Bacterial lateral flagella: an inducible flagella system

M I N I R E V I E W

Bacterial lateral£agella: an inducible£agella systemSusana Merino1, Jonathan G. Shaw2 & Juan M. Tomas1

1Departamento de Microbiologıa, Facultad de Biologıa, Universidad de Barcelona, Diagonal 645, Barcelona, Spain; and 2Division of Molecular and

Genetic Medicine, University of Sheffield Medical School, Sheffield, UK

Correspondence: Susana Merino,

Departamento de Microbiologıa, Facultad de

Biologıa, Universidad de Barcelona, Diagonal

645, 08071 Barcelona, Spain. Tel.: 134

934021708; fax: 134 934039047; e-mail:

[email protected]

Received 8 May 2006; revised 26 June 2006;

accepted 4 July 2006.

First published online 2 August 2006.

DOI:10.1111/j.1574-6968.2006.00403.x

Editor: Ian Henderson

Keywords

lateral flagella; swarming; Aeromonas ; Vibrio

parahaemolyticus .

Abstract

Flagella are complex surface organelles that allow bacteria to move towards

favourable environments and that contribute to the virulence of pathogenic

bacteria through adhesion and biofilm formation on host surfaces. There are a

few bacteria that possess functional dual flagella systems, such as Vibrio para-

haemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and

Azospirillum brasilense. These bacteria are able to express both a constitutive polar

flagellum required for swimming motility and a separate lateral flagella system that

is induced in viscous media or on surfaces and is essential for swarming motility.

As flagella synthesis and motility have a high metabolic cost for the bacterium, the

expression of the inducible lateral flagella system is highly regulated by a number

of environmental factors and regulators.

Introduction

Motility provides a survival advantage under a wide variety of

environments, allowing bacteria to respond to favourable or

unfavourable conditions and to compete successfully with other

microorganisms. Bacteria have developed different systems to

move in liquid or over surfaces (Harshey & Matsuyama, 1994;

Harshey, 2003). Flagella-based motility is a major mode of

locomotion for bacteria, including Archaea (Jarrell et al., 1996).

Flagella are one of the most complex and extremely

effective organelles of locomotion, capable of propelling

bacteria through liquids (swimming) and through viscous

environments or over surfaces (swarming) (Manson et al.,

1998). In addition, these organelles play an important role in

adhesion to substrates, biofilm formation and contribute to

the virulence process in pathogenic bacteria (Otteman &

Miller, 1997; Josenhans & Suerbaum, 2002). The number and

arrangement of flagella on the bacterial surface vary among

species. Many bacterial species express single/multiple polar

flagella, for example Pseudomonas aeruginosa, Vibrio cholerae

and Helicobacter pylori, or they express peritrichous (lateral

noninduced) flagella, such as Escherichia coli, Salmonella

enterica and Proteus mirabilis. However, a limited number of

bacteria possess dual flagella systems and are able to express

two entirely distinct flagella systems: a polar flagellum for

swimming and lateral flagella for swarming; these include

Vibrio parahaemolitycus (Shinoda & Okamoto, 1977), Vibrio

alginolyticus (Kawagishi et al., 1995), Aeromonas spp. (Shi-

mada et al., 1985), Azospirillum brasilense (Tarrand et al.,

1978; Hall & Krieg, 1983), Rhodospirillum centenum

(McClain et al., 2002), Helicobacter mustelae (O’Rouke et al.,

1992) and Plesiomonas shigelloides (Inoue et al., 1991).

