Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.     

Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis

The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor. We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively. Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages.     

Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.     

Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus

The switch in export specificity of the type III flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type III secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FlhB. The T3S4 domain of FliK (FliKT3S4) consisting of amino acid residues 265-405 has an unstable and flexible conformation in its last 35 residues (FliKCT). To investigate the role of FliKT3S4 in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301-350 and five amino acids of residues 401-405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Val302, Ile304, Leu335, Val401 and Ala405, are essential, and that the five C terminal residues (401-405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliKT3S4 interacts with the C terminal half of the cytoplasmic domain of FlhB (FlhBC). We propose a two step binding model of FliKT3S4 and FlhBC, in which residues 301-350 of FliK bind to FlhBC upon hook assembly completion at about 55 nm, and then unfolded FliKCT binds to FlhBC to trigger the switch in substrate specificity.     

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor’s direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY‐binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.     

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.     

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB_C2) and Vibrio alginolyticus (PomB_C5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotB_C2, the dimeric conformation of the PomB_C5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.     

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

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The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3

Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths.     

The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx

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Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella

FlhA is an integral membrane component of the Salmonella type III flagellar protein export apparatus. It consists of 692 amino acid residues and has two domains: the N-terminal transmembrane domain consisting of the first 327 amino acid residues, and the C-terminal cytoplasmic domain (FlhAC) comprising the remainder. Here, we have investigated the structure and function of FlhAC. DNA sequence analysis revealed that temperature-sensitive flhA mutations, which abolish flagellar protein export at the restrictive temperature, lie in FlhAC, indicating that FlhAC plays an important role in the protein export process. Limited proteolysis of purified His-FlhAC by trypsin and V8 showed that only a small part of FlhAC near its N terminus (residues 328-351) is sensitive to proteolysis. FlhAC38K, the smallest fragment produced by V8 proteolysis, is monomeric and has a spherical shape as judged by analytical gel filtration chromatography and analytical ultracentrifugation. The far-UV CD spectrum of FlhAC38K showed that it contains considerable amounts of secondary structure. FlhA(Delta328-351) missing residues 328-351 failed to complement the flhA mutant, indicating that the proteolytically sensitive region of FlhA is important for its function. FlhA(Delta328-351) was inserted into the cytoplasmic membrane, and exerted a strong dominant negative effect on wild-type cells, suggesting that it retains the ability to interact with other export components within the cytoplasmic membrane. Overproduced FlhAC38K inhibited both motility and flagellar protein export of wild-type cells to some degree, suggesting that FlhAC38K is directly involved in the translocation reaction. Amino acid residues 328-351 of FlhA appear to be a relatively flexible linker between the transmembrane domain and FlhAC38K.     

Coupling of proton flow and rotation in the bacterial flagellar motor: stochastic simulation of a microscopic model

Summary The rotatory motor of bacterial flagella is driven by a transmembrane electrochemical gradient of protons. A model of the flagellar motor is analysed, which is based on the notion that protons passing through the motor use a channel-like pathway formed by ligand groups located partly on the rotor, partly on the stator. Proton translocation is linked to the displacement of stator elements which are elastically bound to the cell wall. The model is described by a cyclic sequence of translocation steps and proton binding and release reactions. Stochastic simulations of the model are carried out in which transitions between the states of the reaction cycle are treated as random events. In this way the rotation frequency can be predicted as a function of experimental variables such as driving force and viscous load. Furthermore, the effects of microscopic parameters such as the transition frequencies of stator elements and the force constant of elastic coupling on the dynamic properties of the motor can be studied. The model allows for intrinsic uncoupling (“slippage”) resulting from translocation steps without associated rotational movement. It is shown that mechanistic information can be obtained by studying random fluctuations of rotational speed. Offprint requests to: P. Lauger     

Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin‐proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo‐electron microscopy and X‐ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the β‐subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven α‐subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.     

Soluble components of the flagellar export apparatus,FliI, FliJ,and FliH,do not deliver flagellin,the major filament protein,from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.