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Regulation of flagella
Regulation of flagella Linda L McCarter Flagellar gene networks are fascinating, owing to their complexity — they usually coordinate the expression of more than 40 genes — and particular wiring that elicits temporal expression coupled to organelle morphogenesis. Moreover, many of the lessons learned from flagellar regulation are generally applicable to type III secretion systems. Our understanding of flagellar networks is rapidly expanding to include diverse organisms, as well as deepening to enable the development of predictive wiring diagrams. Numerous regulators control the regulation of flagella, and one of the next challenges in the field is to integrate flagellar gene control into master blueprints of global gene expression. Addresses Microbiology Department, The University of Iowa, Iowa City, IA 52242, USA Corresponding author: McCarter, Linda L ([email protected])
Current Opinion in Microbiology 2006, 9:180–186 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 17th February 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.02.001
(lateral) systems, or even beyond the enteric bacteria. In fact, some E. coli strains have been found to possess an additional flagellar system with a gene complement resembling the s54-dependent lateral flagellar system of Vibrio parahaemolyticus [13]. In comparison, control of the polar flagellar gene system of Caulobacter crescentus is also well understood but very different from the enteric model [14]. Early Caulobacter flagellar gene expression is governed by phosphorylation of the cell cycle regulator CtrA, and late gene expression requires the s54-dependent transcription factor FlbD. Many other bacteria seem to coordinate early flagellar gene expression using s54dependent regulators. Late gene expression seems to be mostly, but not exclusively, controlled by the specialized flagellar sigma factor s28 and its cognate anti-sigma factor FlgM. FlgM keeps late, s28-directed gene expression in check until the flagellar export apparatus is competent for FlgM secretion and, hence, its cytoplasmic depletion. Bioinformatic approaches — including candidate gene targeting, microarray analysis and protein-interaction networks — have revealed new and diverse regulatory scenarios, some of which are outlined below. The master regulators for many systems are presented in Table 1. Probably most similar to the enteric cascade of gene control is the three-tiered flagellar system of Sinorhizobium meliloti; however, it is distinguished by having a non-FlhDC master regulator. Interestingly, this flagellar cascade is controlled by two LuxR-type regulators, VisN and VisR, which probably act as a heterodimer and are activated by unknown signals [15].
Introduction Flagella are complex organelles capable of propelling bacteria through liquids (swimming) and through highly viscous environments or along surfaces (swarming). Many genes participate in generating this sort of motility system. The gene products are produced in a hierarchical fashion: the temporal pattern of gene expression, and/or protein production, generally conforms to the order in which the products are assembled. Figure 1 depicts the major parts of a flagellum. This review discusses only some of the many recent flagellar findings since 2002. The reader is also guided toward more in-depth reviews on various aspects of flagellar assembly and regulation [1–3], flagellar type three export [4–6], motor function [7] and swarming [8–12].
Transcriptional hierarchies The most well understood flagella regulation is the transcription hierarchy found in Escherichia coli and Salmonella typhimurium; however, this paradigm, which has three tiers of gene control coordinated by the master regulators FlhD and FlhC, does not generalize to peritrichous Current Opinion in Microbiology 2006, 9:180–186
Pseudomonas aeruginosa has a four-tiered network elucidated by transcriptional profiling of wild type and flagellar mutant strains [16]. This cascade of gene control, consistent with earlier findings for Vibrio species, seems general for the g-proteobacteria. At the top of the hierarchy is s54-dependent FleQ. FleQ controls the expression of the two-component regulators FleS and FleR, as well as expression of genes whose products are necessary for polar site selection, initiation of basal-body formation and assembly of the export apparatus. The s54-dependent regulator FleR controls expression of class 3 genes necessary for completion of the basal-body hook structure. Class 4 genes are transcribed by s28 and encode the flagellin protein and some chemotaxis proteins. Thus, this regulatory scheme contrasts with E. coli, and although it shares some features (i.e. four levels of gene expression with some dependence on s54) with the system of C. crescentus, it is also significantly different. This scheme also pertains to Legionella pneumophila; in this organism, the candidate gene approach was used to identify FleQ www.sciencedirect.com
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Figure 1
Structure and morphogenesis of the bacterial flagellum. (a) Key parts of the bacterial flagellum. The bacterial flagellum is synthesized in an inside-to-out fashion, nucleated in part by insertion of the MS-ring in the membrane. The basal-body (dark blue) comprises the rings and rods that span the membranes of the cell. Situated below the MS-ring, at its cytoplasmic face is the C-ring (or rotor–switch complex; medium blue), which participates in export and assembly, motor function and chemotaxis. Other export proteins are housed within the C-ring, with some contained in the cytoplasmic membrane within the MS ring of the basal-body. Exported proteins (light blue) pass through a central channel and assemble at the distal end of the forming structure. The basal-body transmits torque from the motor to the hook, which serves as a flexible linker with the flagellar filament that acts as a propeller. Not shown are the torque-generating units that surround the flagellum in the membrane and serve as stators. Interaction of the stators with the rotor component of the C-ring causes rotation. (b) The order of gene expression and product assembly is generally conserved, although there are many variations in how timing and morphogenesis are coupled. In many bacteria, completion of the basal-body and/or the basal-body-hook structure serve as major checkpoints coordinating assembly with gene expression or translation.
and demonstrate the s54-dependence of the flagellar system [17]. Similarly to members of the g-proteobacteria, the e-proteobacteria Helicobacter pylori and Campylobacter jejuni are polarly flagellated. Transcriptional profiling in H. pylori suggests a different four-tiered cascade. Interestingly, no master flagellar regulator has been identified; expression of many (35) early flagellar genes is constitutive [18]. Mutant analyses in C. jejuni support a similar pattern of regulation [19]. One s54-interacting transcriptional regulator has been identified; FlgR controls the expression of www.sciencedirect.com
class 2 genes, including rod–hook, flagellin and sheath protein, as well as contributing to the control of intermediate class genes. A s28 factor transcribes late flagellar genes (including the flagellin gene flaA) and also participates in directing the expression of the intermediate class genes. FlgR is remarkable because, although it possesses the s54-interaction domain, an amino-terminal regulatory domain and a cognate sensory partner (FlgS), it does not have a DNA binding domain [20]. Two other genes have been classified as regulators in H. pylori, FlhF (resembling proteins in the signal recognition particle family, which participate in targeting proteins across membranes) and FlhA (an integral membrane export protein), because mutations in these genes were found to prevent full transcription of class 2 and 3 genes [18]; FlhF was also recently implicated as a regulator of class 3 and 4 genes in Vibrio cholerae [21]. However, it should be noted that any flagellar gene product that participates in the completion of a timing checkpoint might have apparent, albeit indirect, regulatory effects. H. pylori FlgR is similar to one of two newly discovered regulators in Rhodobacter sphaeroides [22]. By also using the candidate gene approach, two s54-type regulators were found to control the flagellar hierarchy. FleQ (a class 1 gene product) controls transcription of class 2 genes, which encode some of the assembly and export proteins, and the second regulator FleT. FleT and FleQ, acting together as a hetero-oligomer, control the expression of class 3 genes, which results in completion of the hook basal-body complex, export of FlgM and the ensuing s28-directed expression of late flagellar genes. FleT is similar to FlgR in that it possesses no DNA binding domain; FleQ does possess a DNA binding domain. Both FleT and FleQ also lack amino-terminal regulatory domains. Temporal gene expression seems to be achieved by the modulation of the concentration of FleT, which interacts with FleQ. This interaction alters the binding affinity and specificity of FleQ and produces a hetero-oligomer that recognizes class 3 promoters. Most divergent of the known examples of flagellar gene regulation is the case for the spirochetes. These organisms apparently completely lack a transcriptional cascade; flagellar operons are transcribed by the housekeeping s70, and no identifiable s28 homolog exists. Regulation appears to occur post-transcriptionally [23]. The Bacillus subtilis cascade is also seemingly simpler, with two apparent classes: a large fla–che operon (also containing the gene for the flagellar-specific sigma factor sD) and the subsequently transcribed sD-dependent genes [24].