Flagella synthesis and motility are metabolically very

expensive and therefore, their genes are transcribed in a

hierarchical cascade (Macnab, 1996). The master regulatory

genes, such as flhDC in the Enterobacteriaceae and fleQ or flrA

in P. aeruginosa and V. cholerae, respectively, are highly

regulated in response to environmental changes and by global

regulatory proteins, such as H-NS (histone DNA binding

protein) and the cAMP-CAP (catabolite gene activator pro-

tein) (Soutourina & Bertin, 2003). In this sense, highly

viscous environments or surfaces, which reduce flagella

motility, produce in many peritrichous bacteria swarmer cells

and the overexpression of flagella number that can be seen in

Proteus mirabilis, Serratia marcescens, S. enterica serovar

Typhimurium and E. coli (Harshey & Matsuyama, 1994;

Harshey, 2003). In contrast, polar flagellated bacteria with

dual flagella systems express lateral flagella in viscous envir-

onments or on surfaces and show mixed flagellation consist-

ing of a constitutive polar flagellum and inducible lateral

FEMS Microbiol Lett 263 (2006) 127–135 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

flagella, as is seen in V. parahaemolitycus (McCarter & Silver-

man 1990) and Aeromonas hydrophila (Gavın et al., 2002).

Bacterial species with dual flagellasystems

Expression of dual flagella systems was reported in some

polar flagellated bacteria in response to growth in viscous

environments or surfaces that allows the bacteria to swarm

on solid media by a mixed flagellation (polar and lateral

flagella). Vibrio parahaemolyticus, V. alginolyticus and

R. centenum have constitutive sheathed polar flagella and

differentiate into swarmer cells by the cessation of septation,

resulting in the elongation of cells and expression of

unsheathed lateral flagella upon contact with a surface

(Ulitzur, 1975; McCarter & Silverman, 1990; Ragatz et al.,

1995). In contrast, Aeromonas spp. have a constitutive polar

flagellum and inducible lateral flagella that are both un-

sheathed, although they are glycosylated, and swarmer cell

differentiation does not result in multinucleated cells (Raa-

ban et al., 2001; Gavın et al., 2002; Kirov et al., 2002).

Azospirillum spp. swarmer cells, such as Aeromonas spp, do

not show cell elongation (Alexandre et al., 1999).

The best-studied bacteria with dual functional flagella

systems are V. parahaemolyticus and A. hydrophila. Vibrio

parahaemolyticus polar flagellum requires around 60 genes

distributed in five clusters on chromosome I (Yun-Kyeong &

McCarter, 2000) and the lateral flagella are encoded by 38

genes distributed in two clusters on chromosome II (Stewart

& McCarter, 2003). Aeromonas hydrophila polar flagellum

has 55 genes distributed in five clusters (Altarriba et al.,

2003; Canals et al., 2006b), and the lateral flagella are

encoded by 38 genes distributed in a single chromosomal

region (Gavın et al., 2002; Canals et al., 2006a). In these two

bacterial species, the polar and lateral flagella systems do not

appear to share either structural or regulatory genes (Kirov,

2003; McCarter, 2004), whereas in Azospirillum spp. and

R. centenum they may shared distinct structural and/or regula-

tory genes (Jiang et al., 1998; Scheludko et al., 1998;

McClain et al., 2002). Moreover, the dual flagella systems in

V. parahaemolyticus and V. alginolyticus also have different

energy sources driving motility, as the polar flagellum is

powered by the sodium motive force and the lateral flagella

are driven by the proton motive force (Atsumi et al., 1992).

The same situation has been observed in A. hydrophila

(J.M. Tomas, unpublished data).

Recently, comparative genomic analysis of enteroaggrega-

tive E. coli strain 042 reported a new flagella locus (Flag-2)

with 44 genes, whose gene products are homologous to

those of the V. parahaemolyticus lateral flagella system,

except for the motYL gene encoding a motor component

(Ren et al., 2004). This cluster potentially encodes all gene

products required for a functional lateral flagella system, but

a frameshift mutation in lfgC, which encodes a proximal rod

protein, appears to inactivate the system in this strain. PCR

studies suggested the presence of this cluster in 15 of 72

E. coli reference strains. Similar genomic studies show a

Flag-2 like cluster that lacks the inactivating lfgC frameshift

mutation in Chromobacterium violaceum, Citrobacter roden-

tius and Yersinia pseudotuberculosis. Also, a nonfunctional

Flag-2-like cluster with frameshift mutations or deletions in

some genes was found in different Yersinia pestis strains (Ren

et al., 2004). All these findings suggest that the presence of

dual flagella systems within the same species is more

common than was previously thought.

