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Many bacteria swim by means of flagella that are rotated by a nanoscale motor embedded in the cell membrane. Torque is generated by the interaction between ion-conducting membrane proteins that comprise the stator and ring-shaped structures that form the rotor. Although the structure and function of the motor have been extensively studied, many mysteries remain, including the force-generation mechanism, the path of ion flow through the stator, the activation mechanism of the stator, and the mechanism of switching between clockwise (CW) and counterclockwise (CCW) rotation. We summarize recent knowledge of the Na+-driven flagellar motor, especially the Vibrio polar motor that rotates much faster than the H+-driven motor and provides a useful model system for examining comparative aspects of flagellar function. The rotation of the bacterial flagellar motor is generated by the interaction between two parts in the motor, the rotor and the stator. The rotor is composed of a central rod and surrounding rings, and the stator is a unit that works as an energy convertor to couple ion influx to rotation of the rotor. The Na+-driven flagellar motor in Vibrio alginolyticus , whose rotation speed is faster than that of any H+-driven motor, contains PomA/PomB as stator proteins and some additional ring-like structures, the T-, H-, and O-rings, that are not found in H+-driven motors. Assembly of the PomA/PomB stators into the motor depends on the environmental Na+ concentration. In the presence of sodium, the periplasmic region of the stator changes its conformation to an activated form that binds to peptidoglycan, and charged residues in the cytoplasmic region interact with charged residues in the rotor protein FliG to generate rotational force. FliG dynamically changes its conformation to switch the rotational direction of the motor in response to the chemotactic signal transferred from the other rotor component, FliM, that binds to the chemotaxis response regulator protein CheY. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is a reversible rotary motor powered by an electrochemical-potential difference of specific ions across the cytoplasmic membrane. The H + -driven motor of Salmonella spins at ∼300 Hz, whereas the Na + -driven motor of marine Vibrio spp. can rotate much faster, up to 1700 Hz. A highly conserved motor structure consists of the MS ring, C ring, rod, and export apparatus. The C ring and the export apparatus show dynamic properties for exerting their functional activities. Various additional structures surrounding the conserved motor structure are observed in different bacterial species. In this review we summarize our current understanding of the structure, function, and assembly of the flagellar motor in Salmonella and marine Vibrio . [ABSTRACT FROM AUTHOR]

Bacterial flagellar motor is an ion-driven supramolecular nanomachine embedded in the cell envelope. Rotor-stator interaction that couples to the specific ion translocation through the stator channel is the nature of torque generation. To produce fully functional motor, multiple stator complexes must be incorporated around the rotor at appropriate places. However, such stator assembly mechanism has not been investigated by the structural point of view. Here we describe stator assembly and activation mechanism revealed from the crystal structure of a motor component located in the periplasmic space, suggesting the dynamic conformational changes in the stator during its assembly-coupled activation. [ABSTRACT FROM AUTHOR]

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical. [ABSTRACT FROM AUTHOR]

The proteins that form the bacterial flagellum are translocated to its distal end through the central channel of the growing flagellum by the flagellar-specific protein export apparatus, a family of the type III protein secretion system. FliI and FliJ are soluble components of this apparatus. FliI is an ATPase that has extensive structural similarity to the α and β subunits of FoF1-ATP synthase. FliJ is essential for export, but its function remains obscure. Here we show that the structure of FliJ derived from Salmonella enterica serovar Typhimurium is remarkably similar to that of the two-stranded α-helical coiled-coil part of the γ subunit of FoF1-ATP synthase and that FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring. These results suggest that the type III protein export system and F- and V-type ATPases share a similar mechanism and an evolutionary relationship. [ABSTRACT FROM AUTHOR]

For self-assembly of the bacterial flagellum, most of the flagellar component proteins synthesized in the cytoplasm are exported by the flagellar type Ill export apparatus to the growing, distal end. Flagellar protein export is highly organized and well controlled in every step of the flageilar assembly process. Flagellar-specific chaperones not only facilitate the export of their cognate proteins, as well as prevent their premature aggregation in the cytoplasm, but also play a role in fine- tuning flagellar gene expression to be coupled with the flagellar assembly process. FliT is a flagellar-specific chaperone responsible for the export of the filament-cappWig protein FliD and for negative control of flagellar gene expression by binding to the FIhDC complex. Here we report the crystal structure of Salmonella FliT at 3.2-A resolution. The structural and biochemical analyses clearly reveal that the C-terminal segment of FliT regulates its interactions with the FIhDC complex, Flil ATPase, and Fiji (subunits of the export apparatus), and that its conformational change is responsible for the switch in its binding partners during flagellar protein export. [ABSTRACT FROM AUTHOR]

