional factors that may be involved in the environmental regulation of £agellar gene expression have been
identi¢ed, especially in lateral £agellar systems. For example, the heat shock proteins DnaK, DnaJ and GrpE and
the two-component system OmpR regulator may be involved in the control of motility by temperature [115]
and osmolarity, respectively, in E. coli [62,116,117] (Fig.
2). Moreover, the QseBC two-component system participating in quorum sensing, i.e. the complex communication
mechanism of cell-to-cell signalling linking cell density
with gene expression [118], has been shown to regulate
motility by activating the transcription of £hDC in E. coli
[119]. Similarly quorum-sensing regulators in V. cholerae
and P. aeruginosa also control motility together with the
expression of other virulence factors [120,121].
Nevertheless, the mechanism by which numerous other
environmental factors a¡ect bacterial motility remains
largely unknown, in particular regarding the perception
of external signal and the manner by which this signal
may a¡ect £agellar gene expression. Some adverse conditions have been shown to a¡ect £agellar gene transcription
at the top of the hierarchy in E. coli, as well as in strains
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of Enterobacter and Pseudomonas, in correlation with
changes in DNA topology [34,35,73]. For example, environmental factors, such as high temperature and high osmolarity, are known to induce changes in DNA topology
and regulation of gene expression [122^125] and also a¡ect
bacterial motility. In some cases, the control of gene expression by environmental factors is mediated by regulatory proteins that also a¡ect the DNA conformation of
regulatory regions, such as the nucleoid-associated protein
H-NS [89,126]. It is interesting to note that similarly to
H-NS [127,128], a role in DNA topology has been proposed for many proteins known to participate in the control of £agellum biosynthesis, such as the nucleoid protein
HU [129] and the DNA replication initiation factor DnaA
[130]. Similarly, other proteins of the bacterial nucleoid,
such as FIS and Lrp, participate in the regulation of £agellar motility in S. typhimurium and P. mirabilis [131,132].
These observations suggest that environmental conditions
may indirectly a¡ect bacterial motility via such global regulators.
The H-NS protein participates in chromosome organisation and plays a key role in bacterial responses to environmental cues, including acidic pH resistance [126]. This
regulatory protein is also known to positively control motility in enterobacteria [36,37,133,134] and this regulation
is, at least in some cases, mediated in concert with DNA
supercoiling [35,135]. The extended regulatory region of
the £hDC master operon seems to play a key role in this
process. Indeed, the DNA topology of this region, which
encompasses both promoter and £hDC mRNA 5P end, is
speci¢cally altered by H-NS and the resulting e¡ect on
DNA supercoiling has been shown to correlate remarkably well with the £hDC transcription level [135]. The
importance of this region in the control of £hDC expression is further supported by the existence of such an extended 5P end mRNA in £agellar regulatory genes of different bacteria (Fig. 5). On the other hand, the presence of
a 5P untranslated mRNA region has usually been associated with posttranscriptional regulation mechanisms in
E. coli and B. subtilis [136^138]. The recent data on
CsrA, an RNA binding protein that controls £hDC expression, raises the possibility of a posttranscriptional regulation of £agellar regulatory genes [38,39]. A probable
hypothesis would be that the control of £agellum biosynthesis by environmental factors might include speci¢c
DNA topology alterations of the entire £hDC regulatory
region in concert with various regulatory proteins acting
at the synthesis and/or the stability of the transcript. A
comprehensive analysis of these mechanisms will, however,
require further investigations in the future.
6. Comparison of £agellar cascades
A comparative analysis of bacterial £agellar cascades
highlights the existence of many similarities and important
di¡erences between peritrichous and polar £agellar systems (Fig. 3).
6.1. Functional similarities of £agellar systems
In most £agellated micro-organisms £agellar ¢laments
are composed of homologous £agellin protein, which not
only is functionally equivalent, but also shares conserved
amino acid sequence regions [139]. One exception is
archaeal £agellin having no homology with eubacterial
£agellins, but some similarities to type IV pilins [20]. Eubacterial £agellins in di¡erent Gram-negative and Grampositive micro-organisms, whatever the type of £agellation, have a distinctive domain structure, comprising conserved N- and C-terminal regions, responsible for the quaternary interactions between subunits, and a central
domain that may vary considerably in both amino acid
sequence and size, containing all of the potent antigenic
epitopes and responsible for £agellar antigenic variability
[139].
