Regulation cascade of £agellar expression in Gram-negative bacteria

Olga A. Soutourina a, Philippe N. Bertin b;�

a Laboratoire de Biochimie, UMR 7654, CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, Franceb 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 environmentalconditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellatedbacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largelyunknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of thecascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatoryregions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similarhierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests anevolutionary 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

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5052. Chromosomal organisation of £agellar genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5073. Hierarchical organisation of £agellar systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5074. Master regulators of class I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509

4.1. FlhDC in lateral £agellar systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5094.2. Regulators of polar £agellar systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

5. Motility control by environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5126. Comparison of £agellar cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

6.1. Functional similarities of £agellar systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5146.2. Important di¡erences between £agellar systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

1. Introduction

Bacterial motility and chemotaxis is one of the complex

processes allowing £agellated micro-organisms to surviveunder a wide variety of environmental conditions by a co-ordinated control of their gene expression in response toexternal stimuli [1].Flagellar motility represents an important advantage for

bacteria in moving towards favourable conditions or inavoiding 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

* Corresponding author. Tel. : +33 (3) 90 24 20 08;Fax: +33 (3) 90 24 20 28.

E-mail address: bertin@gem.u-strasbg.fr (P.N. Bertin).

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www.fems-microbiology.org

lated bacteria to successfully compete with other micro-organisms [2]. For example, it has been recently demon-strated that a Fe(III) oxide-reducing bacterium, Geobactermetallireducens, speci¢cally expresses £agella and pili tomove towards the insoluble electron acceptor, whichmay explain the predominance of Geobacter species in awide variety of sedimentary environments [3]. In additionto having locomotive properties, bacterial £agella play acrucial role in adhesion, bio¢lm formation and colonisa-tion of micro-organisms, such as Pseudomonas aeruginosa[4], Escherichia coli [5], Vibrio cholerae [6], Salmonellatyphimurium [7] and Helicobacter pylori [8]. Motility inpathogenic micro-organisms is usually considered a viru-lence factor, essential for colonisation of host organism ortarget organ [9,10]. However, the £agellar ¢lament bearsstrong antigenic properties in contact with animal andplant hosts [11^13]. Furthermore, motility by means of£agella is very expensive for cellular economy in termsof the number of genes and the energy required for £a-gellar biosynthesis and functioning [1]. Consequently, it isnot surprising that the synthesis of £agella is highly regu-lated by external factors, including the interaction of bac-terial cells with their host (Fig. 1). With respect to thisdual pathogenicity/antigenicity e¡ect of £agella, it mustbe mentioned that several highly pathogenic bacteria,such as Bordetella pertussis [14,15], Shigella sp. [16], andYersinia pestis [17], in contrast to their close relatives Bor-detella 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 ofmotility has been associated with a frameshift mutation in£hD master regulatory gene-coding sequence [18], whilenucleotide sequence comparisons of £agellin cryptic genessuggest that loss of motility in Shigella is a recent evolu-tionary event [16]. The absence of motility in these speciesmay re£ect di¡erences in pathogenesis or life cycles and/orthe 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 micro-

bial world, and more than 80% of known bacterial speciespossess these organelles, including various £agellated Ar-chaea [19,20]. The structure and arrangement of £agellaon the cell di¡er from species to species and both seem tobe related to the speci¢c environments in which the cellsreside [21,22]. Flagella can be arranged on the cell body ina variety of con¢gurations, including single polar, multiplepolar, and many peritrichous (or lateral) con¢gurations.Some species, e.g. Vibrio parahaemolyticus [23] and Azo-spirillum brasilense [24] display a mixed £agellation andform two structurally unrelated £agellar types on thesame cell.Bacterial £agellum synthesis genes form an ordered cas-

cade in which the expression of one gene at a given levelrequires the transcription of another gene at a higher level[1]. At the top of the hierarchy is the £hDC master operonin enterobacteria [25^27], and £eQ or £rA master genes inP. aeruginosa [28,29] and V. cholerae [30,31], respectively.The organisation of the £agellar system has been exten-sively studied in enterobacteria and multiple levels of£hDC regulation were observed, such as transcriptionaland posttranscriptional control in E. coli (Fig. 2) and pro-tein stability control in Proteus mirabilis [32]. The expres-sion of the c

70-dependent £hDC operon is controlled bynumerous environmental signals, e.g. temperature, osmo-larity and pH [33^35] and by global regulatory proteins,such as H-NS and the cAMP^CAP (catabolite gene acti-vator protein) complex [36,37]. Moreover, the stability ofits mRNA is controlled by the RNA binding regulatorCsrA [38,39] (Fig. 2). In contrast, the regulation of mastergenes governing the synthesis of polar £agella remainslargely unknown. Based on the recent advances in this¢eld, the present review will focus on a comparative anal-ysis of £agellar regulatory cascades in di¡erent bacterial

Fig. 1. The role of motility in the interactions of bacteria with their natural environment.

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systems, especially in terms of motility gene regulation byenvironmental factors and regulatory proteins.

2. Chromosomal organisation of £agellar genes

Flagellar genes in many systems are grouped in severalclusters on bacterial chromosomes. In E. coli and inS. typhimurium nearly 50 genes, required for £agellum bio-synthesis and functioning, are organised in 15 and 17 op-erons, respectively [40,41], clustered in several regions [1].Cluster I (minute 24 and 23 on the E. coli and S. typhi-murium chromosomes, respectively) includes the genes en-coding £agellar structural proteins. Cluster II (minute 41and 40) includes the genes encoding the proteins partici-pating in the regulation of £agellar assembly, the £agellarmotor genes, motA and motB, and the chemotactic genes.Cluster III contains three regions on minute 43 and 40 onthe E. coli and S. typhimurium chromosomes, respectively,and includes the genes encoding £agellar structural pro-teins, export apparatus proteins and £agellar-speci¢cc factor.The complete analysis of polar £agellar biosynthesis sys-

tem in V. parahaemolyticus has been recently published[42]. The authors have identi¢ed 57 potential £agellargenes showing sequence homology with the componentsof bacterial motility and chemotaxis systems. These genesare grouped into operons in ¢ve regions on the bacterialchromosome, most of the genes being located in two ofthem. Chromosomal organisation of homologous genes isconserved in V. cholerae [31], Pseudomonas putida andP. aeruginosa [42]. In V. cholerae in particular [31], theorganisation of polar £agellar genes is almost identicalto 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 tworegions in V. parahaemolyticus : regions II and III are con-tiguous in this organism, but separated by about 48 kb inV. cholerae. Second, an additional £agellin gene was foundin V. parahaemolyticus, thus having six £agellins as analternative to the ¢ve found in V. cholerae.