Chromosomal organization of inducedlateral flagella systems

The lateral flagella genes of V. parahaemolyticus are arranged

in two different chromosomal regions (region 1 and region

2) on chromosome II (Stewart & McCarter, 2003). In

contrast, the A. hydrophila lateral flagella genes are arranged

in a single chromosomal region (Canals et al., 2006a), as has

been observed for the E. coli 042 Flag-2 cluster (Ren et al.,

2004) (Fig. 1).

Vibrio parahaemolyticus region 1 encodes the anti-sfactor 28 (s28) factor and many structural proteins involved

in hook basal body formation (Table 1). Region 1 genes are

divided among two divergently transcribed set of genes:

flgAMNL and flgBCDEFGHIJKLL. Aeromonas hydrophila and

E. coli 042 homologous lateral flagella genes exhibit the

same distribution and direction of transcription. Vibrio

parahaemolyticus region 2 encodes the specific lateral flagella

s28, switch, export-assembly, motor and flagellin proteins

(Table 1). Genes of region 2 are arranged in four clusters:

fliMNPQRLflhBAL, lafA and fliDSTKLALmotABL transcribed

in the same direction, and motYLlafKfliEFGHIJL transcribed

in the opposite direction. In contrast, homologous

A. hydrophila lateral flagella genes are transcribed in the

same direction and the E. coli 042 lfiMNPQRlfhBA cluster,

homologous to fliMNPQRLflhBAL, is transcribed diver-

gently. In addition, neither A. hydrophila nor E. coli 042

lateral flagella clusters contain a homologous gene to V.

parahaemolyticus motYL, which encodes a protein similar to

the V. alginolyticus outer membrane sodium-drive stator

motor protein MotYP (Okabe et al., 2002).

Azospirillum hydrophila lateral flagella gene cluster con-

tains between flgLL and lafA, a modification accessory factor

gene, maf-5 (Karlinshev et al., 2002), which is transcribed

independently and in the same direction as lafA. In E. coli

042, this region contains four additional genes: the first

three genes, lafW and two contiguous orfs (Ec042-0277 and

Ec042-0278), are transcribed in the same direction, whereas

the last one, lafZ, is transcribed divergently. The lafW-encoded

FEMS Microbiol Lett 263 (2006) 127–135c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

128 S. Merino et al.

protein may represent a novel hook-associated protein. The

first orf Ec042-0277 encodes a protein of unknown function

and Ec042-0278 encodes a protein that contains a helix-turn-

helix domain and exhibits high amino acid identity with

several other putative transcriptional regulators. The lafZ gene

encodes a putative transmembrane transcriptional regulator.

Moreover, the E. coli 042 Flag-2 cluster contains three other

genes between lfiJ (fliJL) and lfgN (flgNL). Downstream of lfgN

and transcribed in the same direction is lafV, which is

predicted to encode a lysine-N-methylase required for post-

translational methylation of lysine residues in some flagellins

(Burnens et al., 1997). Downstream of lafV are located two

divergently transcribed genes (Ec042-0259 and Ec042-0260),

whose homologous genes are found as part of capsule poly-

saccharide biosynthesis clusters (Table 1). It is possible that

these two genes may be involved in post-translational mod-

ification of flagella proteins.

The finding of two variable regions, between flgLL (lfgL)

and lafA and between fliJL (lfiJ) and flgNL (lfgN), in strains

with a single lateral flagella cluster, as well as the presence of

V. parahaemolyticus lateral flagella genes distributed in two

chromosomal regions suggest that there may exist recombi-

nation points in these two variable areas. In contrast

with the polar flagella systems, neither of the lateral

flagella systems described contains the export-assembly gene

fliOL. The role of FliO is poorly understood, even in

S. enterica serovar Typhimurium and E. coli (Schoenhals

et al., 1998).

