RESUMO
The bacterial flagellar motor is a supramolecular protein machine that drives rotation of the flagellum for motility, which is essential for bacterial survival in different environments and a key determinant of pathogenicity. The detailed structure of the flagellar motor remains unknown. Here we present an atomic-resolution cryoelectron microscopy (cryo-EM) structure of the bacterial flagellar motor complexed with the hook, consisting of 175 subunits with a molecular mass of approximately 6.3 MDa. The structure reveals that 10 peptides protruding from the MS ring with the FlgB and FliE subunits mediate torque transmission from the MS ring to the rod and overcome the symmetry mismatch between the rotational and helical structures in the motor. The LP ring contacts the distal rod and applies electrostatic forces to support its rotation and torque transmission to the hook. This work provides detailed molecular insights into the structure, assembly, and torque transmission mechanisms of the flagellar motor.
Assuntos
Flagelos/fisiologia , Flagelos/ultraestrutura , Salmonella typhimurium/fisiologia , Microscopia Crioeletrônica , Conformação Proteica , TorqueRESUMO
Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.
Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Flagelos/metabolismo , Conformação Proteica , TorqueRESUMO
Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.
Assuntos
Proteínas de Bactérias , Fímbrias Bacterianas , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Bactérias , Flagelos/fisiologiaRESUMO
The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor.
Assuntos
Proteínas de Bactérias , Flagelos , Bactérias/metabolismo , Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestrutura , TorqueRESUMO
The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.
Assuntos
Campylobacter jejuni , Helicobacter pylori , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/metabolismo , Sistemas de Secreção Tipo III/metabolismoRESUMO
Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α-helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate-sink response regulator, CheY1~P by enhancing its decay two-fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 µM, which is 25-fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 µM, and a SEC-MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.
RESUMO
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.
Assuntos
Escherichia coli/fisiologia , Flagelos/metabolismo , Locomoção/fisiologia , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Bactérias , Proteínas de Escherichia coli , Ligação Proteica/fisiologiaRESUMO
The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the â¼45-50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.
Assuntos
Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Flagelos/genética , Flagelos/fisiologia , Bactérias/genética , Proteínas de Bactérias/metabolismo , Flagelos/química , MovimentoRESUMO
The flagellar motor stator is an ion channel nanomachine that assembles as a ring of the MotA5MotB2 units at the flagellar base. The role of accessory proteins required for stator assembly and activation remains largely enigmatic. Here, we show that one such assembly factor, the conserved protein FliL, forms an integral part of the Helicobacter pylori flagellar motor in a position that colocalizes with the stator. Cryogenic electron tomography reconstructions of the intact motor in whole wild-type cells and cells lacking FliL revealed that the periplasmic domain of FliL (FliL-C) forms 18 circumferentially positioned rings integrated with the 18 MotAB units. FliL-C formed partial rings in the crystal, and the crystal structure-based full ring model was consistent with the shape of the rings observed in situ. Our data suggest that each FliL ring is coaxially sandwiched between the MotA ring and the dimeric periplasmic MotB moiety of the stator unit and that the central hole of the FliL ring has density that is consistent with the plug/linker region of MotB in its extended, active conformation. Significant structural similarities were found between FliL-C and stomatin/prohibitin/flotillin/HflK/C domains of scaffolding proteins, suggesting that FliL acts as a scaffold. The binding energy released upon association of FliL with the stator units could be used to power the release of the plug helices. The finding that isolated FliL-C forms stable partial rings provides an insight into the putative mechanism by which the FliL rings assemble around the stator units.
Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Helicobacter pylori/fisiologia , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Helicobacter pylori encodes homologues of PilM, PilN and PilO from bacteria with Type IV pili, where these proteins form a pilus alignment complex. Inactivation of pilO changes H. pylori motility in semi-solid media, suggesting a link to the chemosensory pathways or flagellar motor. Here, we showed that mutation of the pilO or pilN gene in H. pylori strain SS1 reduced the mean linear swimming speed in liquid media, implicating PilO and PilN in the function, or regulation of, the flagellar motor. We also demonstrated that the soluble variants of H. pylori PilN and PilO share common biochemical properties with their Type IV pili counterparts which suggests their adapted function in the bacterial flagellar motor may be similar to that in the Type IV pili.
RESUMO
Motility is important for the survival and dispersal of many bacteria, and it often plays a role during infections. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load).
Assuntos
Flagelos/química , Mecanotransdução Celular , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli , Flagelos/metabolismo , Modelos Teóricos , RotaçãoRESUMO
The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.