Linking regulation to assembly The beauty and complexity of flagellar assembly lies in how intimately gene regulation and protein supply are linked to substrate secretion. Built into all type III secretion and assembly systems that have been studied Current Opinion in Microbiology 2006, 9:180–186
182 Cell regulation
Table 1 Flagellar transcriptional regulators Flagellar system
Master flagellar regulators
Secondary regulators
s54-dependence of regulators
References
Lateral (peritrichous) flagella of enteric bacteria Polar system of a-proteobacteria C. crescentus
FlhDC CtrA
No
No No Yes
[56] [55]
Polar system of a-proteobacteria S. meliloti Polar system of the g-proteobacteria (including Pseudomonas, Legionella, and Vibrio spp.)
VisNR (LuxR-type proteins) FleQ Aka: FlrA FlaK Not identified
FlbD
Polar system of the e-proteobacteria Campylobacter and Helicobacter spp. Polar system of a-proteobacteria R. sphaeroides Lateral system of V. parahaemolyticus Endoflagella of the Spirochaeta
FleQ LafK Not identified
are checkpoints of control that couple temporal patterns of gene expression with organelle assembly or function. This strategy results in an ordered procession of substrates incorporated into, or exported by, the organelle and is achieved by numerous mechanisms. Some of the most recent findings are summarized below. Substrate gating and switching: hook-length control
Switching of export substrates from rod and hook components to filament proteins occurs upon completion of the hook and is mediated by interaction of the hooklength control protein FliK with the export-gating protein FlhB. Direct evidence has been obtained demonstrating that conformational changes in FlhB mediate substrate specificity for export; the cytoplasmic domain of FlhB undergoes a proteolytic cleavage at the junction of two subdomains, and interaction of these two subdomains determines substrate specificity [25,26]. Mutant forms of FlhB have been isolated, and these mutant proteins, which fail to respond to FliK and undergo proteolytic cleavage, are locked into the transporting rod and hook components. Thus, somehow the amino-terminus of FliK detects information about appropriate hook length and transmits that information through its own carboxy-terminus to the carboxy-terminus of FlhB [27]. However, exactly how FliK works to monitor hook length is still a mystery, although recent work demonstrates two interesting findings, namely that the absolute size of FliK is not crucial for function, and neither is its export [28]. Thus, FliK seems to act differently to its counterpart in the type III secretion system injectisome that delivers virulence factors to eukaryotic cells: it does not seem to act strictly as a molecular ruler, as has been proposed for the similar event in controlling needle length of the injectisome, nor does it need to be secreted to exert its length-control function [29]. The model for the flagellar apparatus proposes that FliK acts to influence FlhB-substrate gating, only after the hook subunits have been emptied from the C-ring and Current Opinion in Microbiology 2006, 9:180–186
FleSR FlrBC FlaLM
Yes
[15] [16,17,57–59]
FlgRS
Yes (for FlgR)
[18,19]
FleT
Yes Yes
[22] [60] [23]
assembled. Thus, the C-ring itself might function as the measuring device. Feedback regulation by chaperones
Work in S. typhimurium has refined our understanding of how the status of assembly is monitored by revealing that multiple chaperones (specifically FlgN, FliT and FliS) not only protect their cognate substrates from degradation, but also directly participate in the control of flagellar transcription and/or protein production [1,6]. In fact, in some cases, the protection role might be secondary to the regulatory role. For example, although intracellular FlgK and FlgL are protected to some degree by FlgN, FlgN is not absolutely required for FlgKL secretion. However, the FlgKL–FlgN interaction does serve to effectively sequester FlgN. Free FlgN promotes translation of the anti-s28 factor FlgM. Thus, the presence or absence of intracellular FlgKL influences the activity of s28 and late gene expression [30]. Activators and anti-activators
There are two checkpoints during the assembly of the polar flagellum in C. crescentus [14]. Early (class 2) flagellar gene expression results in the assembly of the MS-ring, the switch and the export machinery. The s54-dependent regulator FlbD is subsequently activated for transcription of both class 3 (hook basal-body) and class 4 (flagellin) gene expression. FlbD activity is held in check until completion of the class 2 structure by physical interaction with an anti-activator, FliX [31]. Therefore FliX, which is a small protein (144 amino acids) with homologs in other a-proteobacteria, seems to be another molecule capable of reporting the assembly status of the flagellum and controlling flagellar gene expression. A similar regulatory scenario (i.e. using the binding of an anti-activator to control activity of a s54-type regulator) has been demonstrated for P. aeruginosa. In P. aeruginosa, the flagellar number regulator FleN is a MinD-like prowww.sciencedirect.com
Regulation of flagella McCarter 183
tein which physically interacts to control the activity of the s54-dependent master regulator FleQ [32]. It possesses an ATP-binding site that is necessary for this regulatory effect. FleN homologs occur in Vibrio, Campylobacter and Helicobacter strains, where they are named FlhG. In V. cholerae, FlhG has also been shown negatively to influence early gene expression [21], although physical interaction with a flagellar transcriptional regulator has not been demonstrated. Furthermore, the level at which the V. cholerae homolog appears to act seems to be different from FleN: FlhG affects class 1 transcription.
gated regulators are listed in Table 2. Many are response regulators, capable of integrating sensory input to the control of gene expression [34–36]. Many are global regulators [2,11]; some are quorum-sensing responsive [37]; others are small RNAs — specifically, CsrB and CsrC, small RNAs that control the activity of the global regulator CsrA (RsmA) [38]. Regulation can also be achieved by proteolytic degradation of the master regulator [39] or by the concentration of small signaling molecules, such as acetyl phosphate [40]. Why are there so many regulators?
