OUP user menu

Bacterial lateral flagella: an inducible flagella system

Susana Merino, Jonathan G. Shaw, Juan M. Tomás
DOI: http://dx.doi.org/10.1111/j.1574-6968.2006.00403.x 127-135 First published online: 1 October 2006


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 parahaemolyticus, 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.

  • lateral flagella
  • swarming
  • Aeromonas
  • Vibrio parahaemolyticus


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. (Shimada 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 protein) (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 environments or on surfaces and show mixed flagellation consisting of a constitutive polar flagellum and inducible lateral flagella, as is seen in V. parahaemolitycus (McCarter & Silverman 1990) and Aeromonas hydrophila (Gavín et al., 2002).

Bacterial species with dual flagella systems

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 unsheathed, although they are glycosylated, and swarmer cell differentiation does not result in multinucleated cells (Raaban et al., 2001; Gavín 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 (Gavín 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 regulatory 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. Tomás, unpublished data).

Recently, comparative genomic analysis of enteroaggregative 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 rodentius 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 induced lateral 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).


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.

Vibrio parahaemolyticus region 1 encodes the anti-σ factor 28 (σ28) 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 σ28, 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 divergently. 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).

View this table:

Lateral flagella gene nomenclature and predicted function

Azospirillum hydrophila lateral flagella gene cluster contains 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 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 polysaccharide biosynthesis clusters (Table 1). It is possible that these two genes may be involved in post-translational modification 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 recombination 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. hydrophila 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, upstream 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 (Gavín 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 homologues of MCP, CheA, CheY, CheB, CheR, CheW and two ORFs of unknown function.

Regulation of lateral-induced flagella systems

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, middle and late genes temporally expressed, with specific transcriptional regulators and σ factors controlling the different transcription levels (Aldridge & Hughes, 2002; Soutourina & Bertin, 2003; McCarter, 2006). In E. coli and S. enterica serovar Typhimurium, the middle flagella genes are σ70-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 σ54-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 σ54-dependent transcriptional activators. Polar flagella class II genes that encode structural components of the MS ring, switch, export-assembly apparatus, the σ28 flagella-specific factor and the two-component signal-transducing system FlrBC in V. cholerae (FleQS in P. aeruginosa), are activated by the σ54-associated 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 σ54-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. parahaemolyticus and A. hydrophila are activated by the σ54-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 (peritrichous, polar and lateral-induced flagella), late gene expression seems to be controlled by the σ28 flagella-specific factor, and its cognate anti-σ 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 σ54-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 (Soutourina & 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).


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.

It has been proposed that polar flagella in V. parahaemolyticus and Azospirillum brasilense act as a mechano-sensor by measuring viscosity, and transduce this signal to control 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 formation 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-transducing 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 signalling 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 components or histone-like DNA-binding proteins. The homologous 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 modulate 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 regulatory 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-dependent 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 in colonization and biofilm formation

An essential step for any infection is the encounter of the pathogenic bacteria with the target eukaryotic cell. Swimming, 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 & Suerbaum, 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 human 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 (Gavín et al., 2002; Gavín 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 colonization and contributes to biofilm formation on different surfaces such as borosilicate glass or microtitre plates (Gavín et al., 2003; Kirov et al., 2004).

In the nitrogen-fixing rhizobacterium Azospirillum brasilense, 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 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; Szymanskin & 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 proinflammatory response (Arora et al., 2005), and is responsible for host plant recognition and the hypersensitivity reaction in Pseudomonas syringae infections (Takeuchi et al., 2003). It is involved in Campylobacter jejuni gut colonization (Szymanski et al., 2002) and is responsible for increased adsorption 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 (Gavín et al., 2002; Canals et al., 2006a), in contrast to other lateral flagella systems. Aeromonas hydrophila and A. caviae possess both polar and lateral glycosylated 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. Tomás, unpublished observation). Moreover, two A. hydrophila genes (maf-1 and 5), whose products are homologous to H. pylori and Campylobacter jejuni Maf proteins (Power & 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.


Motility plays a crucial role in bacterial physiology, and bacteria living in different habitats need to possess locomotion 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 interaction and proinflammatory action of lateral flagella are needed to understand their pathogenic importance.


This work was supported by Plan Nacional de I+D and FIS grants (Ministerio de Educación, Ciencia y Deporte and Ministerio de Sanidad, Spain), from Generalitat de Catalunya and the Wellcome Trust.


  • Editor: Ian Henderson


View Abstract