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The flagellum, a rotary engine required for motility in many bacteria, plays key roles in gene expression. It has been known for some time that flagellar substructures serve as checkpoints that coordinate flagellar gene expression with assembly. Less well understood, however, are other more global effects on gene expression. For instance, the flagellum acts as a ‘wetness’ sensor in Salmonella typhimurium and as a mechanosensor in other bacteria. Additionally, it has been implicated in a variety of bacterial processes, including biofilm formation, pathogenesis and symbiosis. Although for many of these processes it may be simply that motility is required, for other cases it seems that the flagellum plays an underappreciated role in regulating gene expression.
The bacterial flagellum is a complex rotary engine comprised of three basic parts: basal body, hook and filament  (Figure 1). The basal body anchors the flagellum in the cell envelope, and also contains the motor and a specialized type III secretion system (T3SS) for exporting flagellar proteins. The motor consists of a motile part, or rotor, that is connected to the hook via the rod, and a stationary part, or stator, which converts the potential energy of an electrochemical gradient to kinetic motion of the rotor. The hook is a flexible coupling that transfers torque from motor to the propeller-like filament
In addition to motility, flagella have roles in other microbial processes, such as adherence to host cells, host cell invasion, protein secretion, autoagglutination (i.e. clumping or self-adherence of bacterial cells) and biofilm formation [2–6]. For some of these processes a direct involvement of flagella is clear. For example, Campylobacter jejuni flagella are responsible for secretion of several non-flagellar proteins [7–9]. Interestingly, some obligate intracellular symbionts of insects (e.g. Buchnera aphidicola) have lost many of the genes needed for assembly of the flagellum and appear to use the remaining flagellar assembly gene products for export of bacterial proteins to the host [10–12].
For other processes, such as biofilm formation, it is difficult to ascertain the specific role of flagella, which can be quite different among bacteria. For example, flagella contribute to biofilm formation by acting as adhesins to promote attachment to surfaces in Aeromonas spp. , but not in Escherichia coli . Additionally, studies with non-motile (Mot−) mutants that make but are unable to rotate flagella, although useful, do not distinguish if a particular trait of a mutant is due to defects in locomotion, chemotaxis or just flagellar rotation (independently of the first two processes). Indeed, recent studies suggest a role for flagellar rotation in the regulation of biofilm formation. In Bacillus subtilis, a molecular clutch that disengages the flagellum from the rotor might facilitate the transition of free-swimming cells to biofilm-associated bacteria . For Vibrio cholerae, it has been proposed that the attachment of the cell body and flagellum to a surface stops the flagellar motor, resulting in decreased ion flow through the motor and a transient increase in membrane potential (ΔΨ), which might initiate the transition for transient to permanent attachment in biofilm formation. This hypothesis is supported by the fact that experimental dissipation of ΔΨ blocks transition from transient to permanent attachment .
Regulatory roles for the flagellum in other processes are clearer. For example, the flagellum is involved in the temporal regulation of genes encoding its own components. It monitors the status of its own assembly and communicates this information to regulatory systems that control flagellar gene expression. Thus, the flagellum displays a responsiveness (or sensibility) in its assembly. Additionally, in some bacteria, it senses environmental conditions leading to regulated expression of other genes. Here, we review recent studies on the role of the flagellum in autoregulation, as well as its role as an environmental sensor in global gene regulation. In addition, we examine a recent report that the flagellum plays a role in interspecies communication .
Flagellar assembly is a highly ordered process that requires the coordinated expression of dozens of genes. During flagellar biogenesis, the basal body is the first structure to be assembled, followed by the hook and then the filament. Flagellar genes are expressed temporally within a transcriptional hierarchy that results in their products being made as they are needed for assembly. The best characterized systems are those of the enteric bacteria Salmonella enterica serovar Typhimurium (Salmonella typhimurium) and E. coli. In this section we present a brief overview of flagellar gene regulation in these two bacteria and refer the reader to recent reviews for more detailed information [18–20].