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N-terminal transmembrane domain (FlhATM) and a C-terminal cytoplasmic domain (FlhAC). FlhAC is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhAC at 2.8 Å resolution. FlhAC consists of four subdomains (ACD1, ACD2, ACD3 and ACD4) and a linker connecting FlhAC to FlhATM. The sites of temperature-sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhAC changes its conformation for its function. Molecular dynamics simulation demonstrated an open-close motion with a 5–10 ns oscillation in the distance between ACD2 and ACD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhAC is essential for protein export. [ABSTRACT FROM AUTHOR]

Torque generation in the Salmonella flagellar motor is coupled to translocation of H+ ions through the proton-conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan-binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C-terminal periplasmic fragment of MotB (MotBC) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton-conducting channel. Drastic conformational changes in the N-terminal portion of MotBC are required both for PG binding and the proton channel activation. [ABSTRACT FROM AUTHOR]

The bacterial flagellum, which is responsible for motility, is a biological nanomachine consisting of a reversible rotary motor, a universal joint, a helical screw, and a protein export apparatus dedicated for flagellar assembly. The motor is fueled by an inward-directed electrochemical gradient of protons or sodium ions across the cytoplasmic membrane. The motor consists of a rotor, a drive shaft, a bushing, and about a dozen stator units. The flagellar protein export apparatus is located at the cytoplasmic side of the rotor. Interactions between the rotor and the stators and those between soluble and membrane components of the export apparatus are highly dynamic. The structures of flagellar basal body components including those of the export apparatus, being revealed at high resolution by X-ray crystallography and electron cryomicroscopy and cryotomography, are giving insights into their mechanisms. [Copyright &y& Elsevier]

Construction of the bacterial flagellum in the cell exterior proceeds at its distal end by highly ordered self-assembly of many different component proteins, which are selectively exported through the central channel of the growing flagellum by the flagellar type III export apparatus. Flil is the ATPase of the export apparatus that drives the export process. Here we report the 2.4 Å resolution crystal structure of Flil in the ADP-bound form. Flil consists of three domains, and the whole structure shows extensive similarities to the α and β subunits of F0F1-ATPsynthase, a rotary motor that drives the chemical reaction of ATP synthesis. A hexamer model of Flil has been constructed based on the F1-ATPase structure composed of the α3β3γ subunits. Although the regions that differ in conformation between Flil and the F1-α/β subunits are all located on the outer surface of the hexamer ring, the main chain structures at the subunit interface and those surrounding the central channel of the ring are well conserved. These results imply an evolutionary relation between the flagellum and F0F1-ATPsynthase and a similarity in the mechanism between Flil and F1-ATPase despite the apparently different functions of these proteins. [ABSTRACT FROM AUTHOR]

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95–108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria. [ABSTRACT FROM AUTHOR]

The axial components of the bacterial flagellum and the scaffolding proteins for its assembly are exported through the flagellar-specific type III protein-export apparatus, which is believed to be located on the cytoplasmic surface of the basal body. FlhA is an essential component of the type III export apparatus of Salmonella and consists of two major portions: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhAC). FlhAC and a 38 kDa fragment of FlhAC (FlhAC38K) were purified and crystallized. The crystals were obtained by the sitting-drop vapour-diffusion technique with PEG 8000 as a precipitant. FlhAC crystals grew in the tetragonal space group I41/I43, with unitcell parameters a = b = 216.6, c = 65.0 Å . FlhAC38K was crystallized in an orthorhombic form, with unit-cell parameters a = 53.0, b = 93.1, c = 186.5 Å . X-ray diffraction data from crystals of FlhAC and the SeMet derivative of FlhAC were collected to 2.9 and 3.2 Å, respectively. [ABSTRACT FROM AUTHOR]

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into β-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2–HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum. [Copyright &y& Elsevier]

Reports a study using the crystal structure of a Salmonella flagellin fragment of relative molecular mass 41,300. Switch between left- and right-handed supercoiled forms when bacteria switch their swimming mode between running and tumbling; How the crystals contain pairs of antiparallel straight protofilaments with R-type repeat; Identification of possible switch regions responsible for the bi-stable mechanical switch that generates a difference in repeat distance; Mutations affecting the supercoiling.

Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Δ1–18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Δ1–18) crystals grew in the monoclinic space group P21, with unit-cell parameters a = 48, b = 73, c = 126 Å, β = 94°, and diffracted to 2.4 Å resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination. [ABSTRACT FROM AUTHOR]

KaiB is a component of the circadian clock oscillator in cyano-bacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynecha coccus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli. purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P21, with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 Å, β = 100.1. While the native crystals diffract to 3.7 Å, osmium derivative, which show an approximately 4Å shrinkage in the b axis, diffract to 2.6 Å. the cyrstals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 Å and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 Å, β = 100.1. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination. [ABSTRACT FROM AUTHOR]

Assembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure. Matsunami et al determine the structure of the flagellar hook cap from Salmonella enterica by X-ray crystallography. The 3.3 Å resolution structure in combination with mutagenesis analysis provides insights into a putative hook assembly mechanism. [ABSTRACT FROM AUTHOR]

Assembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure. Matsunami et al determine the structure of the flagellar hook cap from Salmonella enterica by X-ray crystallography. The 3.3 Å resolution structure in combination with mutagenesis analysis provides insights into a putative hook assembly mechanism. [ABSTRACT FROM AUTHOR]

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure. [ABSTRACT FROM AUTHOR]

Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA 5 MotB 2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA. • The Na-type stator of Aquifex aeolicus , a hyperhermophile, comprises MotA and MotB. • The structure of the hyperthermophilic Aa-MotA was determined at 3.42 Å. • Aa-MotA can form a pentameric ring assembly without MotB. • A simulated structure of Aa-stator is equivalent to known A 5 B 2 stator structures. • Extremely hydrophobic nature of Aa-MotA may be a key for its hyperthermostability. [ABSTRACT FROM AUTHOR]

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C-terminal cytoplasmic domain of FlhA ( FlhAC), a membrane component of the export apparatus, provides a binding-site for these chaperone-substrate complexes but it remains unknown how it co-ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhAC is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhAC for the FlgN/ FlgK complex is slightly higher than that for the FliT/ FliD complex and about 14-fold higher than that for the FliS/ FliC complex, leading to the proposal that the different binding affinities of FlhAC for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation. [ABSTRACT FROM AUTHOR]

Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from nonpreferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY‐phosphate (CheY‐P) to the C‐ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp., have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for a clockwise‐locked phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY‐P. Purified ZomB proteins form multimers, suggesting that ZomB may function as a homo‐oligomer. These observations imply that ZomB interacts with protein(s) involved in either flagellar motor rotation, chemotaxis, or both. We provide the evidence that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus. [ABSTRACT FROM AUTHOR]

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)3, forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine. [ABSTRACT FROM AUTHOR]

Summary FliT is a flagellar type III export chaperone specific for the filament-capping protein FliD. The FliT/FliD complex binds to the FliI ATPase of the flagellar export apparatus. The C-terminal α4 helix of FliT controls its interaction with FliI but it remains unknown how it does so. Here, we analysed the FliI-FliT interaction by pull-down assays using GST affinity chromatography. FliT94, missing the C-terminal α4 helix, bound to the extreme N-terminal region of FliI (FliIEN) with high affinity and to the C-terminal ATPase domain (FliICAT) with low affinity. The C-terminal α4 helix of FliT suppressed the interaction with FliIEN. FliH and FliT94 bound to a common binding site on FliIEN and hence FliH induced the release of FliI from FliT94 in an ATP-independent manner. FliD increased the binding affinity of FliICAT for FliT. These results raise a possible hypothesis that the FliH/FliI complex binds to the FliT/FliD complex through FliICAT to escort it from the cytoplasm to the export gate made up of six integral membrane proteins and that, upon dissociation of FliD from FliT, FliT94 may bind to FliIEN and then FliI may transfer from FliT94 to FliH by the direct competition of FliT94 and FliH for FliIEN. [ABSTRACT FROM AUTHOR]