3. Hierarchical organisation of £agellar systems

In Gram-negative bacteria, the hierarchy of the £agellarregulatory system was ¢rst well characterised in micro-or-ganisms with peritrichous £agella, such as E. coli andS. typhimurium [25^27]. This regulatory cascade possessesthree classes of genes. Class I genes form the £hDC masteroperon at the top of the hierarchy, which encodes aFlhD2C2 transcriptional activator of the second classgene expression. The majority of class II genes encodecomponents of the £agellar export system and the basalbody. The £iA gene at this second level encodes a sigmafactor, c28 (or RpoF), speci¢c for £agellar genes [43]. Theoperons of class III are positively regulated by c

28 andnegatively controlled by an anti-sigma factor, FlgM[44,45]. The anti-sigma factor is retained inside the celluntil the £agellar basal body and hook are completed[46]. At that time, the FlgM protein is exported to allowactivation by c

28 of the transcription of class III genes,which encode the components of £agellar ¢lament (e.g.£agellin, FliC), hook-associated, motor, and chemotaxisproteins (Fig. 3A).Another £agellar system has also been well documented.

In Caulobacter crescentus, which is a polarly £agellatedmicro-organism (see Table 1), £agellar gene expression is

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 ofFlhDC synthesis are indicated: +, positive e¡ect ; 3, negative e¡ect; no indication, positive or negative e¡ect.

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strongly linked to the control of the cell cycle, which isdistinguished by an asymmetric cell division [47]. The reg-ulatory cascade includes four classes of genes. The uniqueclass I gene, ctrA, is situated under the control of cell cyclesignals. CtrA regulates the c

70-dependent transcription ofthe second class of genes encoding the subunits of mem-brane/supramembrane (MS) ring complex and export sys-tem. The gene encoding the regulator FlbD of the NtrCfamily of c54-associated transcriptional activators (see Sec-tion 4.2) [48,49] belongs to this class. In concert with c

54,this regulatory protein is essential for the expression ofclass III genes encoding both the basal body and hookstructure and of the class IV genes encoding the three £a-gellin subunits of £agellar ¢lament.Recently, the regulatory cascade components of bacteria

with one polar £agellum, such as V. cholerae and P. aeru-ginosa, have been characterised [28^31] (Fig. 3B, Table 1).In V. cholerae [31], the class I gene at the top of thehierarchy encodes the FlrA protein, a c

54-associated tran-scription activator of the NtrC family. This regulator, to-gether with c

54, activates the expression of the class II

genes encoding structural components of the MS ring,switch and export apparatus, the c

28 £agellar-speci¢c sig-ma factor, FliA, and FlrB and FlrC, the sensor kinase andtranscriptional regulator, respectively, of a two-componentsignal-transducing system. The FlrC regulator along withc54 activates the expression of the class III genes encodingthe basal body, the hook and the £agellin essential formotility, FlaA. The sigma factor c

28 is required for thetranscription of the class IV genes, including the motorgenes 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 anti-genic and environmental variations in £agellar ¢lamentcomposition [50,51]. Surprisingly, in V. parahaemolyticus,entirely distinct lateral and polar £agellation systems havebeen identi¢ed, the lateral £agellar components being ho-mologous to enterobacterial systems [52] and the polar£agellar system components being homologous to Vibrio-naceae systems [42,51].The implication of c54 in bacterial motility regulation is

widespread in Gram-negative bacteria. Indeed, the ele-

Fig. 3. Flagellation cascades. Lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae, Vibrionaceae families) £agellar cascades are comparedand the factors controlling master regulator expression and the connections with other cellular processes are shown. A: The lateral £agellation cascadediscovered in Enterobacteriaceae with the £hDC master operon at the top (class I) encoding the FlhD2C2 transcriptional activator of class II genes in-cluding £agellar-speci¢c c

28 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 c

54-associated NtrC-typetranscriptional activators, FleQ and FlrA, respectively (see Table 1), which activate the transcription of class II genes, such as £eSR and £rBC. Bothare two-component regulatory systems for class III genes that include £agellar ¢lament genes £iC and £aA, in Pseudomonadaceae and Vibrionaceae, re-spectively. Another class II gene, £iA, encodes the c

28 £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 regu-latory gene expression are shown in ovals at the top. Other cellular processes controlled by £agellar regulators are indicated in boxes at the left and theright sides.

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ments of £agellar regulatory cascades including c54-asso-

ciated regulators have also been recently identi¢ed in mi-cro-organisms, such as Rhodobacter sphaeroides [53^55],H. pylori [56] and Campylobacter jejuni [57], whose ge-nomes have been recently deciphered (http://igweb.integra-tedgenomics.com/GOLD) (Table 1). In R. sphaeroides, the£agellar regulatory system is supposed to be a combina-tion of the elements present in enterobacteria and inC. crescentus systems including the c

54-associated regula-tors of class I, the c

54-dependent class II genes and thec28-dependent class III genes (e.g. £agellin-encoding £iCgene) [55]. Similarly as in C. crescentus, the c

54 and themaster transcriptional activator of the NtrC family, FlgR,are required for the transcription of the genes encoding thebasal body, the hook structures, and the £agellin FlaB inH. pylori. In contrast, the expression of the major £agellin,FlaA in this bacterium, is controlled by the c

28, as inenterobacteria, and repressed by FlgR.A particular £agellar hierarchy, sharing some similar-

ities with that of enterobacteria systems, has been recentlyidenti¢ed in the K-proteobacterium Sinorhizobium (Rhi-zobium) meliloti [58]. At the top of the hierarchy is themaster operon, visNR, encoding the global regulatorsVisNR that control motility and chemotaxis genes inthis organism. The VisN and VisR proteins, belongingto the LuxR family with characteristic DNA and ligandbinding domains, form a heterodimer. For the activationof the transcription by these regulators, the binding of ayet unidenti¢ed e¡ector is necessary. The main di¡erenceswith the system of enterobacteria are the existence of novel

master activators and the position of motor genes in thesecond class rather than in the third class of genes.Finally, in Gram-positive bacteria, such as Bacillus sub-

tilis, some gene families have been characterised thatroughly correspond in their structure to the enteric classII and class III genes, including c

D factor, homologous toc28 in enterobacteria with nearly identical promoter spec-i¢city and function, and FlgM anti-sigma factor, whichantagonises cD activity. The regulatory genes correspond-ing to the class I master £agellar regulators have not yetbeen identi¢ed in this organism and some data indicatethat c

D may occupy a central position in the £agellarregulatory 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. typhi-murium, the £hDC operon is at the top of the cascade andencodes FlhD2C2 activator required for the expression ofall other genes of the £agellar regulon, FlhD alone havinglarger regulatory functions (see below) (for a recent reviewsee [62,63]). The FlhD (13.3 kDa) and FlhC (21.5 kDa)proteins form a heterotetrameric complex for activation oftranscription of £agellar class II genes [64,65], the proteinFlhD alone being not capable of binding to DNA andactivating transcription [64]. The sequence TT(T/A)-GCCGATAACG in their promoter regions has been