Flagellum genes are organised in an ordered cascade
with master regulators at the top of the hierarchy in
both polarly and laterally £agellated bacteria. Such a hierarchical organisation suggests that the ¢rst level might
constitute an important target for motility regulation by
external factors. For example, in C. crescentus, the polar
£agellar system responds to signals related to cellular cycle
[47,140] and in enterobacteria, the lateral £agellar system
responds to environmental factors [33,34]. Recent results
on natural isolates suggest that environmental conditions
a¡ect in a similar way motility of strains of Enterobacter
and Pseudomonas, even though they possess a di¡erent
£agellation type [73]. Moreover, like in E. coli [34], the
presence of a DNA gyrase inhibitor was shown to a¡ect
the expression of genes at the ¢rst level of the £agellar
cascade in both organisms, which suggests a similar link
between DNA supercoiling and environmental factors.
Similarly, a possible relationship between DNA supercoiling and £agellar gene expression has been recently proposed in Gram-negative bacteria, i.e. H. pylori [56] and
Y. enterocolitica [141], and in Gram-positive micro-organisms, i.e. L. monocytogenes [142]. These striking similarities suggest that several important steps in the control of
bacterial motility are evolutionarily conserved in polarly
and laterally £agellated bacteria.
An alignment of the £hDC sequences available in databases highlights a similar organisation of the regulatory
region in several Gram-negative bacteria with regard to
the position and sequence of CAP binding site, promoter
elements 335 and 310, transcriptional start site, ribosome
binding site and ATG initiation codon (Fig. 5A). One
conserved sequence with a palindrome structure was observed just downstream of the transcriptional start site,
e.g. TAGGAtTAtTCCTA in Y. enterocolitica. However,
any attempt to experimentally demonstrate its importance
in the expression of the £hDC operon was unsuccessful, at
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least in E. coli [135]. This does not rule out the possibility
of this sequence having a role in some regulatory processes
of the £hDC master operon, for example in concert with
CsrA posttranscriptional regulation (see above).
A similar organisation does not seem to be conserved in
L-proteobacteria, such as B. bronchiseptica [104], B. pertussis and in Bordetella parapertussis (http://www.sanger.ac.uk/Projects), as well as in Ralstonia solanacearum [143]
and Ralstonia eutropha (http://www.jgi.doe.gov/JGI_microbial/html/ralstonia/ralston_homepage.html), whose genome sequencing is in progress or has been recently completed (Fig. 5B). Indeed, a low similarity was observed in
the £agellar regulator genes of these bacteria. Their regulatory regions carry a putative c70 binding site, but unlike
enterobacterial sequences lack a CAP binding site. This
suggests the existence of di¡erent regulatory mechanisms,
as demonstrated for the negative regulation of £rAB £agellar operon transcription by BvgAS in B. bronchiseptica
[104]. Nevertheless, the presence of a long untranslated
region ranging from 197 bp in E. coli to 261 bp in Xenorhabdus nematophilus (Fig. 5A), and from 116 bp in Bordetella species to 132 bp in R. eutropha (Fig. 5B) in all
analysed sequences, including those from Bordetella and
Ralstonia, should be emphasised. More importantly, an
extended 5P untranslated region was also identi¢ed in master £agellar regulatory gene in polarly £agellated bacteria
of the Pseudomonas genus [73] and may be predicted in
P. £uorescens [75] and P. aeruginosa [28]. However, the
lack of nucleotide sequence conservation does not rule
out the existence of a general mechanism in Gram-negative bacteria, which may play a role in the regulation of
master £agellar genes.