Abstract: FliI, the ATPase involved in bacterial flagellar protein export, forms a complex with its regulator FliH in the cytoplasm and hexamerizes upon docking to the export gate composed of integral membrane proteins. The extreme N-terminal region of FliI is involved not only in its interaction with FliH but also in its oligomerization, but the regulatory mechanism of oligomerization remains unclear. Using in-frame 10-residue deletions within the 100 residues of the N-terminal domain, we demonstrate that the first 20 residues are required for FliH binding and that the conformation of the N-terminal domain is sensitive to the export function, even though the oligomerization and FliH-binding ability are retained and the ATPase activity is maintained in most of the deletion variants. [Copyright &y& Elsevier]

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FIgE, and its function as a nanoasized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FIgE31, a major proteolytic fragment of FIgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function. [ABSTRACT FROM AUTHOR]

Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus , which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signalling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM–FliM interaction. The structural models of wild type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, which is a major pathogen of the periodontal disease, secrets virulence factors such as gingipain proteases via the type IX secretion system (T9SS). T9SS consists of a trans-periplasmic core complex, the outer membrane translocon complex and the cell-surface complex attached on the outer membrane. PorM is a major component of the trans-periplasmic core complex and is believed to connect the outer membrane component with the inner membrane component. Recent structural studies have revealed that the periplasmic region of GldM, a PorM homolog of a gliding bacterium, consist of four domains and forms a dimer with a straight rod shape. However, only fragment structures are known for PorM. Moreover, one of the PorM fragment structure shows a kink. Here we show the structure of the entire structure of the periplasmic region of PorM (PorMp) at 3.7 Å resolution. PorMp is made up of four domains and forms a unique dimeric structure with an asymmetric, kinked-rod shape. The structure and the following mutational analysis revealed that R204 stabilizes the kink between the D1 and D2 domains and is essential for gingipains secretion, suggesting that the kinked structure of PorM is important for the functional T9SS formation. • PorM is the largest component of the trans-periplasmic core complex of T9SS. • The entire structure of the periplasmic region of PorM has been determined at 3.7 Å. • PorMp forms a dimer and adopts a unique, asymmetric kinked rod structure. • R204 involved in the kink formation is essential for gingipains secretion. • The kink is suggested to be essential for the functional T9SS formation. [ABSTRACT FROM AUTHOR]

A core fragment of the bacterial flagellar hook protein FlgE was overexpressed, purified and crystallized. The crystal diffracted to 1.6 Å resolution using synchrotron X-radiation. The crystal belongs to the orthorhombic crystal system, with space group P21212 and unit-cell parameters a = 128.4, b = 48.8, c = 96.7 Å. SeMet protein was also overexpressed, purified, crystallized and a set of 2.3 Å MAD data was collected. [ABSTRACT FROM AUTHOR]

Environmental sensing is crucial for bacterial survival and pathogenicity. Bacteria sense environmental chemicals using chemoreceptor proteins, such as Methyl-accepting Chemotaxis Proteins (MCPs). Vibrio cholerae , the etiological agent of cholera, has at least 44 chemoreceptor proteins homologous to MCP-Like Proteins (MLPs). Mlp24 and Mlp37 are dCACHE type chemoreceptors that senses various amino acids. Mlp24 is important for cholera toxin production, whereas Mlp37 is related to biofilm formation. The periplasmic ligand binding regions of Mlp24 and Mlp37 (Mlp24p and Mlp37p, respectively) share similar amino acid sequences, tertiary and quaternary structures, and a common mechanism for the ligand amino acid backbone recognition. However, Mlp37p recognizes various l -amino acids and taurine with similar affinity whereas Mlp24p shows different binding affinities for various l -amino acids and does not bind taurine. Here we solved the crystal structure of Mlp37p in complex with l -arginine and compared it with previously determined structures of Mlp37p, Mlp24p and their ligand complexes. We found that Mlp37p changes the conformation of the loop that forms the upper wall of the ligand binding pocket according to size and shape of the ligand, and thereby show similar affinity for various ligands. Image 1 • Mlp24 and Mlp37 are chemoreceptors with similar amino acid sequences and structures. • Unlike Mlp24, Mlp37 binds various ligands with similar affinity without Ca2+ ions. • Structure of Mlp37p in complex with l -arginine has been determined at 2.35 Å. • The PGP motif in the UJ-loop provides flexibility to the ligands binding site. • The UJ loop flexibility of Mlp37 is a key to the broad ligand specificity. [ABSTRACT FROM AUTHOR]

Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein–protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function. [ABSTRACT FROM AUTHOR]