Table 1Polar £agellar genes and their function

Species Gene function

Master c54

regulatory geneTwo-component c54

regulatory systemaSpeci¢c c

28

factorAnti-c28 factor Filament £agellinb

Pseudomonas aeruginosa £eQ £eSR (FleS: S, FleR: R) £iA £gM £iCVibrio cholerae £rA £rBC (FlrB: S, FlrC: R) £iA £gM £aA (c54) ; £aB, £aC, £aD,

£aE (c28)Vibrio parahaemolyticus £aK £aLM (FlaL: S, FlaM: R) £iA £gM £aA (c28) ; £aB (c28), £aC,

£aD (c28), £aE (c28)Pseudomonas £uorescens adnA £eSR (FleS: S, FleR: R) £iA Contig 458 50% PA3351

of P. aeruginosa,28% £gM of V. cholerae

£iC

Helicobacter pylori ^ £gR (HP703) : R, HP244: S £iA £gM £aA (c28), £aB (c54)Campylobacter jejuni £gR (Cj1024c) : R, Cj073: SCaulobacter crescentus ^ £bD, £bE (FlbE: S, FlbD: R) ^ ^ £jJ, £jK, £jLPseudomonas putidaKT2440

82% £eQ ofP. aeruginosa

74% £eS and £eR ofP. aeruginosa

82% £iA ofP. aeruginosa

53% hypothetical £gMof P. aeruginosa ; 35%£gM of V. cholerae

40% £iC of P. aeruginosa

Pseudomonas syringae 81% £eQ ofP. aeruginosa

75% £eS and 76% £eR ofP. aeruginosa

81% £iA ofP. aeruginosa

53% hypothetical £gMof P. aeruginosa ; 32%£gM of V. cholerae

24% £iC of P. aeruginosa

Desulfovibrio vulgaris 38% £rA ofV. cholerae

25^28% £rB and 38% £rC ofV. cholerae

34% £iA ofV. cholerae

30% £gM of V. cholerae 38% £aA of V. cholerae

The % numbers mean the percentage identity of homologous sequence in a given organism with the indicated gene sequence.aFor two-component regulatory systems in parentheses, S is the sensor kinase protein component, R is the regulatory protein component.bFor ¢lament £agellin in parentheses is indicated a c factor participating in £agellin gene transcription.

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suggested to serve as a binding site for the complexFlhD2C2 [66]. However, footprinting analysis did not al-low identi¢cation of any consensus sequence in E. coli [64].Nevertheless, a recent study on FlhD2C2 binding to theP. mirabilis class II £agellar promoters combined withanalysis of several sequences of the class II £agellar pro-moter regions of E. coli and S. typhimurium [67] identi¢edan imperfect palindrome with two 17^18-bp inverted re-peats (FlhD2C2 boxes: TNAA(C/T)G(C/G)N2=3AAA-TA(A/G)CG) separated by a 10^11-bp spacer showingno consensus. Such a symmetric structure of binding sitesis in accordance with the proposed model of heterotetra-meric FlhD2C2/DNA complex formation. The di¡erencesbetween class II promoter DNA targets may explain thedi¡erential a⁄nity of FlhD2C2 contributing to the selec-tion of promoters to be activated in a temporal sequence[67]. FlhD and FlhC proteins belong to the transcriptionalfactors of class I, the C-terminal domain of the K-subunitof RNA polymerase being essential for the activation oftranscription [68]. Recently, the crystal structure of FlhDwas obtained at 1.8 AO resolution [69] showing that thisprotein is present as a homodimer with a disul¢de bridgebetween Cys65 residues. The presence of such a disul¢debridge in the cytoplasmic protein is surprising, consideringthe reducing environment of cytoplasm in E. coli. Thereplacement of Cys65 by alanine a¡ects neither the ca-pacity of FlhD protein to control transcription of £agellargenes, nor the stability of FlhD dimer. As supposed by theauthors [69], this disul¢de bridge may be necessary foranother intra- or extracellular yet unknown function ofFlhD. The conservation of this residue Cys65 in di¡erententerobacteria favours this hypothesis [69]. The C-terminalpart (residues 83^116) of the protein is £exible and carriesa putative helix-turn-helix (H-T-H) motif responsible forDNA binding. The conformation of this motif is stabilisedonly when FlhD is complexed with other proteins, such asFlhC. 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 con-formation and the speci¢city of binding via the H-T-Hmotif. The extensive alanine scanning analysis allowedthe identi¢cation of residues implicated in the interactionsbetween FlhD and FlhC, as well as in the interactions withDNA [70]. In contrast, Claret and Hughes [71] suggestthat the protein FlhC, and not FlhD, is the DNA bindingcomponent of the FlhD2C2 complex. This suggestion isbased on the fact that, unlike FlhD, the FlhC proteinalone is capable of binding DNA but with a 10-fold lowera⁄nity than in complex with FlhD. These authors alsoidenti¢ed a potential H-T-H motif in the FlhC proteinsequence. However, the results of Campos et al. [69,70]do not rule out the possibility that FlhC also participatesin DNA recognition and/or binding. More recent data ofClaret and Hughes [67] suggest that FlhC and FlhD sub-units contact DNA and contribute together to stabilityand speci¢city of the interaction. Determination of the

FlhD/FlhC crystal structure, the initial steps of whichhave been recently reported [72], will contribute to a betterunderstanding of these protein^protein and protein^DNAinteractions.Recently, sequences encoding FlhDC homologous pro-

teins have been identi¢ed in di¡erent enterobacteria[70,73]. A sequence alignment of FlhD proteins suggestsa high degree of structural conservation among di¡erentspecies, in particular for several important residues identi-¢ed by crystallographic analysis, for example Cys65 form-ing a disul¢de bridge or Gly93 in the H-T-H motif (seeabove). This further supports the essential role that theseresidues might play in the functioning of this type of pro-tein, in particular in dimer formation and in DNA bind-ing.