Interestingly, some di¡erences exist in the transcriptional initiation of the £hDC operon among Enterobacteriaceae. A unique transcriptional start site was identi¢ed in
the £hDC promoter region of Enterobacter spp. [73], consistent with the £hDC single major start site observed in
E. coli [37] and in P. mirabilis [144] (Fig. 5A). In contrast,
six transcriptional start sites were identi¢ed within the upstream region of the S. typhimurium £hDC operon, one of
them corresponding to the start site identi¢ed in E. coli
[65] and in Enterobacter strains [73]. This could indicate a
possible di¡erential control between these organisms. Similarly, multiple transcriptional start sites were identi¢ed by
primer extension analysis in a natural isolate of Pseudomonas, including two major £eQ transcription initiation
sites [73]. In this respect, it should be emphasised that
transcription from the major transcriptional start site
was more sensitive to all adverse conditions tested, including the presence of gyrase inhibitor, than the second major
transcription start site. This could result from a di¡erent
rate of transcription from both promoters and/or mRNA
stability of the corresponding transcripts. Such a complex
promoter structure makes possible multiple regulations of
these genes in response to speci¢c environmental conditions.
515
Some particularities were also observed in £hDC master
operon regulation in di¡erent enterobacteria. In P. mirabilis, for example, new regulatory elements that a¡ect
£hDC master operon expression have been identi¢ed,
such as the £agellar export apparatus component FlhA,
UmoA, B, C, D, the Lon protease and the ¢mbrial gene
product MrpG [32,144^146]. In E. coli, new motility regulators, such as the RNA binding regulator CsrA and
HdfR and LrhA, two regulators of the LysR family,
were recently discovered [38,39,109,147,148]. In S. typhimurium, the ClpXP ATP-dependent protease was shown
to a¡ect the expression of £agellar regulon [149]. Moreover, in S. typhimurium, the Fur protein was shown to
positively regulate the expression of the £hDC master operon [150]. Between closely related enterobacterial species,
such as E. coli and S. typhimurium, some particularities
were also reported concerning the motility control by
H-NS protein and cAMP^CAP complex. In E. coli, each
of these regulators is essential for motility [37], whereas in
S. typhimurium, single mutants in the corresponding gene,
i.e. hns or crp, showed a reduced motility, while a complete lack of motility was only observed in an hns crp
double mutant [151].
The presence of numerous regulatory proteins controlling motility has been demonstrated mainly in enterobacterial systems, i.e. E. coli and S. typhimurium. The question arises whether it is possible to extrapolate such a
complex regulatory network to other bacterial £agellar
systems. The extensive sequencing of bacterial genomes
(http://igweb.integratedgenomics.com/GOLD) makes it
possible to search for regulatory genes homologous to
known £agellar regulators and to try to answer this question. In fact, some proteins homologous to known regulators may be identi¢ed in other organisms, but their role
in motility control remains to be demonstrated. In contrast, sequence homologous to known regulators are not
present in all bacteria, which suggests that other proteins
may ful¢l these functions.
For example, the presence of cAMP^CAP homologues
as well as the conservation of the cAMP^CAP binding site
in £hDC promoter regions (Fig. 5A) in several enterobacterial sequences make plausible the conservation of the
molecular mechanism of £hDC activation demonstrated
in E. coli [37]. More generally, cAMP is also necessary
for £agellar formation in V. cholerae [152] and both the
presence of CAP protein in this organism [153] and the
identi¢cation by genome sequencing of a CAP-like protein
(PA0652) in P. aeruginosa [154] allow us to speculate that
such a complex may also mediate the catabolite repression
of motility in polar £agellar systems. In fact, recent data
suggest a downregulation of the £eQ master regulatory
gene by Vfr, a CAP protein homologue in P. aeruginosa
[155].
Another example is the implication of nucleoid-associated proteins, including HU (see above), in the control of
bacterial motility. HU homologous proteins have been
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identi¢ed in many motile bacteria, such as laterally £agellated enterobacteria [156] and polarly £agellated P. aeruginosa [157] and P. putida [158], and also exist in P. £uorescens, V. cholerae, C. crescentus (SwissProt accession
numbers Q9KHS6, Q9KV83, Q9KQS9, O87475). Moreover, a HU homologue was identi¢ed in motile Grampositive bacteria, such as B. subtilis [159]. Thus, HU-like
proteins are conserved in many micro-organisms, even
though their implication in the motility control in all of
them remains to be determined.