The earliest flagellar genes expressed — flhD and flhC — are referred to as class I genes and encode a master regulator FlhDC that activates transcription of the class II flagellar genes [21, 22]. Class II genes encode components of the basal body and hook, the alternative sigma factor FliA (or σ28) and the corresponding anti-sigma factor FlgM (or anti-σ28) [21–23]. Class III genes encode flagellin and other proteins needed late in flagellar biogenesis and require σ28 for their transcription [21, 24]. FlgM binds σ28 and prevents expression of class III genes . Upon completion of the hook–basal body (HBB) complex, FlgM is secreted from the cell via the flagellar protein export apparatus, which allows the class III genes to be expressed . Thus, the flagellum has a direct role in regulating its own biosynthesis, with formation of the HBB complex serving as a key checkpoint for controlling flagellar gene expression (Figure 2a).
How does formation of the HBB complex trigger secretion of FlgM? The flagellar protein export apparatus undergoes a conformational change upon completion of the HBB complex that causes a switch in secretion of rod- and hook-type substrates to filament-type substrates. Two flagellar proteins, FlhB and FliK, are involved directly in the substrate specificity switch [27. 28]. FlhB is a membrane-bound component of the export apparatus that functions as an export switch. The protein consists of a hydrophobic N-terminal domain and a large cytoplasmic C-terminal domain (FlhBC) . FlhBC undergoes autocleavage between the asparagine and proline residues of a specific motif (NPTH) that is conserved among FlhB proteins and T3SS FlhB homologues of plant and animal pathogens [30–34]. Amino acid substitutions within the NPTH motif prevent autocleavage and lock the export apparatus in the rod/hook-type specificity state, indicating that autocleavage is essential for the switch in substrate specificity . Recent reports on the crystal structures of the cytoplasmic domains of four FlhB T3SS homologs have shown that autocleavage does not affect protein folding but rather results in localized electrostatic and conformational changes that might influence interactions with other export apparatus components [32–34].
FliK, the hook length control protein, is also required for the switch in substrate specificity [36–38]. FliK proteins and their homologs in virulence-associated T3SS (such as Yersinia YscP) act as molecular rulers to control the length of the flagellar hook and T3SS needle, respectively [39–42]. The length of the hook is well controlled by FliK at about 55 nm in S. typhimurium, and disruption of fliK results in abnormally long hook structures referred to as ‘polyhooks’ . FliK contains an unstable N-terminal region (FliKN), a stably folded region near the C-terminus (FliKC) and a partially unfolded region at the C-terminus (FliKCT). FliKC and FliKCT, which interact with FlhBC to control export specificity [38, 43, 44], are conserved among FliK/YscP family members and form a type III secretion substrate specificity switch (T3S4) domain .
The model illustrated in Figure 3 accounts for how FliK controls the length of the hook . FliK is secreted by the flagellar protein export apparatus during hook assembly, with the export signal located within FliKN . Within the lumen of the nascent HBB structure the N-terminus of the unfolded FliKN domain can bind the hook cap . If the hook is very short, the T3S4 domain of FliK cannot approach FlhBC, FliK is secreted into the surrounding medium, and hook assembly continues. When the hook length is around 55 nm, binding of the N-terminus of FliK to the hook cap stretches the unfolded FliKN domain out fully, allowing the T3S4 domain to interact with FlhBC and facilitating the switch in export substrate specificity.
Several bacteria, including C. crescentus, Helicobacter pylori, Campylobacter jejuni, V. cholerae and Pseudomonas aeruginosa, employ a particular sigma factor known as σ54 (or RpoN) for expression of specific sets of flagellar genes [19, 20]. Expression of σ54-dependent flagellar genes is responsive to specific flagellar checkpoints and/or a functional flagellar protein export apparatus in some of these bacteria (Figure 2b).