4.2. Regulators of polar £agellar systems

The regulators at the top of the polar £agellar hierarchybelong to the NtrC family of c54-associated transcriptionactivators. In addition to its role in polar £agellar geneexpression in P. aeruginosa, V. cholerae, C. crescentus,Pseudomonas £uorescens [30,74^76], this alternative sigmafactor participates in the transcription of genes havingdi¡erent physiological functions, such as nitrogen assimi-lation in E. coli and S. typhimurium [48,77] and pilin syn-thesis in P. aeruginosa and Neisseria gonorrhoeae [78]. TheRNA polymerase in complex with c

54 (RpoN) requires anactivator protein for transcription initiation [49]. Such reg-ulators usually bind to the promoter region and activatetranscription via the direct contact with RNA polymerasein complex with c

54 [48]. The activity of these activators ismodulated in response to environmental changes. Recentstudies on FleQ regulatory mechanism in P. aeruginosa[79] revealed the absence of consensus sequences for acti-vation of transcription and the atypical location of prob-ably the majority of enhancer binding sites on FleQ-regu-lated promoters.The complete sequence determination of many bacterial

genomes provides new insight into £agellar regulators(http://igweb.integratedgenomics.com/GOLD). In additionto the recently identi¢ed master £agellar regulators inV. cholerae, V. parahaemolyticus, P. aeruginosa, P. £uores-cens, genes showing high sequence homology with themaster £agellar regulatory gene £eQ also exist in P. putidaand P. syringae (Table 1). The homology search for otherc54-associated £agellar regulators in some polarly £agel-lated bacteria, whose genome sequencing is in progress,e.g. Desulfovibrio vulgaris, makes it possible to identifyproteins having only limited sequence homology withknown polar £agellar regulators (Table 1). Such an anal-ysis may be complicated by high sequence homology with-in the NtrC family of c

54-associated regulators, whichcontrol various physiological processes. This observationmay also suggest the existence of entirely new but yetunidenti¢ed £agellar regulatory proteins.

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Unlike FlhD protein, structural data on polar £agellarregulators are limited. However, some information isavailable on the structure of both the DNA binding andthe receiver domains of related NtrC proteins [80,81]. Allproteins identi¢ed until now share structural and function-al domains conserved among the NtrC family of transcrip-tional activators that work in concert with RpoN [28].Despite a relatively low homology in the N-terminal re-gion (Fig. 4), these proteins possess residues believed to beinvolved in their phosphorylation [82], e.g. the acid pock-et-forming residues Asp11 and Asp12 present in all se-quences with the exception of FlbD of C. crescentus. Incontrast, two other sites corresponding to the phosphory-lation site Asp54 and the salt bridge-forming residueLys104 in other NtrC family proteins are not conservedin most of the master polar £agellar regulators except for

the FlgR regulators of H. pylori and C. jejuni, and theFlbD regulator of C. crescentus. This is in agreementwith the presumed absence of a cognate kinase for theregulators at the top of the hierarchy of polar £agellarsystems [28]. The activity of these regulators may bechanged by phosphorylation at a serine or a threonineresidue present at position 54 instead of Asp54 in otherNtrC family proteins or may be controlled by a novelsignal-transducing mechanism. But the absence of exper-imental evidence of such an e¡ect and of any cognate ki-nase favours the hypothesis that these regulators probablydo not require phosphorylation for their activation. Alter-natively, external signals may in£uence motility at the levelof master regulatory gene expression, as recently demon-strated for the regulation of £eQ transcription by environ-mental factors in Pseudomonas sp. [73]. In contrast, FlgR

Fig. 4. Protein sequence alignment of regulators of the polar £agellar gene system. The multiple alignment was performed by the CLUSTALW methodand 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. £uores-cens (GenBank accession number AF312695); FlrA_vibch, FlrA of V. cholerae (GenBank accession number AF014113); FlaK_vibpa, FlaK of V. para-haemolyticus (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). Thearrowheads indicate the position corresponding to conserved residues involved in the phosphorylation of the NtrC family transcriptional activators. Theconserved amino acids constituting the ATP-binding site and the DNA-binding site (H-T-H motif) are boxed.

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proteins in H. pylori and C. jejuni contain all four con-served residues known to be involved in phosphorylationby a speci¢c kinase suggesting a classical phosphorelaymechanism. In fact, the cognate sensor kinase HP244 pro-tein 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 theATP binding site and/or the H-T-H DNA binding elementof the various analysed proteins (Fig. 4). Nevertheless, theabsence in the FlgR sequence of H. pylori of the H-T-Hmotif conserved in all analysed regulators must be em-phasised. This protein has been proposed to be the speci¢cmaster activator of transcription of c54-regulated £agellargenes in H. pylori [56]. This could be related to the exis-tence in FlgR of a DNA-binding domain di¡erent fromthat present in regulators playing a more general role invarious micro-organisms.

5. Motility control by environmental factors

Motility and the chemotaxis system play a crucial rolein the adaptation of micro-organisms to multiple environ-mental conditions. These external factors a¡ect not onlythe physiology of motility via the chemotaxis system andthe functioning of £agella (for a recent review see [84^86]),but also the process of £agellum biosynthesis via the con-trol of expression of £agellar genes at the top of the reg-ulatory cascade. In E. coli, £agellum biosynthesis is inhib-ited by catabolite repression in the presence of D-glucose,under conditions of high temperature or high concentra-tion of salts, in the presence of carbohydrates or of low-molecular-mass alcohols, at extreme pH and in the pres-ence of DNA gyrase inhibitors [33,35,87] (Fig. 2). More-over, the £agellin expression in E. coli is known to beregulated by various metal ions [88], while oxygen-limitedconditions were shown to induce £agellar gene transcrip-tion in E. coli [89]. In the enteric pathogen Salmonellaserotype enteritidis, pH and temperature a¡ect £agellumproduction [90]. In Campylobacter coli, the expression of£agellar genes is also known to be modulated by severalgrowth conditions such as pH, temperature, the composi-tion of the growth atmosphere, the concentration of inor-ganic salts and divalent ions [91]. Similarly, motility inV. cholerae is a¡ected by temperature, NaCl, pH and or-ganic nutrients [92]. In addition, sodium salicylate inhibitsmotility in E. coli, Proteus, Providencia and Pseudomonasspp. in relation with osmoregulation [93]. A negative e¡ectof sodium deoxycholate on £agellum production and mo-tility 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 recentlyrevealed as a new environmental signal molecule involvedin £agellar gene regulation in Halobacillus halophilus [95]while the role of inorganic phosphate was demonstrated in

the motility of several pathogenic bacteria, such asP. aeruginosa, V. cholerae, S. enterica, E. coli and Kleb-siella pneumoniae [96]. Finally, the control of motility bytemperature, in relation with the transition betweengrowth outside and inside the host, has been reported invarious pathogenic bacteria, such as Y. enterocolitica [97],Listeria monocytogenes [98], B. bronchiseptica [14], Legio-nella pneumophila [99] and Actinobacillus pleuropneumoniae[100].A possible mechanism of environmental regulation of