The role in motility regulation of another nucleoid-associated protein family, H-NS, seems to be quite general.
The positive role of H-NS in enterobacteria and phylogenetically related micro-organisms [36,37,133,134], and the
implication of an H-NS-like protein, VicH, in the positive
control of polar £agellar synthesis in V. cholerae [160]
provide evidence that such proteins participate in the motility control of bacteria having di¡erent types of £agella
and so di¡erent types of regulatory cascades. However,
until now no H-NS-like proteins have been identi¢ed in
many other motile bacteria, in any Gram-positive bacterium, e.g. B. subtilis [161], and in some Gram-negative
bacteria, such as H. pylori and Pseudomonas spp. We
may hypothesise either that in these micro-organisms the
mechanism of motility control is entirely di¡erent from
enterobacterial systems, or more probably that other proteins play the role of H-NS-like proteins. In this respect,
the link between DNA topology and £agellar gene expression in di¡erent bacteria, including H. pylori [56] and
Pseudomonas spp. [73], suggests a possible implication of
functional homologues of nucleoid-associated proteins in
motility control in these micro-organisms.
Flagellar biosynthesis and cell division are known to be
co-regulated in E. coli [162,163] and FlhD is involved in
this process [162]. Similarly, it has been recently proposed
that FlrC, a polar £agellum regulatory protein, a¡ects cell
division in V. cholerae [30] and the existence of several
mechanisms that couple £agellar biosynthesis to cell cycle
was demonstrated in C. crescentus [47]. Moreover, recent
results [73] showed that the transcription of master regulator genes located at the top of the hierarchy is growth
phase-dependent in Enterobacter and Pseudomonas strains.
Similarly, £agellar expression in L. pneumophila is dependent on growth phase [164]. This provides evidence that
£agellar regulation is linked to the cell cycle in many micro-organisms.
Finally, in addition to its role in bacterial motility, FlhD
protein participates in the transcriptional regulation of
other cellular processes, such as anaerobic and aerobic
respiration [62,165,166], and FlhD2 C2 participates in the
synthesis of the £agellar export apparatus also used in
export of virulence factors, for example in Y. enterocolitica
[167^169] and in Serratia liquefaciens [170,171]. Likewise,
the master £agellar regulator plays a role in the control of
lipolysis, extracellular haemolysis, and virulence in insects
in X. nematophilus [172], in the expression of nuclease in
Serratia marcescens [173] and in the di¡erentiation into
swarmer cells in S. liquefaciens and P. mirabilis
[144,174]. The master polar £agellar regulators FleQ in
P. aeruginosa and AdnA in P. £uorescens play an important role not only in motility control, but also in the regulation of adhesion of these bacteria to substrates
[28,75,175]. The role of master £agellar regulator therefore
seems to be more general in bacterial physiology than the
sole control of bacterial motility.
6.2. Important di¡erences between £agellar systems
Several di¡erences in the structural organisation of £agellar systems must also be underlined (Fig. 3). Indeed,
these systems di¡er from each other by the existence of
speci¢c sigma factors and transcriptional activators, by
motive force and the e⁄ciency of motors. The involvement
of c54 and transcriptional activators associated with this
sigma factor in the regulatory cascade of the polar £agellar system must be emphasised, in addition to the existence of c28 £agellar-speci¢c factor also present in the
lateral £agellar regulatory system. In this respect, the polar
£agellar hierarchy in V. cholerae [31] constitutes a combination of both enterobacterial c28 -dependent and C. crescentus c54 -dependent systems.