Most of the sequenced lateral flagella clusters (A. hydro-

phila AH-3, E. coli O42 and V. parahameolyticus) show a

single flagellin, but analysis of the partially sequenced

Aeromonas caviae Sch3N lateral flagella cluster shows, up-

stream of the capping flagella gene lafB, two flagellin subunit

genes (lafA1 and lafA2) that are transcribed in the same

direction and an fliU gene, which encodes an N-lysine

methylase involved in flagella biosynthesis (LafV), which is

transcribed divergently (Gavın et al., 2002) (Fig. 1). PCR

analysis has shown that some A. hydrophila strains also

possess two lateral flagellin genes.

Although the R. centenum lateral flagella system has not

been completely sequenced, a second chemotactic operon

(che2) that is involved in polar and lateral flagella formation

has been reported recently (Berleman & Bauer, 2005), in

addition to the che1 operon that controls the chemotactic

and phototactic behaviour of both swim and swarm cells

(Jiang & Bauer, 1997). This second chemotactic operon

contains eight genes whose encoded proteins are homolo-

gues of MCP, CheA, CheY, CheB, CheR, CheW and two

ORFs of unknown function.

Regulation of lateral-induced flagellasystems

Flagella generation requires many genes that are organized

in a hierarchical manner with master regulators at the top

of the hierarchy. The regulatory cascade includes early,

Vibrio parahaemolyticus

Aeromonas hydrophila

flgNMA flgBCDEFGHIJKL fliJIHGFElafK motY

Region Region

fliMNPQRflhBA lafA fliDSTKLAmotAB

fliMNPQRflhBA lafKfliEFGHIJ flgNMA flgBCDEFGHIJKL maf-5 lafA lafBCXEFSTU

Escherichia coli 042

lfhABlf iRQPNM lafKlf iEFGHIJ lafV lfgNMA lfgBCDEFGHIJKL lafW lafZ lafA lafBCDEFSTU

Aeromonas caviae

fliU lafA1 lafA2BCXEFSTU

0259 0260 0277 0278

Fig. 1. Comparative schematic representation of Vibrio parahaemolyticus, Aeromonas hydrophila AH-3, Escherichia coli O42 and Aeromonas caviae Sch3N

lateral flagella regions. Arrows of the same colour indicate homologous genes among these bacteria, and nonsolid colour indicates a frameshift-mutated gene.


129Bacterial lateral flagella: an inducible flagella system

middle and late genes temporally expressed, with specific

transcriptional regulators and s factors controlling the dif-

ferent transcription levels (Aldridge & Hughes, 2002; Sou-

tourina & Bertin, 2003; McCarter, 2006). In E. coli and

S. enterica serovar Typhimurium, the middle flagella genes are

s70-dependent and activated by the master regulators FlhD

and FlhC, whereas the homologous polar flagella genes from

different bacterial species, as well as V. parahaemolyticus and A.

hydrophila lateral-induced flagella genes are s54-dependent

(Stewart & McCarter, 2003; Canals et al., 2006b). Moreover,

the middle polar flagella-expressed genes are divided into two

subclasses (class II and class III) with different s54-dependent

transcriptional activators. Polar flagella class II genes that

encode structural components of the MS ring, switch, export-

assembly apparatus, the s28 flagella-specific factor and the

two-component signal-transducing system FlrBC in V. choler-

ae (FleQS in P. aeruginosa), are activated by the s54-asso-

ciated transcriptional activator FlrA in V. cholerae (FleQ in

P. aeruginosa). The polar flagellar class III genes that encode

the basal body, hook and some flagellins are activated by the

s 54-dependent response regulator FlrC in V. cholerae (FleS in

P. aeruginosa) (Prouty et al., 2001; Dasgupta et al., 2003). For

the lateral flagella system, the middle genes of V. parahaemo-

lyticus and A. hydrophila are activated by the s54-associated

transcriptional activator LafK, which is homologous to the

V. cholerae FlrA and P. aeruginosa FleQ polar flagella regulators

(Stewart & McCarter, 2003). In most flagella systems (peritri-

chous, polar and lateral-induced flagella), late gene expression

seems to be controlled by the s28 flagella-specific factor, and

its cognate anti-s factor FlgM (Aldridge & Hughes, 2002;

Soutourina & Bertin 2003).