Regulators that influence message stability
Although both class 3 and 4 genes in C. crescentus are induced by FlbD, the class 4 flagellin gene products are produced only upon completion of the second checkpoint — that is, upon hook assembly. The hook checkpoint somehow influences the balance between the opposing activities of two other regulatory proteins: FlbT, which prevents flagellin mRNA translation, and FlaF, which acts to derepress the translational block and stabilize flagellin mRNA [33].
Regulating the master flagellar regulators A rapidly expanding list of regulators acts to coordinate flagellar production. Some of the most recently investi-
The production of flagella requires a sizable investment of cellular reserves — many genes are expressed, and some at very high levels. Moreover, operation of the flagellar system is costly in terms of the energy commitment to power flagellar rotation. Thus, there are clearly times when the ability to swim is advantageous and times when it is not advantageous. For example, CodY, a global regulator in B. subtilis and other Gram-positive bacteria, controls the expression of many genes in response to nutrient availability and growth rate. CodY represses flagellar operons in nutrient-rich conditions [41]. Under highly favorable nutrient conditions, B. subtilis appears to prefer to remain stationary; whereas less favorable conditions induce the formation of a motility and chemotaxis
Table 2 Some examples of global regulators known to influence flagellar number* Regulators of the master flagellar regulators
Target (organisms)
References
Lrp
FlhDC (E. coli, S. typhimurium, Proteus mirabilis, Xenorhabdus nematophila, Serratia marcescens)
[2,11,34–37,40,44–47,51,52,61]
CtrA (C. crescentus) FleQ (P. aeruginosa)
[62] [63]
CsrA (RsmA) and CsrBC RcsCDB (RcsD, a.k.a. YojN and RsbA) RssAB CAP H-NS ClpX/P QseBC (quorum sensing) OmpR LrhA FimZ (fimbrial regulator) RtsAB Acetyl phosphate USPs (universal stress proteins) GcrA Vfr
Other regulators known to influence flagellar number CodY B. subtilis DegU Listeria monocytogenes MogR SinR SwrA ExoRS ScrABC
S. meliloti V. parahaemolyticus
[24,41,42,53,64,65]
[43] [48]
* In some cases, the target is known (e.g. the flagellar master regulators and/or class 1 gene expression) and in other cases, the level at which control is exerted is not known.
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system enabling movement into new, more attractive environments. However, unfavorable nutrient conditions might also not be conducive or capable of sustaining energy-intensive flagellar production, and two-component regulators such as DegSU can serve to prevent flagellar biosynthesis and induce the production of nutrient-scavenging extracellular enzymes [24].The decision to swarm seems to be an even larger commitment because swarming involves differentiation into an elongated cell type, hyperflagellation and the production of slime (or friction-reducing molecules). Some pivotal swarming regulators have been identified (e.g. the enteric RcsBCD regulators [11] and the B. subtilis swrA locus [42]) but what they sense or how they modulate regulation is not yet clear. Many of the regulators listed in Table 2 act to balance adhesiveness and motility. Examples of such regulators include: S. meliloti ExoRS, which inversely controls the production of succinoglycan and flagella [43]; S. typhimurium FimZ and E. coli LrhA, which inversely control the production of fimbriae and flagella [44,45]; the RcsCDB phosphorelay controlling capsular polysaccharide, flagella and swarming [46]; and the universal stress proteins of E. coli which coordinate oxidative stress resistance, adhesion and motility [47]. Cyclic di-GMP, whose level is controlled by GGDEF and EAL-type proteins acting as diguanylate cyclases and/or phosphodiesterases, might also serve to coordinate the switch between sticking and swimming by regulating flagella, capsular polysaccharide, pili, curli and cellulose production [48–50]. Altering cell surface properties and motility can have profound consequences on the pathogenicity of the organism. In addition, other virulence genes are often co-regulated or inversely regulated with motility [10,51–53].
Figure 2
To swim, swarm, or stick? One of the next challenges in understanding regulation of flagella is mapping the global regulatory networks that coordinate the flight or sticking responses. How do bacteria make decisions about whether to swim, to induce the swarmer cell program and greatly upregulate flagella, or to downregulate flagella production and stick by upregulating other extracellular features including polysaccharide (EPS), pili and other components of the extracellular matrix (such as cellulose)?.
Acknowledgements Work in my laboratory is supported by NSF grant MCB0315617.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
Conclusions: to swim, swarm or stick? As evidenced by the numerous regulators influencing flagellar gene expression, the cell integrates multiple sensory inputs before committing to carefully programmed expression of flagellar genes. This program of flagellar gene expression has been particularly and elegantly dissected in E. coli and C. crescentus [54,55]. One important and exciting consequence is that these flagellar gene networks can serve as models to study gene expression in silico. For E. coli, it has resulted in the development of a quantitative wiring diagram, a blueprint modeling regulation [54]. This wiring pattern is hallmarked by a feed-forward loop that accommodates the observed temporal pattern of gene expression. One of the next challenges in deciphering flagellar regulation is the formulation of wiring diagrams connecting the numerous sensory and crosstalking inputs that govern the regulation of flagella. Such connectivity blueprints will provide insight (and testable models) into how bacteria make the decision to swim, swarm or stick (Figure 2). Current Opinion in Microbiology 2006, 9:180–186
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40. Wolfe AJ, Chang DE, Walker JD, Seitz-Partridge JE, Vidaurri MD, Lange CF, Pruss BM, Henk MC, Larkin JC, Conway T: Evidence that acetyl phosphate functions as a global signal during biofilm development. Mol Microbiol 2003, 48:977-988. 41. Bergara F, Ibarra C, Iwamasa J, Patarroyo JC, Aguilera R, Marquez-Magana LM: CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J Bacteriol 2003, 185:3118-3126. 42. Calvio C, Celandroni F, Ghelardi E, Amati G, Salvetti S, Ceciliani F, Galizzi A, Senesi S: Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J Bacteriol 2005, 187:5356-5366. This study characterized a key regulatory locus controlling swimming and swarming in B. subtilis. 43. Yao SY, Luo L, Har KJ, Becker A, Ruberg S, Yu GQ, Zhu JB, Cheng HP: Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production. J Bacteriol 2004, 186:6042-6049. 44. Clegg S, Hughes KT: FimZ is a molecular link between sticking and swimming in Salmonella enterica serovar Typhimurium. J Bacteriol 2002, 184:1209-1213. 45. Blumer C, Kleefeld A, Lehnen D, Heintz M, Dobrindt U, Nagy G, Michaelis K, Emody L, Polen T, Rachel R et al.: Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli. Microbiology 2005, 151:3287-3298. 46. Francez-Charlot A, Laugel B, Van Gemert A, Dubarry N, Wiorowski F, Castanie-Cornet MP, Gutierrez C, Cam K: RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol 2003, 49:823-832. 47. Nachin L, Nannmark U, Nystrom T: Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility. J Bacteriol 2005, 187:6265-6272.