motility may be proposed in some cases. In pathogenicbacteria, two-component signal-transducing systems areusually involved in the control of both motility and viru-lence. In these systems environmental signalling is per-formed via a phosphorylation cascade, i.e. the sensing ofexternal factors is mediated by a sensor kinase activitythat phosphorylates and, thus, activates the correspondingtranscriptional regulator. The polar £agellar regulatorycascade contains such two-component regulatory systems.One example is the co-ordinated control of the expres-sion of virulence and motility genes in V. cholerae bythe ToxR transcriptional regulatory system in responseto environmental changes in pH, osmolarity and temper-ature [6,101^103]. In other pathogenic bacteria, such asB. bronchiseptica, motility and virulence genes are co-ordinately regulated by the two-component system BvgASin response to environmental stimuli, including tem-perature. Inside the host, this system represses £agellarmaster operon transcription and activates the expressionof virulence factors [14,15,104^106]. Another member oftwo-component signal-transducing systems, SirA, partici-pates in the control of motility and virulence in variousbacteria of genera Pseudomonas, Vibrio and Erwinia [107]and in L. pneumophila [108]. Its orthologue UvrY has asimilar function in E. coli [109]. In V. parahaemolyticus, anovel operon scrABC has been shown to inversely a¡ecttwo gene systems that are pertinent for colonisation ofsurfaces, i.e. swarming and capsular polysaccharides[110].On the other hand, the molecular mechanism by which

catabolite repression a¡ects £agellar biosynthesis in E. colihas been recently elucidated. This phenomenon is ob-served in the presence of D-glucose, when the concentra-tion of cAMP in the cell is low and CAP is present essen-tially in a form that is unable to activate the transcriptionof speci¢c genes without its cAMP ligand. With respect tomotility, the catabolite repression of £agellar gene expres-sion was observed many years ago [87,111], and implica-tion of the cAMP^CAP complex in this process has beenproposed on the basis of the non-motile phenotypes ofE. coli and S. typhimurium strains mutated in cya and/orcrp genes encoding adenylate cyclase and CAP, respec-tively [111^114]. Recent experimental data provide evi-dence that the cAMP^CAP complex positively controlsthe biosynthesis of £agella by activation of £hDC masteroperon transcription via binding to its promoter and a

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direct interaction with the K-subunit of RNA polymerase[37].Additional factors that may be involved in the environ-

mental regulation of £agellar gene expression have beenidenti¢ed, especially in lateral £agellar systems. For exam-ple, the heat shock proteins DnaK, DnaJ and GrpE andthe two-component system OmpR regulator may be in-volved 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 partici-pating in quorum sensing, i.e. the complex communicationmechanism of cell-to-cell signalling linking cell density

with gene expression [118], has been shown to regulatemotility by activating the transcription of £hDC in E. coli[119]. Similarly quorum-sensing regulators in V. choleraeand P. aeruginosa also control motility together with theexpression of other virulence factors [120,121].Nevertheless, the mechanism by which numerous other

environmental factors a¡ect bacterial motility remainslargely unknown, in particular regarding the perceptionof external signal and the manner by which this signalmay a¡ect £agellar gene expression. Some adverse condi-tions have been shown to a¡ect £agellar gene transcriptionat the top of the hierarchy in E. coli, as well as in strains

Fig. 5. Nucleotide sequence alignment of regions encompassing the promoters and the +1 translational start site of £hDC-like genes (A) and £rA-likegenes (B). The multiple alignment was performed by the CLUSTALW method and re¢ned manually [191]. A: Enterobacterial £hDC nucleotide sequen-ces are indicated as follows: £hD-ecoli, E. coli (GenBank accession number AE005411); £hD-salty, S. typhimurium (GenBank accession numberD43640); £hD-entsp, Enterobacter strain 22 (GenBank accession number AJ308469); £hD-erwca, Erwinia carotovora (GenBank accession numberAF130387); £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-Proteobacteri-al sequences are indicated as follows: £rA-borbr, B. bronchiseptica (GenBank accession number U17998, http://www.sanger.ac.uk/Projects/B_bronchisep-tica), £rA-borpe, B. pertussis (http://www.sanger.ac.uk/Projects/B_pertussis), £rA-borpa, B. parapertussis (http://www.sanger.ac.uk/Projects/B_parapertus-sis), £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 indicatesthe length in bp of missing region. The positions of the CRP (cAMP^CAP complex) binding site, the 310 and 335 promoter sequences and the tran-scriptional start sites (+1) are boxed. The transcriptional start sites have been identi¢ed experimentally in E. coli [37], S. typhimurium (one of six tran-scriptional start sites) [65], Enterobacter spp. [73], P. mirabilis [144], B. bronchiseptica [104]. RBS indicates a putative ribosome binding site, Start codonindicates the translation initiation codon.

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of Enterobacter and Pseudomonas, in correlation withchanges in DNA topology [34,35,73]. For example, envi-ronmental factors, such as high temperature and high os-molarity, are known to induce changes in DNA topologyand regulation of gene expression [122^125] and also a¡ectbacterial motility. In some cases, the control of gene ex-pression by environmental factors is mediated by regula-tory proteins that also a¡ect the DNA conformation ofregulatory regions, such as the nucleoid-associated proteinH-NS [89,126]. It is interesting to note that similarly toH-NS [127,128], a role in DNA topology has been pro-posed for many proteins known to participate in the con-trol of £agellum biosynthesis, such as the nucleoid proteinHU [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 £ag-ellar motility in S. typhimurium and P. mirabilis [131,132].These observations suggest that environmental conditionsmay indirectly a¡ect bacterial motility via such global reg-ulators.The H-NS protein participates in chromosome organi-

sation and plays a key role in bacterial responses to envi-ronmental cues, including acidic pH resistance [126]. Thisregulatory protein is also known to positively control mo-tility in enterobacteria [36,37,133,134] and this regulationis, at least in some cases, mediated in concert with DNAsupercoiling [35,135]. The extended regulatory region ofthe £hDC master operon seems to play a key role in thisprocess. Indeed, the DNA topology of this region, whichencompasses both promoter and £hDC mRNA 5P end, isspeci¢cally altered by H-NS and the resulting e¡ect onDNA supercoiling has been shown to correlate remark-ably well with the £hDC transcription level [135]. Theimportance of this region in the control of £hDC expres-sion is further supported by the existence of such an ex-tended 5P end mRNA in £agellar regulatory genes of dif-ferent bacteria (Fig. 5). On the other hand, the presence ofa 5P untranslated mRNA region has usually been associ-ated with posttranscriptional regulation mechanisms inE. coli and B. subtilis [136^138]. The recent data onCsrA, an RNA binding protein that controls £hDC ex-pression, raises the possibility of a posttranscriptional reg-ulation of £agellar regulatory genes [38,39]. A probablehypothesis would be that the control of £agellum biosyn-thesis by environmental factors might include speci¢cDNA topology alterations of the entire £hDC regulatoryregion in concert with various regulatory proteins actingat the synthesis and/or the stability of the transcript. Acomprehensive analysis of these mechanisms will, however,require further investigations in the future.

