Flagellar Switch Protein Research Articles

A putative spermidine synthase interacts with flagellar switch protein FliM and regulates motility in Helicobacter pylori.

The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull-down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE-null mutant was generated and its motility behavior was evaluated. We found that deletion of SpeE did not affect flagellar formation, but induced clockwise rotation bias. We further determined the crystal structure of the FliM-SpeE complex at 2.7 Å resolution. SpeE dimer binds to FliM with micromolar binding affinity, and their interaction is mediated through the β1' and β2' region of FliM middle domain. The FliM-SpeE binding interface partially overlaps with the FliM surface that interacts with FliG and is essential for proper flagellar rotational switching. By a combination of protein sequence conservation analysis and pull-down assays using FliM and SpeE orthologues in E. coli, our data suggest that FliM-SpeE association is unique to Helicobacter species.

New Architecture for Reagentless, Protein-Based Electrochemical Biosensors.

Here we demonstrate a new class of reagentless, single-step sensors for the detection of proteins and peptides that is the electrochemical analog of fluorescence polarization (fluorescence anisotropy), a versatile optical approach widely employed to this same end. Our electrochemical sensors consist of a redox-reporter-modified protein (the "receptor") site-specifically anchored to an electrode via a short, flexible polypeptide linker. Interaction of the receptor with its binding partner alters the efficiency with which the reporter approaches the electrode surface, thus causing a change in redox current upon voltammetric interrogation. As our first proof-of-principle we employed the bacterial chemotaxis protein CheY as our receptor. Interaction with either of CheY's two binding partners, the P2 domain of the chemotaxis kinase, CheA, or the 16-residue "target region" of the flagellar switch protein, FliM, leads to easily measurable changes in output current that trace Langmuir isotherms within error of those seen in solution. Phosphorylation of the electrode-bound CheY decreases its affinity for CheA-P2 and enhances its affinity for FliM in a manner likewise consistent with its behavior in solution. As expected given the proposed sensor signaling mechanism, the magnitude of the binding-induced signal change depends on the placement of the redox reporter on the receptor. Following these preliminary studies with CheY, we also developed and characterized additional sensors aimed at the detection of specific antibodies using the relevant protein antigens as the receptor. These exhibit excellent detection limits for their targets without the use of reagents or wash steps. This novel, protein-based electrochemical sensing architecture provides a new and potentially promising approach to sensors for the single-step measurement of specific proteins and peptides.

Salmonella STM1697 coordinates flagella biogenesis and virulence by restricting flagellar master protein FlhD4C2 from recruiting RNA polymerase.

Salmonella reduces flagella biogenesis to avoid detection within host cells by a largely unknown mechanism. We identified an EAL-like protein STM1697 as required and sufficient for this process. STM1697 surges to a high level after Salmonella enters host cells and restrains the expression of flagellar genes by regulating the function of flagellar switch protein FlhD4C2, the transcription activator of all other flagellar genes. Unlike other anti-FlhD4C2 factors, STM1697 does not prevent FlhD4C2 from binding to target DNA. A 2.0 Å resolution STM1697–FlhD structure reveals that STM1697 binds the same region of FlhD as STM1344, but with weaker affinity. Further experiments show that STM1697 regulates flagella biogenesis by restricting FlhD4C2 from recruiting RNA polymerase and the regulatory effect of STM1697 on flagellar biogenesis and virulence are all achieved by interaction with FlhD. Finally, we describe a novel mechanism mediated by STM1697 in which Salmonella can inhibit the production of flagella antigen and escape from the host immune system.

Rescuing chemotaxis of the anticancer agent Salmonella enterica serovar Typhimurium VNP20009

The role of chemotaxis and motility in Salmonella enterica serovar Typhimurium tumor colonization remains unclear. We determined through swim plate assays that the well-established anticancer agent S. Typhimurium VNP20009 is deficient in chemotaxis, and that this phenotype is suppressible. Through genome sequencing, we revealed that VNP20009 and four selected suppressor mutants had a single nucleotide polymorphism (SNP) in cheY causing a mutation in the conserved proline residue at position 110. CheY is the response regulator that interacts with the flagellar motor-switch complex and modulates rotational bias. The four suppressor mutants additionally carried non-synonymous SNPs in fliM encoding a flagellar switch protein. The CheY-P110S mutation in VNP20009 likely rendered the protein unable to interact with FliM, a phenotype that could be suppressed by mutations in FliM. We replaced the mutated cheY in VNP20009 with the wild-type copy and chemotaxis was partially restored. The swim ring of the rescued strain, VNP20009 cheY+, was 46% the size of the parental strain 14028 swim ring. When tested in capillary assays, VNP20009 cheY+ was 69% efficient in chemotaxis towards the attractant aspartate as compared to 14028. Potential reasons for the lack of complete restoration and implications for bacterial tumor colonization will be discussed.