54. Kalir S, Alon U: Using a quantitative blueprint to reprogram the dynamics of the flagella gene network. Cell 2004, 117:713-720. Based upon experimental data, a quantitative blueprint of flagellar gene expression was mathematically modeled and tested by using controlled expression of regulators. This work represents an exciting advance to probing and understanding complex genetic circuits. 55. McAdams HH, Shapiro L: A bacterial cell-cycle regulatory network operating in time and space. Science 2003, 301:1874-1877. This work also describes a complex wiring diagram, in particular the CtrA regulatory network of C. crescentus. CtrA is a key regulator controlling the Caulobacter cell cycle and the determining regulator for the flagellar network. 56. Claret L, Hughes C: Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy. J Mol Biol 2002, 321:185-199. This study showed that the binding affinities of FlhD2C2 to the various flagellar promoters correlate with promoter strength and the timing of gene expression. 57. Kim YK, McCarter LL: Cross-regulation in Vibrio parahaemolyticus: compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK. J Bacteriol 2004, 186:4014-4018. 58. Correa NE, Klose KE: Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC. J Bacteriol 2005, 187:3158-3170. 59. Millikan DS, Ruby EG: FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization. J Bacteriol 2003, 185:3547-3557. 60. Stewart BJ, McCarter LL: Lateral flagellar gene system of Vibrio parahaemolyticus. J Bacteriol 2003, 185:4508-4518. This study mapped the three-tiered lateral flagellar transcriptional hierarchy by using mutant and fusion analyses.
48. Boles BR, McCarter LL: Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation. J Bacteriol 2002, 184:5946-5954.
61. Tomoyasu T, Takaya A, Isogai E, Yamamoto T: Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol 2003, 48:443-452.
49. Simm R, Morr M, Kader A, Nimtz M, Romling U: GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 2004, 53:1123-1134.
62. Crosson S, McAdams H, Shapiro L: A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus. Cell Cycle 2004, 3:1252-1254.
50. Jenal U: Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? Curr Opin Microbiol 2004, 7:185-191.
63. Dasgupta N, Ferrell EP, Kanack KJ, West SE, Ramphal R: fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein. J Bacteriol 2002, 184:5240-5250.
51. Ellermeier CD, Slauch JM: RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium. J Bacteriol 2003, 185:5096-5108. 52. Teplitski M, Goodier RI, Ahmer BM: Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella. J Bacteriol 2003, 185:7257-7265. 53. Grundling A, Burrack LS, Bouwer HG, Higgins DE: Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. Proc Natl Acad Sci USA 2004, 101:12318-12323.
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64. Williams T, Joseph B, Beier D, Goebel W, Kuhn M: Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes. FEMS Microbiol Lett 2005, 252:287-298. 65. Kearns DB, Chu F, Rudner R, Losick R: Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol 2004, 52:357-369. The authors describe the identification of a key regulatory locus swrA controlling flagellar regulation and swarming in B. subtilis.
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Current Opinion in Microbiology 2006, 9:180–186 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 17th February 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.02.001
(lateral) systems, or even beyond the enteric bacteria. In fact, some E. coli strains have been found to possess an additional flagellar system with a gene complement resembling the s54-dependent lateral flagellar system of Vibrio parahaemolyticus [13]. In comparison, control of the polar flagellar gene system of Caulobacter crescentus is also well understood but very different from the enteric model [14]. Early Caulobacter flagellar gene expression is governed by phosphorylation of the cell cycle regulator CtrA, and late gene expression requires the s54-dependent transcription factor FlbD. Many other bacteria seem to coordinate early flagellar gene expression using s54dependent regulators. Late gene expression seems to be mostly, but not exclusively, controlled by the specialized flagellar sigma factor s28 and its cognate anti-sigma factor FlgM. FlgM keeps late, s28-directed gene expression in check until the flagellar export apparatus is competent for FlgM secretion and, hence, its cytoplasmic depletion. Bioinformatic approaches — including candidate gene targeting, microarray analysis and protein-interaction networks — have revealed new and diverse regulatory scenarios, some of which are outlined below. The master regulators for many systems are presented in Table 1. Probably most similar to the enteric cascade of gene control is the three-tiered flagellar system of Sinorhizobium meliloti; however, it is distinguished by having a non-FlhDC master regulator. Interestingly, this flagellar cascade is controlled by two LuxR-type regulators, VisN and VisR, which probably act as a heterodimer and are activated by unknown signals [15].
Introduction Flagella are complex organelles capable of propelling bacteria through liquids (swimming) and through highly viscous environments or along surfaces (swarming). Many genes participate in generating this sort of motility system. The gene products are produced in a hierarchical fashion: the temporal pattern of gene expression, and/or protein production, generally conforms to the order in which the products are assembled. Figure 1 depicts the major parts of a flagellum. This review discusses only some of the many recent flagellar findings since 2002. The reader is also guided toward more in-depth reviews on various aspects of flagellar assembly and regulation [1–3], flagellar type three export [4–6], motor function [7] and swarming [8–12].
Transcriptional hierarchies The most well understood flagella regulation is the transcription hierarchy found in Escherichia coli and Salmonella typhimurium; however, this paradigm, which has three tiers of gene control coordinated by the master regulators FlhD and FlhC, does not generalize to peritrichous Current Opinion in Microbiology 2006, 9:180–186
Pseudomonas aeruginosa has a four-tiered network elucidated by transcriptional profiling of wild type and flagellar mutant strains [16]. This cascade of gene control, consistent with earlier findings for Vibrio species, seems general for the g-proteobacteria. At the top of the hierarchy is s54-dependent FleQ. FleQ controls the expression of the two-component regulators FleS and FleR, as well as expression of genes whose products are necessary for polar site selection, initiation of basal-body formation and assembly of the export apparatus. The s54-dependent regulator FleR controls expression of class 3 genes necessary for completion of the basal-body hook structure. Class 4 genes are transcribed by s28 and encode the flagellin protein and some chemotaxis proteins. Thus, this regulatory scheme contrasts with E. coli, and although it shares some features (i.e. four levels of gene expression with some dependence on s54) with the system of C. crescentus, it is also significantly different. This scheme also pertains to Legionella pneumophila; in this organism, the candidate gene approach was used to identify FleQ www.sciencedirect.com
Regulation of flagella McCarter 181
Figure 1
Structure and morphogenesis of the bacterial flagellum. (a) Key parts of the bacterial flagellum. The bacterial flagellum is synthesized in an inside-to-out fashion, nucleated in part by insertion of the MS-ring in the membrane. The basal-body (dark blue) comprises the rings and rods that span the membranes of the cell. Situated below the MS-ring, at its cytoplasmic face is the C-ring (or rotor–switch complex; medium blue), which participates in export and assembly, motor function and chemotaxis. Other export proteins are housed within the C-ring, with some contained in the cytoplasmic membrane within the MS ring of the basal-body. Exported proteins (light blue) pass through a central channel and assemble at the distal end of the forming structure. The basal-body transmits torque from the motor to the hook, which serves as a flexible linker with the flagellar filament that acts as a propeller. Not shown are the torque-generating units that surround the flagellum in the membrane and serve as stators. Interaction of the stators with the rotor component of the C-ring causes rotation. (b) The order of gene expression and product assembly is generally conserved, although there are many variations in how timing and morphogenesis are coupled. In many bacteria, completion of the basal-body and/or the basal-body-hook structure serve as major checkpoints coordinating assembly with gene expression or translation.