6. Comparison of £agellar cascades

A comparative analysis of bacterial £agellar cascadeshighlights the existence of many similarities and important

di¡erences between peritrichous and polar £agellar sys-tems (Fig. 3).

6.1. Functional similarities of £agellar systems

In most £agellated micro-organisms £agellar ¢lamentsare composed of homologous £agellin protein, which notonly is functionally equivalent, but also shares conservedamino acid sequence regions [139]. One exception isarchaeal £agellin having no homology with eubacterial£agellins, but some similarities to type IV pilins [20]. Eu-bacterial £agellins in di¡erent Gram-negative and Gram-positive micro-organisms, whatever the type of £agella-tion, have a distinctive domain structure, comprising con-served N- and C-terminal regions, responsible for the qua-ternary interactions between subunits, and a centraldomain that may vary considerably in both amino acidsequence and size, containing all of the potent antigenicepitopes and responsible for £agellar antigenic variability[139].Flagellum genes are organised in an ordered cascade

with master regulators at the top of the hierarchy inboth polarly and laterally £agellated bacteria. Such a hier-archical organisation suggests that the ¢rst level mightconstitute an important target for motility regulation byexternal 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 systemresponds to environmental factors [33,34]. Recent resultson natural isolates suggest that environmental conditionsa¡ect in a similar way motility of strains of Enterobacterand Pseudomonas, even though they possess a di¡erent£agellation type [73]. Moreover, like in E. coli [34], thepresence of a DNA gyrase inhibitor was shown to a¡ectthe expression of genes at the ¢rst level of the £agellarcascade in both organisms, which suggests a similar linkbetween DNA supercoiling and environmental factors.Similarly, a possible relationship between DNA supercoil-ing and £agellar gene expression has been recently pro-posed in Gram-negative bacteria, i.e. H. pylori [56] andY. enterocolitica [141], and in Gram-positive micro-organ-isms, i.e. L. monocytogenes [142]. These striking similar-ities suggest that several important steps in the control ofbacterial motility are evolutionarily conserved in polarlyand laterally £agellated bacteria.An alignment of the £hDC sequences available in data-

bases highlights a similar organisation of the regulatoryregion in several Gram-negative bacteria with regard tothe position and sequence of CAP binding site, promoterelements 335 and 310, transcriptional start site, ribosomebinding site and ATG initiation codon (Fig. 5A). Oneconserved sequence with a palindrome structure was ob-served just downstream of the transcriptional start site,e.g. TAGGAtTAtTCCTA in Y. enterocolitica. However,any attempt to experimentally demonstrate its importancein the expression of the £hDC operon was unsuccessful, at

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least in E. coli [135]. This does not rule out the possibilityof this sequence having a role in some regulatory processesof the £hDC master operon, for example in concert withCsrA posttranscriptional regulation (see above).A similar organisation does not seem to be conserved in

L-proteobacteria, such as B. bronchiseptica [104], B. per-tussis 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_mi-crobial/html/ralstonia/ralston_homepage.html), whose ge-nome sequencing is in progress or has been recently com-pleted (Fig. 5B). Indeed, a low similarity was observed inthe £agellar regulator genes of these bacteria. Their regu-latory regions carry a putative c70 binding site, but unlikeenterobacterial sequences lack a CAP binding site. Thissuggests the existence of di¡erent regulatory mechanisms,as demonstrated for the negative regulation of £rAB £a-gellar operon transcription by BvgAS in B. bronchiseptica[104]. Nevertheless, the presence of a long untranslatedregion ranging from 197 bp in E. coli to 261 bp in Xeno-rhabdus nematophilus (Fig. 5A), and from 116 bp in Bor-detella species to 132 bp in R. eutropha (Fig. 5B) in allanalysed sequences, including those from Bordetella andRalstonia, should be emphasised. More importantly, anextended 5P untranslated region was also identi¢ed in mas-ter £agellar regulatory gene in polarly £agellated bacteriaof the Pseudomonas genus [73] and may be predicted inP. £uorescens [75] and P. aeruginosa [28]. However, thelack of nucleotide sequence conservation does not ruleout the existence of a general mechanism in Gram-nega-tive bacteria, which may play a role in the regulation ofmaster £agellar genes.Interestingly, some di¡erences exist in the transcription-

al initiation of the £hDC operon among Enterobacteria-ceae. A unique transcriptional start site was identi¢ed inthe £hDC promoter region of Enterobacter spp. [73], con-sistent with the £hDC single major start site observed inE. coli [37] and in P. mirabilis [144] (Fig. 5A). In contrast,six transcriptional start sites were identi¢ed within the up-stream region of the S. typhimurium £hDC operon, one ofthem corresponding to the start site identi¢ed in E. coli[65] and in Enterobacter strains [73]. This could indicate apossible di¡erential control between these organisms. Sim-ilarly, multiple transcriptional start sites were identi¢ed byprimer extension analysis in a natural isolate of Pseudo-monas, including two major £eQ transcription initiationsites [73]. In this respect, it should be emphasised thattranscription from the major transcriptional start sitewas more sensitive to all adverse conditions tested, includ-ing the presence of gyrase inhibitor, than the second majortranscription start site. This could result from a di¡erentrate of transcription from both promoters and/or mRNAstability of the corresponding transcripts. Such a complexpromoter structure makes possible multiple regulations ofthese genes in response to speci¢c environmental condi-tions.

Some particularities were also observed in £hDC masteroperon regulation in di¡erent enterobacteria. In P. mira-bilis, 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 geneproduct MrpG [32,144^146]. In E. coli, new motility reg-ulators, such as the RNA binding regulator CsrA andHdfR and LrhA, two regulators of the LysR family,were recently discovered [38,39,109,147,148]. In S. typhi-murium, the ClpXP ATP-dependent protease was shownto a¡ect the expression of £agellar regulon [149]. More-over, in S. typhimurium, the Fur protein was shown topositively regulate the expression of the £hDC master op-eron [150]. Between closely related enterobacterial species,such as E. coli and S. typhimurium, some particularitieswere also reported concerning the motility control byH-NS protein and cAMP^CAP complex. In E. coli, eachof these regulators is essential for motility [37], whereas inS. typhimurium, single mutants in the corresponding gene,i.e. hns or crp, showed a reduced motility, while a com-plete lack of motility was only observed in an hns crpdouble mutant [151].The presence of numerous regulatory proteins control-

ling motility has been demonstrated mainly in enterobac-terial systems, i.e. E. coli and S. typhimurium. The ques-tion arises whether it is possible to extrapolate such acomplex regulatory network to other bacterial £agellarsystems. The extensive sequencing of bacterial genomes(http://igweb.integratedgenomics.com/GOLD) makes itpossible to search for regulatory genes homologous toknown £agellar regulators and to try to answer this ques-tion. In fact, some proteins homologous to known regu-lators may be identi¢ed in other organisms, but their rolein motility control remains to be demonstrated. In con-trast, sequence homologous to known regulators are notpresent in all bacteria, which suggests that other proteinsmay ful¢l these functions.For example, the presence of cAMP^CAP homologues