Master regulators FlhDC, FlrA or FleQ of bacteria with

single flagella systems are essential for flagella expression, but

this situation seems to be different in bacteria with dual flagella

systems. In V. parahaemolyticus, the s54-dependent polar

flagella response regulator FlaK (FlrA, FleQ equivalent) is

dispensable for polar flagella expression, as the lateral flagella

transcriptional activator LafK, which is essential for lateral

flagella generation, compensates for its loss (Kim & McCarter,

2004) (Fig. 2). Flagella systems are transcriptionally and post-

transcriptionally regulated by a number of environmental

conditions, global regulators and the growth phase (Soutour-

ina & Bertin, 2003; McCarter, 2006). In general, the increase in

the media viscosity restricts polar flagella swimming and

induces lateral flagella expression. However, viscosity is not

the only lateral flagellar induction signal, as iron-depleted

growth medium is a second signal in V. parahaemolyticus

(McCarter & Silverman, 1989) and static liquid growth was

reported to induce lateral flagella in Azospirillum brasilense Cd

but not in other Azospirillum species (Madi et al., 1988).

It has been proposed that polar flagella in V. parahaemo-

lyticus and Azospirillum brasilense act as a mechano-sensor

by measuring viscosity, and transduce this signal to control

Table 1. Lateral flagella gene nomenclature and predicted function

Gene nomenclature

Predicted function

A.

hydrophila�E. coli

042wV.

parahaemolyticusz

fliML lfiM fliML Switch (C ring)

fliNL lfiN fliNL Switch (C ring)

fliPL lfiP fliPL Export/assembly

fliQL lfiQ fliQL Export/assembly

fliRL lfiR fliRL Export/assembly

flhBL lfhB flhBL Export/assembly

flhAL lfhA flhAL Export/assembly

motYL Proton motor

lafK lafK lafK Regulatory

fliEL lfiE fliEL Basal body component

fliFL lfiF fliFL M-ring

fliGL lfiG fliGL Switch (C ring)

fliHL lfiH fliHL Export/assembly

fliIL lfiI fliIL Export ATP synthase

fliJL lfiJ fliJL Export/assembly

Ec042-0259 Cytidylyl transferase

Ec042-0260 Glycosyl transferase

lafV Lysine-N-methylase

flgNL lfgN flgNL Chaperone

flgML lfgM flgML Anti-s28

flgAL lfgA flgAL P-ring assembly

flgBL lfgB flgBL Rod

flgCL lfgC flgCL Rod

flgDL lfgD flgDL Rod

flgEL lfgE flgEL Hook

flgFL lfgF flgFL Rod

flgGL lfgG flgGL Rod

flgHL lfgH flgHL L-ring

flgIL lfgI flgIL P-ring

flgJL lfgJ flgJL Peptidoglycan hydrolase

flgKL lfgK flgKL Hook-associated protein 1

flgLL lfgL flgLL Hook-associated protein 3

maf-5 Motility accesory factor

lafW Possible hook-associated

protein

Ec042-0277 Unknown

Ec042-0278 Regulator

lafZ Transmembrane regulator

lafA lafA lafA Flagellin

lafB lafB fliDL Hook-associated protein 2

lafC lafC fliSL Chaperone

lafX lafD fliTL Chaperone

lafE lafE fliKL Hook length control

lafF lafF fliLL Unknown

lafS lafS fliAL s28

lafT lafT motAL Proton motor

lafU lafU motBL Proton motor

�A. hydrophila gene designation according to accession number

DQ124694-5 (Canals et al., 2006a, b).wE. coli 042 gene designation according to the nomenclature suggested

by Ren et al. (2004). Underlined genes contain a frameshift mutation.zV. parahaemolyticus gene designation according to the nomenclature

suggested by Stewart & McCarter (2003). Boldface indicates genes

located in lateral flagella region 1, whereas nonboldface genes are

located in region 2.