In the α-proteobacterium C. crescentus, the expression of late flagellar genes is regulated by the σ54-dependent transcriptional activator FlbD [47–49]. The activity of FlbD is controlled by FliX, which acts as both a repressor and activator of FlbD [50–52]. FliX forms a stable complex with FlbD and prevents it from activating transcription of σ54-dependent flagellar genes, presumably by interfering with the ability of FlbD to bind DNA sites at target genes . Upon assembly of a nascent flagellar substructure consisting of the MS-ring, motor switch and flagellar protein export apparatus, FliX responds to an unknown signal to activate FlbD, resulting in transcriptional activation of σ54-dependent genes [50–52]. Following completion of the remainder of the flagellum, FliX returns to its role as a repressor of FlbD. Orthologs of flbD and fliX, which always appear to be present together, are confined to a subset of α-Proteobacteria that possess polar flagella . FlbD and FliX link flagellum biosynthesis with cell division in C. crescentus. Evidence for this comes from the observation that class II flagellar mutants exhibit a delay in cytokinesis, but this phenotype can be suppressed by flbD or fliX alleles that allow transcription of late flagellar genes in the class II flagellar mutants .
Assembly of the HBB complex also serves as a checkpoint for flagellar assembly in C. crescentus. The two major C. crescentus flagellin genes, fljK and fljL, are transcribed in the absence of the completed HBB complex but these mRNAs are not translated . A regulatory protein, FlbT, inhibits translation and decreases the stability of the fljK transcript. The 5′-untranslated region (UTR) of the fljK mRNA is predicted to adopt either of two conformations: one favors translation and the other interferes with translation by obscuring the ribosome-binding site through base pairing . Prior to completion of the HBB complex, FlbT is thought to bind the 5′-UTR of the fljK transcript to stabilize the conformation that inhibits translation, which results in decreased stability of the transcript. It is not clear how the inhibitory effect of FlbT on translation of fljK mRNA is alleviated following completion of the HBB complex, but other proteins might be involved. Translation of fljK mRNA is positively regulated by FlaF , which is a small protein (120 amino acid residues) in C. crescentus. The flaF gene is immediately downstream of flbT, a gene arrangement that is conserved in many other bacterial species. Mutant strains that lack flaF are able to assemble the HBB complex but are unable to translate fljK mRNA. A mutant strain deficient in both flbT and flaF is able to synthesize flagellin protein but is non-motile, which suggests that FlaF might play an additional role in flagellum assembly. Orthologs of flbT and flaF occur in many other members of the α-Proteobacteria, and FlbT and FlaF are proposed to control a regulatory checkpoint linking hook assembly and flagellin translation in these bacteria .
Flagellar biogenesis in the ε-Proteobacteria C. jejuni and H. pylori requires three distinct sigma factors. Genes required early in flagellar assembly rely on the housekeeping sigma factor (σ80) for their transcription, while genes needed next in assembly require σ54 for their expression, and those needed at the end of flagellar biogenesis utilize σ28 [19,20] (Figure 2c). Disrupting any one of several genes encoding components of the flagellar protein export apparatus in C. jejuni or H. pylori interferes with expression of the σ54-dependent flagellar genes [57–59]. The export apparatus of C. jejuni appears to modulate the activity of the FlgS/FlgR two-component system required for transcription of the RpoN regulon, perhaps by contributing to signal sensing through the sensor kinase FlgS . The nature of the signal sensed by FlgS is not known, but it is possible that FlgS detects conformational changes within the export apparatus (Box 1). Because FlgS is a soluble protein, it could approach the export apparatus readily from the cytoplasm.
