Flagellar Switch Research Articles

Binding of the Chemotaxis Response Regulator CheY to the Isolated, Intact Switch Complex of the Bacterial Flagellar Motor: LACK OF COOPERATIVITY

In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY approximately P) was 4.0 +/- 1.1 microm, well in line with the published range of CheY approximately P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient approximately 1). This lack of CheY approximately P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY approximately P binding.

Biomechanics: bacterial flagellar switching under load.

Flagellated bacteria swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, but how this switching mechanism works is not understood. Here we show that the probability of the motor rotating in the clockwise direction increases under high load, when the motor spins slowly (at less than 50 Hz). We suggest that either the switch is responding to small changes in torque — the torque increases only fractionally between 50 Hz and stall — or it senses motor speed, perhaps by means of proton flux.

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.

The N terminus of FliM is essential to promote flagellar rotation in Rhodobacter sphaeroides.

FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of Rhodobacter sphaeroides. Strains expressing the FliM protein with substitutions in residues previously reported in Escherichia coli as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that R. sphaeroides cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMDelta13 and FliMDelta20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed.

Novel putative photoreceptor and regulatory genes Required for the positive phototactic movement of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803.

Synechocystis: sp. PCC 6803 is a unicellular motile cyanobacterium, which shows positive or negative phototaxis on agar plates under lateral illumination. By gene disruption in a substrain showing of positive phototaxis, it was demonstrated that mutants defective in sll0038, sll0039, sll0041, sll0042 or sll0043 lost positive phototaxis but showed negative phototaxis away from the light source. Mutants of sll0040, which is located within the cluster of these genes, retained the capacity of positive phototaxis but to a lesser extent than the parent cells. These genes are homologous to che genes, which are involved in flagellar switching for bacterial chemotaxis. Interestingly, sll0041 (designated pisJ1) is predicted to have a chromophore-binding motif of phytochrome-like proteins and a signaling motif of chemoreceptors for bacterial chemotaxis. It is strongly suggested that the positive phototactic response was mediated by a phytochrome-like photoreceptor and CheA/CheY-type signal transduction system.

Characterization of the chemotaxis fliY and cheA genes in Bacillus cereus

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in Gram-positive bacteria.

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus.

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.

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.

The flagellar switch genes fliM and fliN of Rhodobacter sphaeroides are contained in a large flagellar gene cluster.

In this work, the genes that encode the FliM and FliN proteins of Rhodobacter sphaeroides were characterized. These genes are part of a large flagellar gene cluster in which six additional open reading frames encoding products homologous to FliL, FliO, FliP, FliQ, FliR, and FlhB proteins from other bacteria were identified. The inactivation of the fliM gene gave a nonflagellate phenotype (Fla-), suggesting that FliM is required for flagellar assembly. Complementation analysis of this fliM mutant indicated that fliM and fliN transcription starts beyond the 5' end of fliK and terminates after fliN.

Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet.

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

Bacterial chemotaxis: unsolved mystery of the flagellar switch.

Impressive progress has been made in understanding the mechanism of bacterial chemotaxis and function of the flagellar motor, but how the direction of rotation is reversed by the 'flagellar switch'--a central step in chemotaxis--remains obscure and calls for new experimental approaches.

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

This article was published in Mol Microbiol (1998) 27(5), 1065–1075. The headings of columns 2 and 4 of Table 1 were mistakenly transposed. The complete, correct table is reproduced below.

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.

The chemotaxis system, but not chemotaxis, is essential for swarming motility in Escherichia coli

The chemotaxis system plays an essential role in swarm cell differentiation and motility. We show in this study that two (Tsr and Tar) of the four chemoreceptors in Escherichia coli can support swarming individually, but sensing their most powerful chemoattractants is not necessary. Conditions that abolish chemotaxis toward serine (presence of serine concentrations that saturate Tsr, or mutations in Tsr that destroy serine binding) have no effect on swarming. Similar results were obtained for the aspartate and maltose chemoreceptor Tar. We also show that although a mutation in the signaling domain of Tsr that inhibits CheA kinase abolishes swarming, nonchemotactic flagellar switch mutants can swarm. Our results suggest that during swarming, the chemoreceptors signal through the chemotaxis pathway and induce swarmer cell differentiation in response to signals other than their known chemoeffectors.

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.

Architectural Features of theSalmonella typhimuriumFlagellar Motor Switch Revealed by Disrupted C-Rings

The three-dimensional surface topology of rapid-frozenSalmonella typhimuriumflagellar hook basal body complexes was studied by stereo-examination of thin-film metal replicas. The complexes contained the extended cytoplasmic structure, composed of the switch complex proteins; FliG, FliM, and FliN. Distinct nanometer-scale element arrays, separated by grooves, defined the outer surface of the cytoplasmic (C-) ring. The number of array elements was comparable to previously determined FliG and FliM copy numbers in the basal body. In addition to basal body complexes lacking C-rings, complexes containing incomplete C-rings were identified. The incomplete C-rings had lost segments of the proximal array. Basal bodies with the distal C-ring array alone were not found. These findings are compatible with the spatial organization of the flagellar switch suggested by previous biochemical data.

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 response of halobacteria to single light stimuli: a theoretical analysis

Halobacteria are propelled by rotating flagella. Under stationary environmental conditions, the direction of rotation of the flagella switches spontaneously at intervals ranging from 5 to 50 s. Sudden changes of light intensity induce (repellent response) or transiently suppress (attractant response) a switching event. For repellent and non-saturating attractant stimuli, the average time between stimulus application and succeeding switching event apparently varies as a function of the time of stimulation relative to the preceding switching event. However, thorough evaluation of the experimental data suggests that the way in which a stimulus changes the internal state of a cell is independent of both the time of stimulation and the spinning direction of the flagellar motors. Flagellar switching is described in terms of a switch complex of the flagellar motor apparatus. It is assumed that a switching event is triggered with constant probability per unit time if the switch complex is in its active state and no stimulus was applied. After a switching event, the switch complex regains activity in a multi-step process. Stimuli modulate the switching step through an intracellular signal which is interpreted as the effective output of the sensory chain (including adaptation processes). This signal is assumed to be independent of the status of the motor apparatus. The model yields a quantitative description of spontaneous switching behavior. Attractant responses can be understood by assuming a transient block of the switching step. The explanation of repellent responses requires the assumption of an oscillating signal and a concomitant acceleration of reactivation of the switch complex.