and demonstrate the s54-dependence of the flagellar system [17]. Similarly to members of the g-proteobacteria, the e-proteobacteria Helicobacter pylori and Campylobacter jejuni are polarly flagellated. Transcriptional profiling in H. pylori suggests a different four-tiered cascade. Interestingly, no master flagellar regulator has been identified; expression of many (35) early flagellar genes is constitutive [18]. Mutant analyses in C. jejuni support a similar pattern of regulation [19]. One s54-interacting transcriptional regulator has been identified; FlgR controls the expression of www.sciencedirect.com
class 2 genes, including rod–hook, flagellin and sheath protein, as well as contributing to the control of intermediate class genes. A s28 factor transcribes late flagellar genes (including the flagellin gene flaA) and also participates in directing the expression of the intermediate class genes. FlgR is remarkable because, although it possesses the s54-interaction domain, an amino-terminal regulatory domain and a cognate sensory partner (FlgS), it does not have a DNA binding domain [20]. Two other genes have been classified as regulators in H. pylori, FlhF (resembling proteins in the signal recognition particle family, which participate in targeting proteins across membranes) and FlhA (an integral membrane export protein), because mutations in these genes were found to prevent full transcription of class 2 and 3 genes [18]; FlhF was also recently implicated as a regulator of class 3 and 4 genes in Vibrio cholerae [21]. However, it should be noted that any flagellar gene product that participates in the completion of a timing checkpoint might have apparent, albeit indirect, regulatory effects. H. pylori FlgR is similar to one of two newly discovered regulators in Rhodobacter sphaeroides [22]. By also using the candidate gene approach, two s54-type regulators were found to control the flagellar hierarchy. FleQ (a class 1 gene product) controls transcription of class 2 genes, which encode some of the assembly and export proteins, and the second regulator FleT. FleT and FleQ, acting together as a hetero-oligomer, control the expression of class 3 genes, which results in completion of the hook basal-body complex, export of FlgM and the ensuing s28-directed expression of late flagellar genes. FleT is similar to FlgR in that it possesses no DNA binding domain; FleQ does possess a DNA binding domain. Both FleT and FleQ also lack amino-terminal regulatory domains. Temporal gene expression seems to be achieved by the modulation of the concentration of FleT, which interacts with FleQ. This interaction alters the binding affinity and specificity of FleQ and produces a hetero-oligomer that recognizes class 3 promoters. Most divergent of the known examples of flagellar gene regulation is the case for the spirochetes. These organisms apparently completely lack a transcriptional cascade; flagellar operons are transcribed by the housekeeping s70, and no identifiable s28 homolog exists. Regulation appears to occur post-transcriptionally [23]. The Bacillus subtilis cascade is also seemingly simpler, with two apparent classes: a large fla–che operon (also containing the gene for the flagellar-specific sigma factor sD) and the subsequently transcribed sD-dependent genes [24].
Linking regulation to assembly The beauty and complexity of flagellar assembly lies in how intimately gene regulation and protein supply are linked to substrate secretion. Built into all type III secretion and assembly systems that have been studied Current Opinion in Microbiology 2006, 9:180–186
182 Cell regulation
Table 1 Flagellar transcriptional regulators Flagellar system
Master flagellar regulators
Secondary regulators
s54-dependence of regulators
References
Lateral (peritrichous) flagella of enteric bacteria Polar system of a-proteobacteria C. crescentus
FlhDC CtrA
No
No No Yes
[56] [55]
Polar system of a-proteobacteria S. meliloti Polar system of the g-proteobacteria (including Pseudomonas, Legionella, and Vibrio spp.)
VisNR (LuxR-type proteins) FleQ Aka: FlrA FlaK Not identified
FlbD
Polar system of the e-proteobacteria Campylobacter and Helicobacter spp. Polar system of a-proteobacteria R. sphaeroides Lateral system of V. parahaemolyticus Endoflagella of the Spirochaeta
FleQ LafK Not identified
are checkpoints of control that couple temporal patterns of gene expression with organelle assembly or function. This strategy results in an ordered procession of substrates incorporated into, or exported by, the organelle and is achieved by numerous mechanisms. Some of the most recent findings are summarized below. Substrate gating and switching: hook-length control
Switching of export substrates from rod and hook components to filament proteins occurs upon completion of the hook and is mediated by interaction of the hooklength control protein FliK with the export-gating protein FlhB. Direct evidence has been obtained demonstrating that conformational changes in FlhB mediate substrate specificity for export; the cytoplasmic domain of FlhB undergoes a proteolytic cleavage at the junction of two subdomains, and interaction of these two subdomains determines substrate specificity [25,26]. Mutant forms of FlhB have been isolated, and these mutant proteins, which fail to respond to FliK and undergo proteolytic cleavage, are locked into the transporting rod and hook components. Thus, somehow the amino-terminus of FliK detects information about appropriate hook length and transmits that information through its own carboxy-terminus to the carboxy-terminus of FlhB [27]. However, exactly how FliK works to monitor hook length is still a mystery, although recent work demonstrates two interesting findings, namely that the absolute size of FliK is not crucial for function, and neither is its export [28]. Thus, FliK seems to act differently to its counterpart in the type III secretion system injectisome that delivers virulence factors to eukaryotic cells: it does not seem to act strictly as a molecular ruler, as has been proposed for the similar event in controlling needle length of the injectisome, nor does it need to be secreted to exert its length-control function [29]. The model for the flagellar apparatus proposes that FliK acts to influence FlhB-substrate gating, only after the hook subunits have been emptied from the C-ring and Current Opinion in Microbiology 2006, 9:180–186
FleSR FlrBC FlaLM
Yes
[15] [16,17,57–59]
FlgRS
Yes (for FlgR)
[18,19]
FleT
Yes Yes
[22] [60] [23]
assembled. Thus, the C-ring itself might function as the measuring device. Feedback regulation by chaperones