Stoichiometry and turnover of the bacterial flagellar switch protein FliN.

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.

De- and repolarization mechanism of flagellar morphogenesis during a bacterial cell cycle

Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger--bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)--that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.

Are “Power-Packs” Associated with the Switch-Motor Complex of Bacterial Flagella?

Bacterial flagella are rotated by a motor embedded in the cytoplasmic membrane, the direction of rotation being dictated by a switch at the base of the motor. In the case of bacteria such as E.coli and Salmonella, the motor is driven by the proton-motive force (PMF) across the membrane. The PMF is also used, inter alia, to synthesize ATP by means of the reversible membrane enzyme F0F1ATPase, whose mechanism involves the rotary action of a spindle. Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in E.coli. Employing Forster resonance energy transfer in vivo and pull-down assays in vitro, we provided evidence for the interaction of F0F1ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F0F1ATP synthase with FliG is important for the function of the switch of bacterial flagella.

Energy Complexes Are Apparently Associated with the Switch–Motor Complex of Bacterial Flagella

Recently, the switch–motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch–motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F0F1 ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch–motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F0F1 ATP synthase with FliG is important for the function of the switch of bacterial flagella.

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY

The silent information regulator (Sir2) family proteins are NAD+-dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2-like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains ΔcobB, Δacs, Δ(cobB) Δ(acs), Δ(cheA) Δ(cheZ), Δ(cheA) Δ(cheZ) Δ(cobB) and Δ(cheA) Δ(cheZ) Δ(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the ΔcobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Δacs mutant was indistinguishable from the parental strain, (ii) CheY from the ΔcobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Δ(cheA) Δ(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.

2P185 双方向及び一方向回転型べん毛モータースイッチタンパク質FliMのクローニング及び機能解析(細胞生物的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

2P185 双方向及び一方向回転型べん毛モータースイッチタンパク質FliMのクローニング及び機能解析(細胞生物的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

A New Perspective on Response Regulator Activation

Two-component signaling pathways are the most prevalent of strategies used by bacteria for coupling environmental signals to adaptive responses (10). The response regulator component typically functions at the end of the pathway, with the conserved regulatory/receiver domain acting as a phosphorylation-activated switch to control an associated or attached effector domain that elicits the output response (24). In the simplest mechanistic model, these domains are considered to exist in two conformations, an inactive (“off”) state and an active (“on”) state, with phosphorylation at a conserved active-site aspartic acid residue serving to shift the equilibrium toward the active state (22). In this issue of the Journal of Bacteriology, Dyer and Dahlquist provide direct structural evidence that this model is overly simplistic (8). Their crystallographic characterization of the unphosphorylated Escherichia coli chemotaxis response regulator CheY bound to a peptide of its effector protein target, the flagellar switch protein FliM, reveals a conformation that is intermediate between inactive and active states. This finding provides evidence against a strict two-state mechanism in which the orientations of key residues that distinguish the inactive and active states are presumed to be obligatorily coupled. The intermediate state reported in their study not only provides insight into the mechanism of switching between inactive and active states but also has potential physiological relevance for some response regulators.

Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export

The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed.

Agrobacterium flagellar switch gene fliG is liquid inducible and important for virulence.

Agrobacterium tumefaciens C58 was mutagenized with a mini-Tn5 transposon containing a promoterless gene encoding the green fluorescent protein (GFP). A mutant, CGS74, exhibited a higher GFP expression level in liquid media than on solid media. The ability of the mutant to cause tumors on plants was attenuated. Sequence analysis showed that the transposon was inserted at the fliG gene, which encodes a flagellar motor switch protein required for flagellar movement. Studies of the fliG-gfp fusion gene indicated that the promoter activity of the fliG gene was higher in liquid than in solid media. Electron microscopy studies demonstrated that the mutant was nonflagellate. This suggests that the A. tumefaciens motility is important for virulence and that bacterial flagellar synthesis occurs at a higher level in a liquid environment than in a solid environment, perhaps resulting in a higher motility.

CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis.

Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.

Deletion analysis of the flagellar switch protein FliG of Salmonella.

The flagellar motor/switch complex, consisting of the three proteins FliG, FliM, and FliN, plays a central role in bacterial motility and chemotaxis. We have analyzed FliG, using 10-amino-acid deletions throughout the protein and testing the deletion clones for their motility and dominance properties and for interaction of the deletion proteins with the MS ring protein FliF. Only the N-terminal 46 amino acids of FliG (segments 1 to 4) were important for binding to FliF; consistent with this, an N-terminal fragment consisting of residues 1 to 108 bound FliF strongly, whereas a C-terminal fragment consisting of residues 109 to 331 did not bind FliF at all. Deletions in the region from residues 37 to 96 (segments 4 to 9), 297 to 306 (segment 30), and 317 to 326 (segment 32) permitted swarming, though not at wild-type levels; all other deletions caused paralyzed or, more commonly, nonflagellate phenotype. Except for those near the N terminus, deletions had a dominant negative effect on wild-type cells.

Identification of the binding interfaces on CheY for two of its targets the phosphatase CheZ and the flagellar switch protein FliM

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.

Acetylation at Lys-92 enhances signaling by the chemotaxis response regulator protein CheY.

When Escherichia coli cells lacking all chemotaxis proteins except the response regulator CheY are exposed to acetate, clockwise flagellar rotation results, indicating the acetate stimulus has activated signaling by CheY. Acetate can be converted to acetyl-CoA by either of two different metabolic pathways, which proceed through acetyl phosphate or acetyl-AMP intermediates. In turn, CheY can be covalently modified by either intermediate in vitro, leading to phosphorylation or acetylation, respectively. Either pathway is sufficient to support the CheY-mediated response to acetate in vivo. Whereas phosphorylation of Asp-57 is a recognized mechanism for activation of CheY to stimulate clockwise flagellar rotation, acetylation of CheY is less well characterized. We found evidence for multiple CheY acetylation sites by mass spectrometry and directly identified Lys-92 and Lys-109 as acetylation sites by Edman degradation of peptides from [14C]acetate-labeled CheY. Replacement of CheY Lys-92, the preferred acetylation site, with Arg has little effect on chemotaxis but completely prevents the response to acetate via the acetyl-AMP pathway. Thus acetylation of Lys-92 activates clockwise signaling by CheY in vivo. The mechanism by which acetylation activates CheY apparently is not simple charge neutralization, nor does it involve enhanced binding to the FliM flagellar switch protein. Thus acetylation probably affects signal generation by CheY at a step after switch binding.

Chemotactic response regulator mutant CheY95IV exhibits enhanced binding to the flagellar switch and phosphorylation-dependent constitutive signalling.

CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95--in close proximity to residue 106--results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction.

Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY

The FliG, FliM, and FliN proteins of the bacterial flagellar motor are believed to interact with one another to form the switch complex, which in turn is thought to interact with one of the chemotaxis proteins, CheY. In particular, FliM appears to be an intermediary between CheY and FliG: the current model suggests that CheY, when phosphorylated (CheY-P), binds to FliM and produces a conformational change in FliM that is propagated to FliG. The result of these interactions is to induce clockwise rotation of the flagellar motors and tumbling of the cell. Various genetic and biochemical studies have provided evidence that the switch proteins associate with each other and that CheY-P binds to FliM. Here, we have used affinity blotting to obtain direct evidence of interaction between Salmonella typhimurium FliM and FliN, FliM and FliG, and FliM and CheY-P. We have also examined the ability of various FliM deletion and truncation mutant proteins to bind to FliN, FliG, and CheY-P. From these data, we conclude that distinct regions of the FliM protein bind to each of these other proteins. We propose a model in which the N-terminal region of FliM binds to CheY-P, the middle region of FliM binds to FliG, and the C-terminal region binds to FliN.

The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN

We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.© 1997 Elsevier Science B.V. All rights reserved.