as well as the conservation of the cAMP^CAP binding sitein £hDC promoter regions (Fig. 5A) in several enterobac-terial sequences make plausible the conservation of themolecular mechanism of £hDC activation demonstratedin E. coli [37]. More generally, cAMP is also necessaryfor £agellar formation in V. cholerae [152] and both thepresence of CAP protein in this organism [153] and theidenti¢cation by genome sequencing of a CAP-like protein(PA0652) in P. aeruginosa [154] allow us to speculate thatsuch a complex may also mediate the catabolite repressionof motility in polar £agellar systems. In fact, recent datasuggest a downregulation of the £eQ master regulatorygene by Vfr, a CAP protein homologue in P. aeruginosa[155].Another example is the implication of nucleoid-associ-

ated proteins, including HU (see above), in the control ofbacterial motility. HU homologous proteins have been

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identi¢ed in many motile bacteria, such as laterally £agel-lated enterobacteria [156] and polarly £agellated P. aeru-ginosa [157] and P. putida [158], and also exist in P. £uo-rescens, V. cholerae, C. crescentus (SwissProt accessionnumbers Q9KHS6, Q9KV83, Q9KQS9, O87475). More-over, a HU homologue was identi¢ed in motile Gram-positive bacteria, such as B. subtilis [159]. Thus, HU-likeproteins are conserved in many micro-organisms, eventhough their implication in the motility control in all ofthem remains to be determined.The role in motility regulation of another nucleoid-as-

sociated protein family, H-NS, seems to be quite general.The positive role of H-NS in enterobacteria and phyloge-netically related micro-organisms [36,37,133,134], and theimplication of an H-NS-like protein, VicH, in the positivecontrol of polar £agellar synthesis in V. cholerae [160]provide evidence that such proteins participate in the mo-tility control of bacteria having di¡erent types of £agellaand so di¡erent types of regulatory cascades. However,until now no H-NS-like proteins have been identi¢ed inmany other motile bacteria, in any Gram-positive bacte-rium, e.g. B. subtilis [161], and in some Gram-negativebacteria, such as H. pylori and Pseudomonas spp. Wemay hypothesise either that in these micro-organisms themechanism of motility control is entirely di¡erent fromenterobacterial systems, or more probably that other pro-teins play the role of H-NS-like proteins. In this respect,the link between DNA topology and £agellar gene expres-sion in di¡erent bacteria, including H. pylori [56] andPseudomonas spp. [73], suggests a possible implication offunctional homologues of nucleoid-associated proteins inmotility 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 inthis process [162]. Similarly, it has been recently proposedthat FlrC, a polar £agellum regulatory protein, a¡ects celldivision in V. cholerae [30] and the existence of severalmechanisms that couple £agellar biosynthesis to cell cyclewas demonstrated in C. crescentus [47]. Moreover, recentresults [73] showed that the transcription of master regu-lator genes located at the top of the hierarchy is growthphase-dependent in Enterobacter and Pseudomonas strains.Similarly, £agellar expression in L. pneumophila is depen-dent on growth phase [164]. This provides evidence that£agellar regulation is linked to the cell cycle in many mi-cro-organisms.Finally, in addition to its role in bacterial motility, FlhD

protein participates in the transcriptional regulation ofother cellular processes, such as anaerobic and aerobicrespiration [62,165,166], and FlhD2C2 participates in thesynthesis of the £agellar export apparatus also used inexport 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 oflipolysis, extracellular haemolysis, and virulence in insectsin X. nematophilus [172], in the expression of nuclease in

Serratia marcescens [173] and in the di¡erentiation intoswarmer cells in S. liquefaciens and P. mirabilis[144,174]. The master polar £agellar regulators FleQ inP. aeruginosa and AdnA in P. £uorescens play an impor-tant role not only in motility control, but also in the reg-ulation of adhesion of these bacteria to substrates[28,75,175]. The role of master £agellar regulator thereforeseems to be more general in bacterial physiology than thesole control of bacterial motility.

6.2. Important di¡erences between £agellar systems

Several di¡erences in the structural organisation of £ag-ellar systems must also be underlined (Fig. 3). Indeed,these systems di¡er from each other by the existence ofspeci¢c sigma factors and transcriptional activators, bymotive force and the e⁄ciency of motors. The involvementof c54 and transcriptional activators associated with thissigma factor in the regulatory cascade of the polar £a-gellar system must be emphasised, in addition to the exis-tence of c

28 £agellar-speci¢c factor also present in thelateral £agellar regulatory system. In this respect, the polar£agellar hierarchy in V. cholerae [31] constitutes a combi-nation of both enterobacterial c28-dependent and C. cres-centus c

54-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 sodium-driven [178]. These di¡erent motors allow £agellum rota-tion speeds of about 15 000 rpm [179] and 100 000 rpm[180], respectively. In V. parahaemolyticus, which possessesboth £agellar systems, the polar £agellum uses a sodiumtransmembrane potential, whereas the lateral £agellar sys-tem is proton-driven [181]. The functional di¡erence be-tween polar and lateral £agella was demonstrated in aviscous environment for Vibrio alginolyticus, lateral £ag-ella being more e⁄cient than polar ones under the high-viscosity conditions [182]. A possibility to create hybridmotors, in which the motor proteins of V. cholerae,PomA, PomB, MotX, and MotY, are replaced by theE. coli proteins, MotA and MotB [183], has been recentlypublished. In V. cholerae, such a hybrid system runs usingthe proton transmembrane potential, as in E. coli. Theanalysis of this hybrid motor may help to understandthe mechanism of high-speed sodium-driven £agellar rota-tion. Indeed, the MotA and MotB proteins may serve tostabilise the motor in the membrane resulting in an in-crease in rotation speed or to form a PomAB-independentcanal 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 £ag-ellar biosynthesis. In this case the control of motility ismade possible by the capacity to synthesise the £agellumwith properties adapted to speci¢c environmental condi-tions [50] and to avoid a strong immune response in the

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host by antigenic modi¢cations [51]. Di¡erent £agellingenes are transcribed with di¡erent sigma factors (Table1). For example, in H. pylori, two £agellin genes, £aA and£aB, are regulated by two di¡erent sigma factors (c28 andc54, respectively), suggesting that these genes may be dif-ferently expressed depending on environmental conditions[56]. Thus, H. pylori may be able to produce £agella suitedfor motility in a given environment by changing £agellarparameters. Instead of a single £agellin, i.e. FliC in E. coli,the polar £agella of V. parahaemolyticus [23,42], Vibrioanguillarum [184], and V. cholerae [50] are composed of¢ve or six di¡erent £agellin subunits. Despite their highamino acid identity, only one £agellin, i.e. FlaA, is essen-tial for motility and is transcribed with c