Work in S. typhimurium has refined our understanding of how the status of assembly is monitored by revealing that multiple chaperones (specifically FlgN, FliT and FliS) not only protect their cognate substrates from degradation, but also directly participate in the control of flagellar transcription and/or protein production [1,6]. In fact, in some cases, the protection role might be secondary to the regulatory role. For example, although intracellular FlgK and FlgL are protected to some degree by FlgN, FlgN is not absolutely required for FlgKL secretion. However, the FlgKL–FlgN interaction does serve to effectively sequester FlgN. Free FlgN promotes translation of the anti-s28 factor FlgM. Thus, the presence or absence of intracellular FlgKL influences the activity of s28 and late gene expression [30]. Activators and anti-activators
There are two checkpoints during the assembly of the polar flagellum in C. crescentus [14]. Early (class 2) flagellar gene expression results in the assembly of the MS-ring, the switch and the export machinery. The s54-dependent regulator FlbD is subsequently activated for transcription of both class 3 (hook basal-body) and class 4 (flagellin) gene expression. FlbD activity is held in check until completion of the class 2 structure by physical interaction with an anti-activator, FliX [31]. Therefore FliX, which is a small protein (144 amino acids) with homologs in other a-proteobacteria, seems to be another molecule capable of reporting the assembly status of the flagellum and controlling flagellar gene expression. A similar regulatory scenario (i.e. using the binding of an anti-activator to control activity of a s54-type regulator) has been demonstrated for P. aeruginosa. In P. aeruginosa, the flagellar number regulator FleN is a MinD-like prowww.sciencedirect.com
Regulation of flagella McCarter 183
tein which physically interacts to control the activity of the s54-dependent master regulator FleQ [32]. It possesses an ATP-binding site that is necessary for this regulatory effect. FleN homologs occur in Vibrio, Campylobacter and Helicobacter strains, where they are named FlhG. In V. cholerae, FlhG has also been shown negatively to influence early gene expression [21], although physical interaction with a flagellar transcriptional regulator has not been demonstrated. Furthermore, the level at which the V. cholerae homolog appears to act seems to be different from FleN: FlhG affects class 1 transcription.
gated regulators are listed in Table 2. Many are response regulators, capable of integrating sensory input to the control of gene expression [34–36]. Many are global regulators [2,11]; some are quorum-sensing responsive [37]; others are small RNAs — specifically, CsrB and CsrC, small RNAs that control the activity of the global regulator CsrA (RsmA) [38]. Regulation can also be achieved by proteolytic degradation of the master regulator [39] or by the concentration of small signaling molecules, such as acetyl phosphate [40]. Why are there so many regulators?
Regulators that influence message stability
Although both class 3 and 4 genes in C. crescentus are induced by FlbD, the class 4 flagellin gene products are produced only upon completion of the second checkpoint — that is, upon hook assembly. The hook checkpoint somehow influences the balance between the opposing activities of two other regulatory proteins: FlbT, which prevents flagellin mRNA translation, and FlaF, which acts to derepress the translational block and stabilize flagellin mRNA [33].
Regulating the master flagellar regulators A rapidly expanding list of regulators acts to coordinate flagellar production. Some of the most recently investi-
The production of flagella requires a sizable investment of cellular reserves — many genes are expressed, and some at very high levels. Moreover, operation of the flagellar system is costly in terms of the energy commitment to power flagellar rotation. Thus, there are clearly times when the ability to swim is advantageous and times when it is not advantageous. For example, CodY, a global regulator in B. subtilis and other Gram-positive bacteria, controls the expression of many genes in response to nutrient availability and growth rate. CodY represses flagellar operons in nutrient-rich conditions [41]. Under highly favorable nutrient conditions, B. subtilis appears to prefer to remain stationary; whereas less favorable conditions induce the formation of a motility and chemotaxis
Table 2 Some examples of global regulators known to influence flagellar number* Regulators of the master flagellar regulators
Target (organisms)
References
Lrp
FlhDC (E. coli, S. typhimurium, Proteus mirabilis, Xenorhabdus nematophila, Serratia marcescens)
[2,11,34–37,40,44–47,51,52,61]
CtrA (C. crescentus) FleQ (P. aeruginosa)
[62] [63]
CsrA (RsmA) and CsrBC RcsCDB (RcsD, a.k.a. YojN and RsbA) RssAB CAP H-NS ClpX/P QseBC (quorum sensing) OmpR LrhA FimZ (fimbrial regulator) RtsAB Acetyl phosphate USPs (universal stress proteins) GcrA Vfr
Other regulators known to influence flagellar number CodY B. subtilis DegU Listeria monocytogenes MogR SinR SwrA ExoRS ScrABC
S. meliloti V. parahaemolyticus
[24,41,42,53,64,65]
[43] [48]
* In some cases, the target is known (e.g. the flagellar master regulators and/or class 1 gene expression) and in other cases, the level at which control is exerted is not known.
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184 Cell regulation
system enabling movement into new, more attractive environments. However, unfavorable nutrient conditions might also not be conducive or capable of sustaining energy-intensive flagellar production, and two-component regulators such as DegSU can serve to prevent flagellar biosynthesis and induce the production of nutrient-scavenging extracellular enzymes [24].The decision to swarm seems to be an even larger commitment because swarming involves differentiation into an elongated cell type, hyperflagellation and the production of slime (or friction-reducing molecules). Some pivotal swarming regulators have been identified (e.g. the enteric RcsBCD regulators [11] and the B. subtilis swrA locus [42]) but what they sense or how they modulate regulation is not yet clear. Many of the regulators listed in Table 2 act to balance adhesiveness and motility. Examples of such regulators include: S. meliloti ExoRS, which inversely controls the production of succinoglycan and flagella [43]; S. typhimurium FimZ and E. coli LrhA, which inversely control the production of fimbriae and flagella [44,45]; the RcsCDB phosphorelay controlling capsular polysaccharide, flagella and swarming [46]; and the universal stress proteins of E. coli which coordinate oxidative stress resistance, adhesion and motility [47]. Cyclic di-GMP, whose level is controlled by GGDEF and EAL-type proteins acting as diguanylate cyclases and/or phosphodiesterases, might also serve to coordinate the switch between sticking and swimming by regulating flagella, capsular polysaccharide, pili, curli and cellulose production [48–50]. Altering cell surface properties and motility can have profound consequences on the pathogenicity of the organism. In addition, other virulence genes are often co-regulated or inversely regulated with motility [10,51–53].