Any model to explain how the export apparatus affects expression of the σ54 (RpoN) regulon in ε-Proteobacteria must take into account the following observations. First, σ54-dependent reporter genes are expressed at enhanced levels in fliK mutants of H. pylori and C. jejuni, whereas they are expressed at wild-type levels in strains that produce FlhB variants defective in autocleavage [59, 60]. This suggests that FliK and the processing of FlhB affect the export apparatus differently with regard to how the apparatus influences expression of the RpoN regulon. Second, H. pylori possesses a protein (FlhX) that shares homology with the C-terminal end of FlhBC (FlhBCC) and can functionally replace it . Interestingly, flhX is required for optimal expression of σ54-dependent reporter genes in a wild-type background but not in strains expressing non-cleavable FlhB variants . A possible explanation for this observation is that FlhBCC might be able to dissociate from the export apparatus upon autocleavage of FlhB and be replaced with FlhX, whereas FlhBCC or FlhX must remain associated with the export apparatus for optimal expression of the RpoN regulon. Finally, mutations in flhA, which encodes a membrane component of the export apparatus, inhibit expression of the RpoN regulon in H. pylori, but this inhibition is alleviated by disrupting flgM. It is not known if FlgM influences the RpoN regulon directly or indirectly. Clearly, additional studies are needed in order to clarify the role of the export apparatus in regulating the RpoN regulon in the ε-Proteobacteria.
Expression of σ54-dependent flagellar genes is stimulated in H. pylori and C. jejuni by deletion of fliK, which supports the hypothesis that the FlgS/FlgR signal transduction system is responsive to conformational changes in the export apparatus [59, 61, 62]. The fliK gene is part of the RpoN regulon in these bacteria, and so FliK might be part of a negative feedback loop that turns off expression of σ54-dependent genes upon completion of the HBB complex. Disrupting fliK in a H. pylori flhB mutant does not restore expression of σ54-dependent reporter genes , suggesting that the inhibition of the RpoN regulon in export apparatus mutants is not due solely to the inability to secrete FliK.
In addition to communicating the status of its assembly to the transcriptional regulatory machinery, the flagellum functions as an environmental sensor in some bacteria. Recent studies revealed that the S. typhimurium flagellum acts as a wetness sensor to initiate swarmer cell differentiation . Swarming is a specialized form of flagellum-mediated motility on moist surfaces. Swarmer cells secrete surfactants and wetting agents that assist in their movement across surfaces in coordinated groups.
Although chemotaxis is not required for the outward migration of S. typhimurium swarmer cells, chemotaxis (che) mutants fail to swarm . Wang and co-workers noted that lawns of S. typhimurium che mutants on swarm agar were drier than wild-type cells, which prompted them to try to rescue the swarming phenotype by rehydrating the lawns with a fine mist of water . Interestingly, this approach worked. DNA microarray studies revealed that only a small set of genes are down-regulated in the che mutants when propagated on swarm agar. These genes include class III motility genes and T3SS genes within the SPI-1 pathogenicity island required by pathogenic Salmonella strains for invasion of epithelial cells. The sequences of the promoters of the virulence genes do not obviously resemble those of class III motility genes, and so the molecular basis that links expression of these genes is not known. Because the class III genes are negatively regulated by FlgM, the researchers examined the ability of the che mutants to secrete FlgM. When grown in broth, che mutants exported FlgM at wild-type levels, but failed to export FlgM when grown on swarm agar. Rehydration of the che mutant lawn restored the ability of the mutants to export FlgM and up-regulate the class III and SPI-1 T3SS genes with the concomitant rescue of swarming . The researchers proposed that when external conditions are too dry, flagellin subunits fail to polymerize at the growing end of the filament and become stuck within the filament. They hypothesize that this blocks secretion of FlgM which leads to repression of the class III genes.
Why are lawns of che mutants dry? The authors speculate that the flagellar filament sticks to swarm agar, and che mutants (which cannot change the direction of the motor) are unable to free their filaments. Consistent with this hypothesis, substitutions in FliM (a component of the motor switch) that increase the frequency of motor reversal suppress the swarming defect of a cheY mutant . Rotation of the filament is thought to aid in external hydration by knocking lipopolysaccharide off neighboring cells which can act as a surfactant or wetting agent [63, 64].
Swarming has been observed for members of several other genera of bacteria, including Aeromonas, Bacillus, Proteus, Serratia and Vibrio. Bacteria that swarm on high agar concentrations, such as Proteus mirabilis, increase their number of flagella for swarming. Other bacteria, such as Aeromonas spp. and Vibrio parahaemolyticus, possess two distinct flagellar systems: a polar flagellum that is expressed constitutively and used for swimming, and an inducible lateral flagellar system used for swarming. Several environmental factors, cellular cues and regulatory proteins have been implicated in swarmer cell differentiation in these bacteria and we refer the reader to recent reviews [65, 66]. We focus here on studies in V. parahemolyticus and P. mirabilis that show that the flagellum acts as a mechanosensor that interprets changes in environmental conditions leading to swarmer cell differentiation.