A. hydrophila, Aeromonas hydrophila; E. coli, Escherichia coli;

V. parahaemolyticus, Vibrio parahaemolyticus.



lateral flagella expression. In these bacterial species, both

flagella types are intimately linked and their regulation

systems seem to interact, as defects in polar flagella forma-

tion or motility allow lateral flagella expression to be

constitutive (McCarter et al., 1988; Kawagishi et al., 1996;

Alexandre et al., 1999). However, the molecular mechanism

for sensing polar flagella inhibition and the signal-transdu-

cing pathway regulating lateral flagella expression are not

known. In contrast, polar flagellum defects in Aeromonas

spp. or R. centenum do not induce constitutive lateral

flagella formation (Jiang et al., 1998; Altarriba et al., 2003;

Canals et al., 2006a), demonstrating that lateral flagella

expression is still under its natural control, and suggesting

that polar flagella do not interact with lateral flagella

regulation.

Components of the second Che2 of R. centenum, however,

do not appear to affect directly or indirectly chemotaxis, and

instead appear to encode components of the Che-like

transducing cascade involved in lateral and polar flagella

post-transcriptional or post-translational regulation. This

regulation suggests that polar flagella and induced lateral

flagella must be coordinatedly regulated (Berleman & Bauer,

2005). The two-component signal-transducing systems,

which are capable of integrating sensory input to the control

of gene expression, are important regulatory mechanisms in

response to environmental changes. In this sense, the

scrABC operon of V. parahaemolitycus inversely affects two

different genes systems important to life on a surface: lateral

flagella and capsule. The periplasmic-binding protein ScrB

receives an input signal and interacts with the periplasmic

domain of ScrC. This interaction could modulate the

activity of the cytoplasmic GGDEF and EAL domains of

ScrC, which possibly control levels of an intracellular signal-

ling molecule (cyclic di-GMP). ScrA contains a domain

shared with pyridoxal-phosphate-dependent enzymes and

is required for signal transduction, but its role is unclear.

Levels of the small signalling molecule (cyclic di-GMP)

modulate expression of lateral flagella and capsule inversely.

The loss of the scrABC operon reduces but does not abolish

swarm differentiation, suggesting that it may play a role in

mediating the switch between lateral flagella and capsule

expression during surface colonization (Boles & McCarter,

2002). Other V. parahameolyticus lateral flagella-regulatory

mechanisms are homologues to quorum-sensing compo-

nents or histone-like DNA-binding proteins. The homolo-

gous quorum-sensing components OpaR, SwrT and SwrZ

modulate lateral flagella expression. OpaR is the central

transcriptional regulator involved in the opaque-translucent

switching by upregulation of capsule expression, but it also

represses lateral flagella expression. SwrT appears to mod-

ulate swarming by repressing transcription of the lateral

flagella repressor SwrZ. Interestingly, an SwrZ mutant does

not display constitutive lateral flagella, suggesting that SwrZ

is not responsible for transmitting either the iron starvation

or the polar flagella inhibition signal (Jaques & McCarter,

2006). Lateral flagella regulation is also mediated by regula-

tory proteins that affect DNA conformation. Thus,

V. parahemolyticus trh-positive strains express the VpaH

protein, a homologue of the histone DNA-binding protein

H-NS that positively regulates the enterobacterial flhDC

operon by DNA supercoiling. VpaH positively regulates

lateral flagella biogenesis, whereas no effect was observed

on polar flagella expression (Park et al., 2005) (Fig. 2).

In addition, regulation by proteolytic degradation of

master regulators is a rapid mechanism to control critical

regulatory proteins. Vibrio parahaemolyticus ATP-depen-

dent protease LonS inhibits the swarmer cell phenotype by

degrading a transcriptional activator of lateral flagella genes

and a cell division inhibitor. LonS mutants express lateral

flagella and produce elongated cells in liquid medium

(Stewart et al., 1997) (Fig. 2).