Figure 2
To swim, swarm, or stick? One of the next challenges in understanding regulation of flagella is mapping the global regulatory networks that coordinate the flight or sticking responses. How do bacteria make decisions about whether to swim, to induce the swarmer cell program and greatly upregulate flagella, or to downregulate flagella production and stick by upregulating other extracellular features including polysaccharide (EPS), pili and other components of the extracellular matrix (such as cellulose)?.
Acknowledgements Work in my laboratory is supported by NSF grant MCB0315617.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
Conclusions: to swim, swarm or stick? As evidenced by the numerous regulators influencing flagellar gene expression, the cell integrates multiple sensory inputs before committing to carefully programmed expression of flagellar genes. This program of flagellar gene expression has been particularly and elegantly dissected in E. coli and C. crescentus [54,55]. One important and exciting consequence is that these flagellar gene networks can serve as models to study gene expression in silico. For E. coli, it has resulted in the development of a quantitative wiring diagram, a blueprint modeling regulation [54]. This wiring pattern is hallmarked by a feed-forward loop that accommodates the observed temporal pattern of gene expression. One of the next challenges in deciphering flagellar regulation is the formulation of wiring diagrams connecting the numerous sensory and crosstalking inputs that govern the regulation of flagella. Such connectivity blueprints will provide insight (and testable models) into how bacteria make the decision to swim, swarm or stick (Figure 2). Current Opinion in Microbiology 2006, 9:180–186
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10. Kirov SM: Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis. FEMS Microbiol Lett 2003, 224:151-159. 11. Rather PN: Swarmer cell differentiation in Proteus mirabilis. Environ Microbiol 2005, 7:1065-1073. 12. McCarter LL: Dual flagellar systems enable motility under different circumstances. J Mol Microbiol Biotechnol 2004, 7:18-29. 13. Ren CP, Beatson SA, Parkhill J, Pallen MJ: The Flag-2 locus, an ancestral gene cluster, is potentially associated with a novel flagellar system from Escherichia coli. J Bacteriol 2005, 187:1430-1440. Multiple strain sequencing has led to the discovery of a second flagellar system in some enteric genomes. 14. England JC, Gober JW: Cell cycle control of cell morphogenesis in Caulobacter. Curr Opin Microbiol 2001, 4:674-680. 15. Sourjik V, Muschler P, Scharf B, Schmitt R: VisN and VisR are global regulators of chemotaxis, flagellar, and motility genes in Sinorhizobium (Rhizobium) meliloti. J Bacteriol 2000, 182:782-788. 16. Dasgupta N, Wolfgang MC, Goodman AL, Arora SK, Jyot J, Lory S, Ramphal R: A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol Microbiol 2003, 50:809-824. Transcriptional profiling using wild type and mutant strains was implemented to elucidate the P. aeruginosa flagellar hierarchy. 17. Jacobi S, Schade R, Heuner K: Characterization of the alternative sigma factor sigma54 and the transcriptional regulator FleQ of Legionella pneumophila, which are both involved in the regulation cascade of flagellar gene expression. J Bacteriol 2004, 186:2540-2547. 18. Niehus E, Gressmann H, Ye F, Schlapbach R, Dehio M, Dehio C, Stack A, Meyer TF, Suerbaum S, Josenhans C: Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori. Mol Microbiol 2004, 52:947-961. Similar to methods first used in P. aeruginosa [16], this work also used transcriptional profiling to determine the unique flagellar hierarchy of H. pylori; this hierarchy is pertinent to regulation in C. jejuni. 19. Hendrixson DR, DiRita VJ: Transcription of sigma54-dependent but not sigma28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus. Mol Microbiol 2003, 50:687-702. 20. Brahmachary P, Dashti MG, Olson JW, Hoover TR: Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme. J Bacteriol 2004, 186:4535-4542. 21. Correa NE, Peng F, Klose KE: Roles of the regulatory proteins FlhF and FlhG in the Vibrio cholerae flagellar transcription hierarchy. J Bacteriol 2005, 187:6324-6332. 22. Poggio S, Osorio A, Dreyfus G, Camarena L: The flagellar hierarchy of Rhodobacter sphaeroides is controlled by the concerted action of two enhancer-binding proteins. Mol Microbiol 2005, 58:969-983. A candidate gene approach was used to identify two s54-dependent flagellar regulators in Rhodobacter sphaeroides. This work proposes that hierarchical transcription occurs in four tiers and is determined by the concentration of the activators and their interaction. These regulators are unusual, in that they both lack an amino-terminal regulatory domain and one lacks a DNA binding domain. 23. Motaleb MA, Sal MS, Charon NW: The decrease in FlaA observed in a flaB mutant of Borrelia burgdorferi occurs posttranscriptionally. J Bacteriol 2004, 186:3703-3711. 24. Amati G, Bisicchia P, Galizzi A: DegU-P represses expression of the motility fla-che operon in Bacillus subtilis. J Bacteriol 2004, 186:6003-6014. 25. Fraser GM, Hirano T, Ferris HU, Devgan LL, Kihara M, Macnab RM: Substrate specificity of type III flagellar protein export in www.sciencedirect.com
Salmonella is controlled by subdomain interactions in FlhB. Mol Microbiol 2003, 48:1043-1057. FlhB acts as a gate in the flagellar export apparatus to control substrate specificity. This work shows that subdomain interactions within FlhB, which are promoted by cleavage of FlhB, determine the specificity of gating. 26. Ferris HU, Furukawa Y, Minamino T, Kroetz MB, Kihara M, Namba K, Macnab RM: FLHB regulates ordered export of flagellar components via autocleavage mechanism. J Biol Chem 2005, 280:41236-41242. 27. Minamino T, Saijo-Hamano Y, Furukawa Y, Gonzalez-Pedrajo B, Macnab RM, Namba K: Domain organization and function of Salmonella FliK, a flagellar hook-length control protein. J Mol Biol 2004, 341:491-502. 28. Hirano T, Shibata S, Ohnishi K, Tani T, Aizawa S: N-terminal signal region of FliK is dispensable for length control of the flagellar hook. Mol Microbiol 2005, 56:346-360. FliK controls hook length; this study found that it does not need to be exported to exert this control, and, moreover, its absolute size is not crucial for function. Thus, FliK is unlikely to function as a molecular ruler. 29. Minamino T, Pugsley AP: Measure for measure in the control of type III secretion hook and needle length. Mol Microbiol 2005, 56:303-308. 30. Aldridge P, Karlinsey J, Hughes KT: The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL. Mol Microbiol 2003, 49:1333-1345. This work shows that the hook-associated proteins FlgK and FlgL can sequester the chaperone FlgN. This has consequences for s28-directed transcription as FlgN promotes translation of the anti-s28 factor FlgM. Thus, this paper raises the interesting question of who is guarding whom, i.e. does the chaperone protect the substrate or does the substrate sequester a regulatory factor? 