P. mirabilis is a member of the family Enterobacteriaceae and is associated with urinary tract infections. It forms short vegetative cells when grown in liquid medium, but differentiates into elongated, highly flagellated swarmer cells (20- to 40-fold increase in both cell length and number of flagella per cell) upon contacting a solid surface . Swarmer cell differentiation involves global changes in gene expression, including the increased expression of several virulence genes. The molecular signals required for swarming in P. mirabilis are not well understood, but inhibition of flagellar rotation is a signal for swarmer cell differentiation . Evidence for this model comes from the observation that restricting the rotation of the flagella, by increasing the viscosity of the medium or tethering the flagellum with antibodies, induces swarmer cell differentiation in cells grown in liquid culture . In addition, mutations in some flagellar genes result in aberrant swarmer cell elongation . For example, mutations in fliL cause abnormal swarmer cell differentiation. FliL is an inner membrane protein within the basal body that is thought to interact with motility-enabling (Mot) proteins and/or the MS ring [68–70]. With regard to a potential role in mechanosensing, FliL has been proposed to sense the torque applied to the basal body and motor components upon stalling of the flagellar motor in response to high viscosity environments, or it could monitor proton flux through the motor .
Although how inhibition of flagellar rotation is sensed by P. mirabilis is unclear, the signaling pathway appears to be mediated through the flagellar master regulator FlhDC. The switch from vegetative to swarmer cells is associated with a sharp transient increase in expression of flhDC , and artificial overproduction of FlhDC results in increased elongation and hyperflagellation of swarmer cells . A recent report identified a novel gene, wosA, whose overexpression causes constitutive swarmer cell differentiation under non-inducing conditions . However, WosA is not essential for swarmer cell differentiation as a wosA null mutant displays only a modest reduction in swarmer motility. Although the function of WosA is not known, it appears to be involved in regulating expression of flhDC and might be part of a signaling pathway from the flagellar motor switch .
Swarmer cell differentiation in V. parahaemolyticus similarly involves signal input from the inhibition of flagellum rotation. V. parahaemolyticus is a γ-Proteobacterium that is common to marine and estuarine environments. When growing planktonically, cells utilize polar flagella for swimming. When grown on a solid or highly viscous medium, V. parahaemolyticus forms elongated swarmer cells (5 to 20 times longer than vegetative cells) that possess large numbers of lateral flagella used for swarming motility . Not only do polar and lateral flagellar (laf) systems in V. parahaemolyticus differ in their function but also in how they are powered. The polar flagellum is driven by a sodium motive force whereas lateral flagella are powered by a proton motive force . Similar to P. mirabilis, impairing rotation of the polar flagellum — by increasing the viscosity of the medium, through sodium channel blockers or by mutations — results in an induction of the laf system [75–78]. The mechanism by which V. parahaemolyticus senses inhibition of polar flagellum rotation is not known, but the signal transduction pathway is mediated through the σ54-dependent activator LafK, which controls transcription of the laf genes . Iron-limitation and the signaling molecule cyclic di-guanosine monophosphate (cyclic di-GMP) also play crucial roles in expression of the laf system [80–81], but it is unclear if or how inhibition of polar flagellum rotation is integrated with these cellular signals to regulate swarmer cell differentiation. Polar flagella of some other bacteria, such as Azospirillum lipoferum , also appear to function as mechanosensors in regulating expression of lateral flagella, but this does not seem to be true for other bacteria that have dual flagellar systems (Box 2).