The motor of peritrichous £agella in E. coli uses the
energy of the proton transmembrane gradient [176,177]
and the motor of polar £agella in V. cholerae is sodiumdriven [178]. These di¡erent motors allow £agellum rotation speeds of about 15 000 rpm [179] and 100 000 rpm
[180], respectively. In V. parahaemolyticus, which possesses
both £agellar systems, the polar £agellum uses a sodium
transmembrane potential, whereas the lateral £agellar system is proton-driven [181]. The functional di¡erence between polar and lateral £agella was demonstrated in a
viscous environment for Vibrio alginolyticus, lateral £agella being more e⁄cient than polar ones under the highviscosity conditions [182]. A possibility to create hybrid
motors, in which the motor proteins of V. cholerae,
PomA, PomB, MotX, and MotY, are replaced by the
E. coli proteins, MotA and MotB [183], has been recently
published. In V. cholerae, such a hybrid system runs using
the proton transmembrane potential, as in E. coli. The
analysis of this hybrid motor may help to understand
the mechanism of high-speed sodium-driven £agellar rotation. Indeed, the MotA and MotB proteins may serve to
stabilise the motor in the membrane resulting in an increase in rotation speed or to form a PomAB-independent
canal to improve the e⁄ciency of energy conversion.
Another di¡erence is the presence of multiple £agellins
especially in polar systems (for example in V. cholerae)
suggesting the existence of a di¡erential regulation of £agellar biosynthesis. In this case the control of motility is
made possible by the capacity to synthesise the £agellum
with properties adapted to speci¢c environmental conditions [50] and to avoid a strong immune response in the
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host by antigenic modi¢cations [51]. Di¡erent £agellin
genes are transcribed with di¡erent sigma factors (Table
1). For example, in H. pylori, two £agellin genes, £aA and
£aB, are regulated by two di¡erent sigma factors (c28 and
c54 , respectively), suggesting that these genes may be differently expressed depending on environmental conditions
[56]. Thus, H. pylori may be able to produce £agella suited
for motility in a given environment by changing £agellar
parameters. Instead of a single £agellin, i.e. FliC in E. coli,
the polar £agella of V. parahaemolyticus [23,42], Vibrio
anguillarum [184], and V. cholerae [50] are composed of
¢ve or six di¡erent £agellin subunits. Despite their high
amino acid identity, only one £agellin, i.e. FlaA, is essential for motility and is transcribed with c54 factor in
V. cholerae [50]. However, some exceptions exist, like
the polar £agellum in P. aeruginosa, which is composed
of a single structural protein, FliC [185], while three £agellin genes, £eA, £eB and £eC, and two £agellin genes,
£iC and £jB, were identi¢ed in the laterally £agellated
bacteria Y. enterocolitica and S. typhimurium, respectively
[1,97].
Finally, the lateral £agellar biosynthesis system in enterobacteria includes three classes of genes, but the polar
£agellar systems in C. crescentus and V. cholerae are organised into four classes of genes. Such a more complex
organisation may provide additional possibilities for a ¢ne
temporal regulation of the £agellar system. Moreover, in
C. crescentus and in V. cholerae, an important step in the
class II^III transition is the phosphorylation of the FlbD
and FlrC regulators, after the completion of MS ring^
switch assembly [31]. In enterobacteria and probably in
V. cholerae, the class II^III and the II^IV transition, respectively, depend on FlgM anti-sigma factor export and,
thus, on the activation of FliA, the speci¢c £agellar c28
factor, after the assembly of the basal body^hook structure [1,31].
The presence of a speci¢c type of £agellar regulatory
cascade in di¡erent bacteria appears to be dependent on
the functional particularity of a given £agellar insertion
type, which seems to be itself related to speci¢c environments rather than to result from phylogenetic di¡erences
between organisms. Indeed, the polar and lateral £agellar
systems are widely distributed among micro-organisms, in
particular within K- to Q-subdivisions of Gram-negative
bacteria. Moreover, these two unrelated £agellar systems
may simultaneously function in some bacteria, such as
A. brasilense [24] and V. parahaemolyticus [23,42,51,52].
In this organism, distinct £agellar cascades control the
continuous expression of polar £agella and the induction
of lateral £agellar expression under speci¢c conditions,
respectively. Such a mixed £agellation is probably related
to the ecological niche and the environments where this
micro-organism resides, polar £agella being especially e⁄cient in liquid environments and lateral £agella being more
suitable for movement on surfaces and in highly viscous
environments [23,182].