54 factor inV. cholerae [50]. However, some exceptions exist, likethe polar £agellum in P. aeruginosa, which is composedof a single structural protein, FliC [185], while three £a-gellin genes, £eA, £eB and £eC, and two £agellin genes,£iC and £jB, were identi¢ed in the laterally £agellatedbacteria Y. enterocolitica and S. typhimurium, respectively[1,97].Finally, the lateral £agellar biosynthesis system in en-

terobacteria includes three classes of genes, but the polar£agellar systems in C. crescentus and V. cholerae are or-ganised into four classes of genes. Such a more complexorganisation may provide additional possibilities for a ¢netemporal regulation of the £agellar system. Moreover, inC. crescentus and in V. cholerae, an important step in theclass II^III transition is the phosphorylation of the FlbDand FlrC regulators, after the completion of MS ring^switch assembly [31]. In enterobacteria and probably inV. cholerae, the class II^III and the II^IV transition, re-spectively, depend on FlgM anti-sigma factor export and,thus, on the activation of FliA, the speci¢c £agellar c

28

factor, after the assembly of the basal body^hook struc-ture [1,31].The presence of a speci¢c type of £agellar regulatory

cascade in di¡erent bacteria appears to be dependent onthe functional particularity of a given £agellar insertiontype, which seems to be itself related to speci¢c environ-ments rather than to result from phylogenetic di¡erencesbetween organisms. Indeed, the polar and lateral £agellarsystems are widely distributed among micro-organisms, inparticular within K- to Q-subdivisions of Gram-negativebacteria. Moreover, these two unrelated £agellar systemsmay simultaneously function in some bacteria, such asA. brasilense [24] and V. parahaemolyticus [23,42,51,52].In this organism, distinct £agellar cascades control thecontinuous expression of polar £agella and the inductionof lateral £agellar expression under speci¢c conditions,respectively. Such a mixed £agellation is probably relatedto the ecological niche and the environments where thismicro-organism resides, polar £agella being especially e⁄-cient in liquid environments and lateral £agella being moresuitable for movement on surfaces and in highly viscousenvironments [23,182].

7. Conclusions

The importance of motility for bacteria can be deducedfrom the cells’ investment in synthesising £agella and fromthe redundancy of these organelles. The constant interestof microbiologists in bacterial £agellar regulatory systemsresults from the recognition of the crucial role that motil-ity plays in bacterial physiology. The research in this ¢eldwas also promoted by the role of £agella in virulence,adhesion, bio¢lm formation and colonisation of host or-ganisms in pathogenic bacteria. Moreover, £agellum bio-synthesis is usually co-regulated together with other viru-lence factors within the same regulatory network.The growing amount of information in the literature on

£agellar regulatory cascades in di¡erent bacteria revealsthe striking similarities in the general organisation of thesesystems. Environmental conditions and growth phase af-fect motility of many bacteria in a similar manner. More-over, the implication of DNA topology, nucleoid-associ-ated proteins and/or the regulatory regions of mastergenes might be general in organisms with di¡erent typesof motility cascades. The motility control may thus haveevolved towards similar mechanisms in di¡erent bacterialsystems. The particularities that exist among £agellar cas-cades may be linked to the speci¢c environments in whichdi¡erent micro-organisms live. One could wonder whysuch a similarity in the regulatory mechanism might ap-pear during the evolutionary process. A plausible answeris that within the same genus, di¡erent related bacteriamay encounter various environmental conditions. For ex-ample, pathogenic species, such as the opportunisticpathogen of humans P. aeruginosa [154], are adapted totheir host environment, and natural isolates adapted towater or soil environment, such as the soil bacteriumP. putida [186], are able to colonise the plant rhizosphere.On the other hand, the same micro-organism encountersdi¡erent environments during its cellular cycle. V. choleraemust be capable of surviving inside humans during thecolonisation phase and in water estuaries during thefree-swimming phase and therefore presents two distinctphases in its life cycle with characteristic physiology adap-tations [187]. In this respect, the appearance of a generalregulatory system during the evolutionary process lieswithin cell economy considerations and survival advan-tages. Because of the multitude of ecological niches, evo-lution gave rise to such a conservation of general mecha-nism, di¡erent bacteria being capable of occupyingnumerous environments requiring adaptation to di¡erentexternal conditions. Therefore, a similar solution must befound by bacterial cells to ensure an adequate and rapidresponse to various environmental conditions. The largenumber of genes involved in both the biosynthesis andfunctioning of £agella, the numerous regulators and envi-ronmental factors implicated in motility control and themultiple interactions with other cellular processes highlightthe extreme complexity of this regulatory network. Despite

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the recent studies, multiple questions remain unansweredregarding the molecular mechanisms that control bacterialmotility in response to environmental cues. For example,the perception of environmental signals such as temper-ature, osmolarity or pH and the transmission of thesestimuli giving rise to an appropriate physiological responseremain poorly understood. In future, an e¡ort should bemade to elucidate these processes, in particular because ofthe important role of motility in colonisation. The studiesin this ¢eld would bring important advances in the com-prehension of bacterial physiology and interactions of bac-teria with humans and plants.During the past years the large development of methods

for global analysis of gene expression and macromolecularinteractions, associated with the sequencing of bacterialgenomes, has provided new tools for studying the regula-tory processes at a molecular level. The application ofthese methods for analysis of £agellar systems mighthelp to improve our understanding of molecular mecha-nisms governing £agellum biosynthesis in bacteria. As anexample, a global transcriptome analysis of suppressormutants has recently made it possible to establish a linkbetween H-NS and acid pH control of motility in E. coli[35]. Similarly, DNA array analysis revealed the transcrip-tional regulation of several genes unrelated to motility byFlhD, showing the general role of this £agellar regulatorin bacterial physiology [165,166]. At present, on the basisof available information, only a preliminary model may bedrawn to describe complex £hDC regulation in E. coli(Fig. 2). Genome-wide approaches are promising andwould allow the integration of multiple factors participat-ing in £agellar control into a unique model.The co-ordination of gene expression by regulatory net-

works is widespread in prokaryotes, as well as in eukary-otes [188,189]. Such regulatory systems are usually orga-nised in a similar way in di¡erent organisms with masterregulators at the top of the hierarchy allowing the integra-tion of external signals to ensure adequate cellular re-sponses [190]. In this respect, the studies of £agellar bio-synthesis, which integrate multiple components ofbacterial physiology, may constitute an excellent modelfor the understanding of complex regulatory networks.

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