The bacterial flagellar motor rotates in both counterclockwise (CCW) and clockwise (CW) directions. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring made of a transmembrane protein, FliF. The C ring acts not only as a rotor but also as a switch of the direction of motor rotation. FliG consists of three domains: FliG N , FliG M and FliG C . FliG N directly binds to FliF. Intermolecular interactions between FliG M and FliG C drive FliG ring formation. FliG M is responsible for the interaction with FliM. FliG C is involved in the interaction with the stator protein MotA. Adaptive remodeling of the C ring occurs when the motor switches between the CCW and CW states. However, it remained unknown how. Here, we report the effects of a CW-locked deletion mutation (ΔPEV) in FliG of Thermotaoga maritia ( Tm -FliG) on FliG–FliG and FliG–FliM interactions. The PEV deletion stabilized the intramolecular interaction between FliG M and FliG C , thereby suppressing the oligomerization of Tm -FliG MC in solution. This deletion also induced a conformational change of Helix MC connecting FliG M and FliG C to reduce the binding affinity of Tm- FliG MC for FliM. We will discuss adaptive remodeling of the C ring responsible for flagellar motor switching. [ABSTRACT FROM AUTHOR]

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. [ABSTRACT FROM AUTHOR]

The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export. [ABSTRACT FROM AUTHOR]

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures ( T m ), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C ( t 1/2 ). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. [ABSTRACT FROM AUTHOR]

The bacterial flagellar proteins are transported via a specific export apparatus to the distal end of the growing structure for their self-assembly. FliP is an essential membrane component of the export apparatus. FliP has an N-terminal signal peptide and is predicted to have four transmembrane (TM) helices and a periplasmic domain (FliPP) between TM-2 and TM-3. In this study, FliPP from Thermotoga maritima (TmFliPP) and its selenomethionine derivative (SeMet-TmFliPP) were purified and crystallized. TmFliPP formed a homotetramer in solution. Crystals of TmFliPP and SeMet-TmFliPP were obtained by the hanging-drop vapour-diffusion technique with 2-methyl-2,4-pentanediol as a precipitant. These two crystals grew in the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 114.9, c = 193.8 Å. X-ray diffraction data were collected from crystals of TmFliPP and SeMet-TmFliPP to 2.4 and 2.8 Å resolution, respectively. [ABSTRACT FROM AUTHOR]

The V1- and F1- rotary ATPases contain a rotor that rotates against a catalytic A3B3 or α3β3 stator. The rotor F1-γ or V1-DF is composed of both anti-parallel coiled coil and globular-loop parts. The bacterial flagellar type III export apparatus contains a V1/F1-like ATPase ring structure composed of FliI6 homo-hexamer and FliJ which adopts an anti-parallel coiled coil structure without the globular-loop part. Here we report that FliJ of Salmonella enterica serovar Typhimurium shows a rotor like function in Thermus thermophilus A3B3 based on both biochemical and structural analysis. Single molecular analysis indicates that an anti-parallel coiled-coil structure protein (FliJ structure protein) functions as a rotor in A3B3. A rotary ATPase possessing an F1-γ-like protein generated by fusion of the D and F subunits of V1 rotates, suggesting F1-γ could be the result of a fusion of the genes encoding two separate rotor subunits. Together with sequence comparison among the globular part proteins, the data strongly suggest that the rotor domains of the rotary ATPases and the flagellar export apparatus share a common evolutionary origin. [ABSTRACT FROM AUTHOR]

FlgG is a bacterial flagellar rod protein and constructs the distal rod connecting to the hook. FlgG of Salmonella enterica serovar Typhimurium is a 260-amino-acid protein composed of a folded core region and N- and C-terminal regions that are unfolded in solution. A core fragment of FlgG (FlgG47-227) was expressed, purified and crystallized. Crystals of native and SeMet-labelled FlgG47-227 were obtained by the sitting-drop vapour-diffusion technique with PEG MME 2000 as precipitant. The native crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 47.78, b = 68.94, c = 110.57 Å. The SeMet crystal also belonged to space group P212121, with unit-cell parameters a = 47.53, b = 67.04, c = 110.27 Å. [ABSTRACT FROM AUTHOR]

Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus, is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coil. The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FIgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FIgT at 2.0-A resolution. FlgT is composed of three domains, the N-terminal domain (FIgT-N), the middle domain (FlgT-M), and the C-terminal domain (FIgT-C). FlgT-M is similar to the N-terminal domain of bIB, and FIgT-C resembles the N-terminal domain of Flil and the a/~ subunits of F1-ATPase. To elucidate the role of each domain, we prepared domain deletion mutants of FIgT and analyzed their effects on the basal-body ring formation. The results suggest that FI9T-N contributes to the construction'of the H-ring structure, and FIgT-M mediates the T-ring association on the LP ring. FIgT-C is not essential but stabilizes the H-ring structure. On the basis of these results, we propose an assembly mechanism for the basal-body rings and the stator units of the sodium-driven flagellar motor. [ABSTRACT FROM AUTHOR]

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliHx-FliI6 ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues--Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83--of FliJ. A structural model of the FliI6-FliJ ring complex suggests that they extend out of the FliI6 ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of Flirt and FliI but considerably reduced motility in their absence. These mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FIiJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA. [ABSTRACT FROM AUTHOR]

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB1-94), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB1-94 interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB1-94 also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB1-94 showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB1-94. [ABSTRACT FROM AUTHOR]

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation-enhancing activity of KaiC, ultimately resulting in genome-wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the domain structure of an orthodox histidine kinase and that its C-terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature- and phosphorylation state-dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N-terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation-enhancing activity of KaiC requires the C-terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night. [ABSTRACT FROM AUTHOR]

Summary FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a Δ fliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A Δ fliH-fliI flhB(P28T) Δ flgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhAC). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhAC. The FlgN-FlgK complex bound to FlhAC and FliJ to form the FlgN-FlgK-FliJ-FlhAC complex. The FlgN-FlhAC interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues still bound to FlgK and FliJ but not to FlhAC. A highly conserved Tyr-122 residue was required for the interaction with FlhAC. These results suggest that FlgN efficiently transfers FlgK/L subunits to FlhAC to promote their export. [ABSTRACT FROM AUTHOR]

Abstract: The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhAC) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone–substrate complexes. FlhAC is composed of a flexible linker region and four compact domains (ACD1–ACD4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhAC blocks the export process after the FliH–FliI–FliJ–substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhAC(G368C), and its pseudorevertant variants FlhAC(G368C/L359F), FlhAC(G368C/G364R), FlhAC(G368C/R370S), and FlhAC(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhAC occurs in a single step, FlhAC(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhAC(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of ACD2 and ACD1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhAC(G368C) and that the loss of function is caused by denaturation of ACD2. We suggest that ACD2 is directly involved in the translocation of export substrates. [Copyright &y& Elsevier]

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhDC activity. FlhDC activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of σ activity. We have found that efficient growth-dependent regulation of FlhDC requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhDC activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhDC circuit. When FlhDC was not bound to DNA, FliT disrupted the FlhDC complex. Interestingly, when FlhDC was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhDC activity by preventing the formation of the FlhDC:DNA complex. Our data would suggest that this level of endogenous regulation of FlhDC activity allows the flagellar system to efficiently respond to external signals. [ABSTRACT FROM AUTHOR]

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhAC) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhAC. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhAC consists of at least three subdomains, a flexible linker, FlhACN, and FlhACC, and that FlhACN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhAC not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure. [ABSTRACT FROM AUTHOR]

The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination. [ABSTRACT FROM AUTHOR]

Rotation of the sodium-driven polar flagellum of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX, and MotY. PomA and PomB form a sodium-ion channel in the cytoplasmic membrane that functions as a stator complex to couple sodium-ion flux with torque generation. MotX and MotY are components of the T-ring, which is located beneath the P-ring of the polar flagellar basal body and is involved in incorporation of the PomA/PomB complex into the motor. Here, we describe the determination of the crystal structure of MotY at 2.9 Å resolution. The structure shows two distinct domains: an N-terminal domain (MotY-N) and a C-terminal domain (MotY-C). MotY-N has a unique structure. MotY-C contains a putative peptidoglycan-binding motif that is remarkably similar to those of peptidoglycan-binding proteins. such as Pal and RmpM, but this region is disordered in MotY. Motility assay of cells producing either of the MotY-N and MotY-C fragments and subsequent biochemical analyses indicate that MotY-N is essential for association of the stator units around the rotor, whereas MotY-C stabilizes the association by binding to the peptidoglycan layer. Based on these observations, we propose a model for the mechanism of stator assembly around the rotor. [ABSTRACT FROM AUTHOR]