Role of lateral induced flagella incolonization and biofilm formation

An essential step for any infection is the encounter of the

pathogenic bacteria with the target eukaryotic cell. Swim-

ming, combined with chemotaxis, enables a fine-tuned

access of pathogens to their target on mucosal tissues, and

flagella are an important structure for adhesion to the

epithelial cell (Otteman & Miller, 1997; Josenhans & Suer-

baum, 2002). Moreover, flagella seem to be an important

stimulator of the host response (Ramos et al., 2004). After

the initial attachment, an important feature for rapid

colonization of the surface is the ability of the bacteria to

move over the surface. In this sense, swarming motility

contributes to the infection process, as reported in Proteus

mirabilis urinary tract infections (Mobley & Belas, 1995).

Colonization usually implies biofilm formation, that is, an

accumulation of microorganisms adhered to a surface

embedded in a polysaccharide matrix of their own making

(Costerton et al., 1999). Bacteria in biofilms are generally

more resistant to host defences and antimicrobial agents,

and also express more virulence factors as a result of gene

activation by quorum sensing.

Vibrio parahaemolyticus, A. hydrophila and A. caviae are

water-borne bacteria involved in different animal and hu-

man infections. In these species, the polar flagellum is

important for motility in liquid media, but after host

attachment, lateral flagella are induced that which form a

linkage between bacteria and surfaces, contributing to

microcolony formation and allowing the bacteria to adhere

more firmly (Belas & Colwell, 1982a; Kirov et al., 2002). In

V. parahaemolyticus, lateral flagella play an important role in

adherence to and colonization of the chitinaceous shells of

crustaceans, probably by a mechanism distinct from that



used by the polar flagellum (Belas & Colwell, 1982b). A

recent report also showed that V. parahaemolyticus lateral

flagella are involved in adhesion to HeLa cells and in biofilm

formation (Park et al., 2005). In mesophilic Aeromonas, at

least 50% of strains commonly associated with diarrheal

illness produce lateral flagella, and various reports have

shown that A. hydrophila and A. caviae lateral flagella and

their motility increase adherence to HEp-2 (Gavın et al.,

2002; Gavın et al., 2003; Canals et al., 2006a) and intestinal

cell lines (Henle 407 and Caco-2) (Kirov et al., 2004).

Nevertheless, swarming motility expands the area of coloni-

zation and contributes to biofilm formation on different

surfaces such as borosilicate glass or microtitre plates (Gavın

et al., 2003; Kirov et al., 2004).

In the nitrogen-fixing rhizobacterium Azospirillum brasi-

lense, migration of bacteria towards the plant roots takes

place by swimming through the water spaces, but is limited

by soil moisture (Bashan, 1986). Adhesion to plant roots

also seems to be a function of the polar flagellum (Croes

et al., 1993), but lateral flagella enable the bacteria to move

along the root and are thought to be important for long-

term colonization (Moens et al., 1995).