V. parahaemolyticus and other bacteria have dual flagellar systems: polar flagella are used for swimming and lateral flagella are used for swarming. While expression of lateral flagella in V. parahaemolyticus is linked to the function of the polar flagellum, this does not appear to be the case for expression of lateral flagella in Rhodospirillum centrum and Aeromonas spp. [86, 87]. However, other signal transduction systems similar to those affecting expression of lateral flagellum genes in V. parahaemolyticus might be used primarily in these other bacteria. For example, expression of the laf system in V. parahaemolyticus is suppressed under high intracellular levels of cyclic di-GMP, the levels of which can be modulated by the products of scrABC . ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains which control, respectively, the formation and degradation of cyclic di-GMP. The activity of ScrC is modulated by the ScrA and ScrB proteins . A recent report identified a potential cyclic di-GMP riboswitch upstream of a lateral flagellar operon in Aeromonas salmonicida, which suggests that Aeromonas spp. might have elaborated upon this signal transduction system . The first gene of this operon encodes the activator LafK, which initiates the transcriptional hierarchy for lateral gene expression. In the same study, the authors identified a cyclic di-GMP riboswitch within the 5′-UTR of a large flagellar operon from Clostridium difficile and showed that this element together with cyclic di-GMP stimulated the premature termination of transcription of the operon in an in vitro transcription assay . Taken together, these observations suggest that cyclic di-GMP regulates expression of the lateral flagellar genes in A. salmonicida by inhibiting transcription of lafK. This mechanism would eliminate the need for additional signal input from the polar flagellum to initiate swarmer cell differentiation and, therefore, might differ from that by which cyclic di-GMP modulates expression of lateral flagellar genes in V. parahaemolyticus.
Anaerobic degradation of organic compounds involves a syntrophy (symbiosis based on nutritional cooperation) between fermentative bacteria (syntrophs) and methanogenic archaea (methanogens), resulting in the conversion of volatile fatty acids to methane . This process requires the transfer of reducing equivalents, in the form of H2, from the syntroph to the methanogen. Because of thermodynamic constraints for some of the reactions in this process, close physical association between the symbionts is needed for efficient H2 transfer . Recent studies on the syntrophy between Pelotomaculum thermopropionicum (syntroph) and Methanothermobacter thermautotrophicus (methanogen) revealed that the filament cap protein, FliD, of P. thermopropionicum specifically alters expression of over 50 genes in M. thermautotrophicus . Several of the genes that are up-regulated by FliD encode enzymes required for methanogenesis, ATP synthesis or hydrogen utilization, suggesting that M. thermautotrophicus perceives P. thermopropionicum FliD to prepare for syntrophy . Thus, the P. thermopropionicum flagellum appears to play an important role in ensuring the proximity of the bacterium with its methanogenic partner as well as helping synchronize the metabolism of the symbionts.
Discoveries on the roles of the flagellum in gene regulation and environmental sensing point to a much broader role for the flagellum in affecting gene expression in bacteria than previously appreciated. In most of the examples discussed here, the molecular basis by which the flagellum communicates with the transcriptional machinery to control gene expression is poorly understood, and many fundamental questions remain unanswered (Box 3).
Reports showing that interrupting flagellar and/or chemotaxis genes impacts bacterial pathogenesis or symbiosis are widespread in the literature. As indicated previously, one difficulty in ascribing specific roles for flagella in these processes is distinguishing between effects directly involving motility and those associated with signal transduction systems responsive to disruptions in flagellar assembly or rotation. In addition, innate immune systems can also recognize flagella, and this may affect host–microbe interactions at yet another level. As we learn more about the mechanisms used by flagella for signal transduction we will be able to discriminate between these possible scenarios more effectively and undoubtedly discover additional examples of flagellum-mediated signaling in bacteria.
We thank Anna Karls and Eric Stabb for their comments on the manuscript. We also thank the reviewers for their insightful comments and the journal editor for his helpful revisions. T.G.S. was supported by fellowships from the University of Georgia Graduate School and the Atlanta Chapter of Achievement Rewards for College Scientists (ARCS) Foundation. Research on flagellar gene regulation in H. pylori in our lab is supported by award AI080923 to T.R.H. from the National Institutes of Health.
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