31. Dutton RJ, Xu Z, Gober JW: Linking structural assembly to gene expression: a novel mechanism for regulating the activity of a sigma transcription factor. Mol Microbiol 2005, 58:743-757. This study showed that activity of the C. crescentus flagellar regulator FlbD can be controlled by direct physical interaction with an anti-activator protein. 32. Dasgupta N, Ramphal R: Interaction of the antiactivator FleN with the transcriptional activator FleQ regulates flagellar number in Pseudomonas aeruginosa. J Bacteriol 2001, 183:6636-6644. 33. Llewellyn M, Dutton RJ, Easter J, O’Donnol D, Gober JW: The conserved flaF gene has a critical role in coupling flagellin translation and assembly in Caulobacter crescentus. Mol Microbiol 2005, 57:1127-1142. Hierarchical transcription is not the sole means by which the assembly of flagella is regulated; translation also can be linked to assembly. This work deciphers how two proteins of C. crescentus influence translation and stability of flagellin mRNA. 34. Lai HC, Soo PC, Wei JR, Yi WC, Liaw SJ, Horng YT, Lin SM, Ho SW, Swift S, Williams P: The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids. J Bacteriol 2005, 187:3407-3414. 35. Liaw SJ, Lai HC, Wang WB: Modulation of swarming and virulence by fatty acids through the RsbA protein in Proteus mirabilis. Infect Immun 2004, 72:6836-6845. 36. Kim DJ, Boylan B, George N, Forst S: Inactivation of ompR promotes precocious swarming and flhDC expression in Xenorhabdus nematophila. J Bacteriol 2003, 185:5290-5294. 37. Clarke MB, Sperandio V: Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. Mol Microbiol 2005, 57:1734-1749. 38. Majdalani N, Vanderpool CK, Gottesman S: Bacterial small RNA regulators. Crit Rev Biochem Mol Biol 2005, 40:93-113. 39. Tomoyasu T, Ohkishi T, Ukyo Y, Tokumitsu A, Takaya A, Suzuki M, Sekiya K, Matsui H, Kutsukake K, Yamamoto T: The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar Typhimurium. J Bacteriol 2002, 184:645-653. Current Opinion in Microbiology 2006, 9:180–186
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40. Wolfe AJ, Chang DE, Walker JD, Seitz-Partridge JE, Vidaurri MD, Lange CF, Pruss BM, Henk MC, Larkin JC, Conway T: Evidence that acetyl phosphate functions as a global signal during biofilm development. Mol Microbiol 2003, 48:977-988. 41. Bergara F, Ibarra C, Iwamasa J, Patarroyo JC, Aguilera R, Marquez-Magana LM: CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J Bacteriol 2003, 185:3118-3126. 42. Calvio C, Celandroni F, Ghelardi E, Amati G, Salvetti S, Ceciliani F, Galizzi A, Senesi S: Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J Bacteriol 2005, 187:5356-5366. This study characterized a key regulatory locus controlling swimming and swarming in B. subtilis. 43. Yao SY, Luo L, Har KJ, Becker A, Ruberg S, Yu GQ, Zhu JB, Cheng HP: Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production. J Bacteriol 2004, 186:6042-6049. 44. Clegg S, Hughes KT: FimZ is a molecular link between sticking and swimming in Salmonella enterica serovar Typhimurium. J Bacteriol 2002, 184:1209-1213. 45. Blumer C, Kleefeld A, Lehnen D, Heintz M, Dobrindt U, Nagy G, Michaelis K, Emody L, Polen T, Rachel R et al.: Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli. Microbiology 2005, 151:3287-3298. 46. Francez-Charlot A, Laugel B, Van Gemert A, Dubarry N, Wiorowski F, Castanie-Cornet MP, Gutierrez C, Cam K: RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol 2003, 49:823-832. 47. Nachin L, Nannmark U, Nystrom T: Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility. J Bacteriol 2005, 187:6265-6272.
54. Kalir S, Alon U: Using a quantitative blueprint to reprogram the dynamics of the flagella gene network. Cell 2004, 117:713-720. Based upon experimental data, a quantitative blueprint of flagellar gene expression was mathematically modeled and tested by using controlled expression of regulators. This work represents an exciting advance to probing and understanding complex genetic circuits. 55. McAdams HH, Shapiro L: A bacterial cell-cycle regulatory network operating in time and space. Science 2003, 301:1874-1877. This work also describes a complex wiring diagram, in particular the CtrA regulatory network of C. crescentus. CtrA is a key regulator controlling the Caulobacter cell cycle and the determining regulator for the flagellar network. 56. Claret L, Hughes C: Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy. J Mol Biol 2002, 321:185-199. This study showed that the binding affinities of FlhD2C2 to the various flagellar promoters correlate with promoter strength and the timing of gene expression. 57. Kim YK, McCarter LL: Cross-regulation in Vibrio parahaemolyticus: compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK. J Bacteriol 2004, 186:4014-4018. 58. Correa NE, Klose KE: Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC. J Bacteriol 2005, 187:3158-3170. 59. Millikan DS, Ruby EG: FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization. J Bacteriol 2003, 185:3547-3557. 60. Stewart BJ, McCarter LL: Lateral flagellar gene system of Vibrio parahaemolyticus. J Bacteriol 2003, 185:4508-4518. This study mapped the three-tiered lateral flagellar transcriptional hierarchy by using mutant and fusion analyses.
48. Boles BR, McCarter LL: Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation. J Bacteriol 2002, 184:5946-5954.
61. Tomoyasu T, Takaya A, Isogai E, Yamamoto T: Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol 2003, 48:443-452.
49. Simm R, Morr M, Kader A, Nimtz M, Romling U: GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 2004, 53:1123-1134.
62. Crosson S, McAdams H, Shapiro L: A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus. Cell Cycle 2004, 3:1252-1254.
50. Jenal U: Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? Curr Opin Microbiol 2004, 7:185-191.
63. Dasgupta N, Ferrell EP, Kanack KJ, West SE, Ramphal R: fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein. J Bacteriol 2002, 184:5240-5250.
51. Ellermeier CD, Slauch JM: RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium. J Bacteriol 2003, 185:5096-5108. 52. Teplitski M, Goodier RI, Ahmer BM: Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella. J Bacteriol 2003, 185:7257-7265. 53. Grundling A, Burrack LS, Bouwer HG, Higgins DE: Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. Proc Natl Acad Sci USA 2004, 101:12318-12323.
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64. Williams T, Joseph B, Beier D, Goebel W, Kuhn M: Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes. FEMS Microbiol Lett 2005, 252:287-298. 65. Kearns DB, Chu F, Rudner R, Losick R: Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol 2004, 52:357-369. The authors describe the identification of a key regulatory locus swrA controlling flagellar regulation and swarming in B. subtilis.
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