Glycosylation

Glycosylation was previously considered to be restricted to

eukaryotes; however, bacteria and particularly mucosal-

associated pathogens have recently been shown to possess

two types of glycosidic linkage: N- and O-glycosides. In

gram-negative bacteria, protein glycosylation is mainly

associated with virulence factors, and several studies have

implied a role in infection and interference with the

inflammatory immune response (Upreti et al., 2003; Szy-

manskin & Wren, 2005). In the last few years, glycosylation

of polar flagellins has been described in an increasing

number of bacterial species. Polar flagellar glycosylation

plays an important role in the P. aeruginosa proinflamma-

tory response (Arora et al., 2005), and is responsible for host

plant recognition and the hypersensitivity reaction in Pseu-

domonas syringae infections (Takeuchi et al., 2003). It is

involved in Campylobacter jejuni gut colonization (Szy-

manski et al., 2002) and is responsible for increased adsorp-

tion of Azospirillum brasilense to plant roots (Moens et al.,

1995). In relation to inducible lateral flagella, only

A. hydrophila and A. caviae lateral flagella are reported to

be glycosylated (Gavın et al., 2002; Canals et al., 2006a), in

contrast to other lateral flagella systems. Aeromonas hydro-

phila and A. caviae possess both polar and lateral glycosy-

lated flagellins (Rabaan et al., 2001; Canals et al., 2006a) and

several genes have been reported in connection with their

glycosylation. Both strains have two genes, flmA and flmB,,

homologous to Campylobacter glycosylation genes (Power &

Jennings, 2002) that are involved in polar and lateral flagella

assembly. In addition to flmA and flmB, three more genes,

neuA-flmD-neuB, are found in the A. caviae cluster.

These five genes in A. caviae Sch3N are also involved in

LPS O-antigen expression, (Gryllos et al., 2001) and are

involved in pseudaminic acid biosynthesis that has been

shown to be present on the polar flagellins (J.M. Tomas,

unpublished observation). Moreover, two A. hydrophila

genes (maf-1 and 5), whose products are homologous to

H. pylori and Campylobacter jejuni Maf proteins (Power &

Polar Flagella Lateral Flagella

Class I lafK motYlafKfliEFGHIJ ?

LafK+σ (RpoN)

Class II f lgAMN f lgBCDEFGHIJ f liDSTKLAmotAB motYlafKfliEFGHI

Class III flgKL lafA fliDSTKLAmotAB flgMN

Class I flaK

Class II f laLM f liEFGHIJKLMNOPQRflhB f lhAFGfliAcheYZABorf1orf2cheWorf3

Class III f lgAMN f lgBCDEFGHIJ f lgKL motY

FlaK + σ (RpoN)

Class IV motAB motX flaAGHIJK flaB flaDE cheVR flgMN

σ (FliA )

Dual f lagella systems Vibrio parahaemolyticus

fliMNPQRflhBA ?

Environmental factors

SwrZ SwrT

ScrABC

OpaR

VpaH

LonS

Regulatory proteins

σ (FliA )

FlaM + σ (RpoN)

Fig. 2. Vibrio parahaemolyticus dual flagella

hierarchy and factors controlling lateral flagella

expression. Blue arrows indicate an activation

effect and red arrows a repression effect.

Transcription of some lateral flagella clusters (?)

remains to be elucidated.



Jennings, 2002), appear to be involved in specific polar and

lateral flagella glycosylation, respectively (Canals et al.,

2006a; Canals et al., 2006b). Little is known about the role

of lateral flagellin glycosylation in aeromonad virulence.

Conclusions

Motility plays a crucial role in bacterial physiology, and

bacteria living in different habitats need to possess locomo-

tion systems adapted to their particular environment. A few

bacteria have dual flagella systems that allow them to adjust

to different environmental circumstances. Currently, all

bacteria known to possess a functional dual flagella system

have a constitutive polar flagellum and an inducible lateral

flagella system that is expressed in highly viscous media or

on a surface. However, dual flagella systems have also been

found in bacteria with constitutive peritrichous flagella, but

their functionality has thus far not been proven. Lateral

flagella expression is highly regulated by environmental

factors and a number of regulators, allowing the bacteria to

swarm. However, the signal by which the bacteria sense

viscosity or a surface remains unknown, although how the

mechanism of induction of lateral flagella expression occurs

is known. In pathogenic bacteria, lateral flagella contribute

to both adhesion to host cells and the formation of biofilms.

Further investigations into the regulation, host cell interac-

tion and proinflammatory action of lateral flagella are

needed to understand their pathogenic importance.

Acknowledgements

This work was supported by Plan Nacional de I1D and FIS

grants (Ministerio de Educacion, Ciencia y Deporte and

Ministerio de Sanidad, Spain), from Generalitat de Catalu-

nya and the Wellcome Trust.

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Bacterial lateral flagella: an inducible flagella system

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