517
7. Conclusions
The importance of motility for bacteria can be deduced
from the cells’ investment in synthesising £agella and from
the redundancy of these organelles. The constant interest
of microbiologists in bacterial £agellar regulatory systems
results from the recognition of the crucial role that motility plays in bacterial physiology. The research in this ¢eld
was also promoted by the role of £agella in virulence,
adhesion, bio¢lm formation and colonisation of host organisms in pathogenic bacteria. Moreover, £agellum biosynthesis is usually co-regulated together with other virulence factors within the same regulatory network.
The growing amount of information in the literature on
£agellar regulatory cascades in di¡erent bacteria reveals
the striking similarities in the general organisation of these
systems. Environmental conditions and growth phase affect motility of many bacteria in a similar manner. Moreover, the implication of DNA topology, nucleoid-associated proteins and/or the regulatory regions of master
genes might be general in organisms with di¡erent types
of motility cascades. The motility control may thus have
evolved towards similar mechanisms in di¡erent bacterial
systems. The particularities that exist among £agellar cascades may be linked to the speci¢c environments in which
di¡erent micro-organisms live. One could wonder why
such a similarity in the regulatory mechanism might appear during the evolutionary process. A plausible answer
is that within the same genus, di¡erent related bacteria
may encounter various environmental conditions. For example, pathogenic species, such as the opportunistic
pathogen of humans P. aeruginosa [154], are adapted to
their host environment, and natural isolates adapted to
water or soil environment, such as the soil bacterium
P. putida [186], are able to colonise the plant rhizosphere.
On the other hand, the same micro-organism encounters
di¡erent environments during its cellular cycle. V. cholerae
must be capable of surviving inside humans during the
colonisation phase and in water estuaries during the
free-swimming phase and therefore presents two distinct
phases in its life cycle with characteristic physiology adaptations [187]. In this respect, the appearance of a general
regulatory system during the evolutionary process lies
within cell economy considerations and survival advantages. Because of the multitude of ecological niches, evolution gave rise to such a conservation of general mechanism, di¡erent bacteria being capable of occupying
numerous environments requiring adaptation to di¡erent
external conditions. Therefore, a similar solution must be
found by bacterial cells to ensure an adequate and rapid
response to various environmental conditions. The large
number of genes involved in both the biosynthesis and
functioning of £agella, the numerous regulators and environmental factors implicated in motility control and the
multiple interactions with other cellular processes highlight
the extreme complexity of this regulatory network. Despite
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518
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the recent studies, multiple questions remain unanswered
regarding the molecular mechanisms that control bacterial
motility in response to environmental cues. For example,
the perception of environmental signals such as temperature, osmolarity or pH and the transmission of these
stimuli giving rise to an appropriate physiological response
remain poorly understood. In future, an e¡ort should be
made to elucidate these processes, in particular because of
the important role of motility in colonisation. The studies
in this ¢eld would bring important advances in the comprehension of bacterial physiology and interactions of bacteria with humans and plants.
During the past years the large development of methods
for global analysis of gene expression and macromolecular
interactions, associated with the sequencing of bacterial
genomes, has provided new tools for studying the regulatory processes at a molecular level. The application of
these methods for analysis of £agellar systems might
help to improve our understanding of molecular mechanisms governing £agellum biosynthesis in bacteria. As an
example, a global transcriptome analysis of suppressor
mutants has recently made it possible to establish a link
between H-NS and acid pH control of motility in E. coli
[35]. Similarly, DNA array analysis revealed the transcriptional regulation of several genes unrelated to motility by
FlhD, showing the general role of this £agellar regulator
in bacterial physiology [165,166]. At present, on the basis
of available information, only a preliminary model may be
drawn to describe complex £hDC regulation in E. coli
(Fig. 2). Genome-wide approaches are promising and
would allow the integration of multiple factors participating in £agellar control into a unique model.
The co-ordination of gene expression by regulatory networks is widespread in prokaryotes, as well as in eukaryotes [188,189]. Such regulatory systems are usually organised in a similar way in di¡erent organisms with master
regulators at the top of the hierarchy allowing the integration of external signals to ensure adequate cellular responses [190]. In this respect, the studies of £agellar biosynthesis, which integrate multiple components of
bacterial physiology, may constitute an excellent model
for the understanding of complex regulatory networks.
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