Assuntos
Bactérias/citologia , Proteínas de Bactérias/metabolismo , Flagelos/ultraestrutura , Bactérias/metabolismo , Bactérias/ultraestrutura , Membrana Externa Bacteriana/metabolismo , Tomografia com Microscopia Eletrônica , Escherichia coli/citologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Flagelos/metabolismo , Legionella pneumophila/citologia , Legionella pneumophila/metabolismo , Legionella pneumophila/ultraestrutura , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestrutura , Shewanella/citologia , Shewanella/metabolismo , Shewanella/ultraestruturaRESUMO
The output of the bacterial chemotaxis signaling pathway, the level of the intracellular regulator CheY-P, modulates the rotation direction of the flagellar motor, thereby regulating bacterial run-and-tumble behavior. The multiple flagellar motors on anE. colicell are controlled by a common cytoplasmic pool of CheY-P. Fluctuation of the CheY-P level was thought to be able to coordinate the switching of multiple motors. Here, we measured the correlation of rotation directions between two motors on a cell, finding that it surprisingly exhibits two well separated timescales. We found that the slow timescale (â¼6 s) can be explained by the slow fluctuation of the CheY-P level due to stochastic activity of the chemotactic adaptation enzymes, whereas the fast timescale (â¼0.3 s) can be explained by the random pulse-like fluctuation of the CheY-P level, due probably to the activity of the chemoreceptor clusters. We extracted information on the properties of the fast CheY-P pulses based on the correlation measurements. The two well-separated timescales in the fluctuation of CheY-P level help to coordinate multiple motors on a cell and to enhance bacterial chemotactic performance.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Escherichia coli/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Quimiotaxia/fisiologiaRESUMO
The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.
Assuntos
Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Flagelos/genética , Especiação Genética , Bactérias/citologia , Bactérias/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/ultraestrutura , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Flagelos/metabolismo , Genes Bacterianos , FilogeniaRESUMO
Molecular motors are found in many living organisms. One such molecular machine, the ion-powered rotary motor (IRM), requires the movement of ions across a membrane against a concentration gradient to drive rotational movement. The bacterial flagellar motor (BFM) is an example of an IRM which relies on ion movement through the stator proteins to generate the rotation of the flagella. There are many ions which can be used by the BFM stators to power motility and different ions can be used by a single bacterium expressing multiple stator variants. The use of ancestral sequence reconstruction (ASR) and functional analysis of reconstructed stators shows promise for understanding how these proteins evolved and when the divergence in ion use may have occurred. In this review, we discuss extant BFM stators and the ions that power them as well as recent examples of the use of ASR to study ion-channel selectivity and how this might be applied to further study of the BFM stator complex.
Assuntos
Proteínas de Escherichia coli , Proteínas Motores Moleculares , Proteínas Motores Moleculares/metabolismo , Íons/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Flagelos/metabolismoRESUMO
Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Flagelos , Proteínas Motores Moleculares , Proteus mirabilis , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Escherichia coli/química , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestrutura , Proteus mirabilis/química , Proteus mirabilis/citologia , Proteus mirabilis/ultraestrutura , Salmonella enterica/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/ultraestruturaRESUMO
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-MotA5MotB2 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.
Assuntos
Proteínas de Bactérias , Flagelos , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/química , Proteínas de Membrana/metabolismo , Proteínas Motores Moleculares/metabolismoRESUMO
FliL is present in nearly all flagellated bacterial species and is associated with the flagellar basal body. This protein was found to be important for the function of the flagellar motor, and its absence led to a variety of motility defects in several species. However, the specific function of FliL in Pseudomonas aeruginosa remains elusive. Here, we studied the effects of FliL on motor output in P. aeruginosa using a bead assay, finding that FliL regulates motor output through its differential effects on the two sets of homologous MotAB and MotCD stators. FliL interacts with the MotCD stators to increase the motor torque and the stability of the motor speed, whereas it works with the MotAB stators to maintain a high motor switching rate. These effects of FliL contribute to enhancing P. aeruginosa's motility and chemotaxis. IMPORTANCE FliL emerged as a modulator of flagellar motor function in several bacterial species, but its function in Pseudomonas aeruginosa was unknown. Here, by performing single-motor studies using a bead assay, we elucidated its effects on the flagellar motor in P. aeruginosa. We found that it differentially interacts with two sets of stators (MotAB and MotCD) to regulate different aspects of bacterial motility (motor switching rate and motor rotation speed), thereby enhancing the ability of P. aeruginosa to explore its environment.
Assuntos
Flagelos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/fisiologia , Flagelos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , QuimiotaxiaRESUMO
Bacterial Flagellar Motor is one of nature's rare rotary molecular machines. It enables bacterial swimming and it is the key part of the bacterial chemotactic network, one of the best studied chemical signalling networks in biology, which enables bacteria to direct its movement in accordance with the chemical environment. The network can sense down to nanomolar concentrations of specific chemicals on the time scale of seconds. Motor's rotational speed is linearly proportional to the electrochemical gradients of either proton or sodium driving ions, while its direction is regulated by the chemotactic network. Recently, it has been discovered that motor is also a mechanosensor. Given these properties, we discuss the motor's potential to serve as a multifunctional biosensor and a tool for characterising and studying the external environment, the bacterial physiology itself and single molecular motor biophysics.
Assuntos
Técnicas Biossensoriais , Flagelos , Bactérias , Proteínas de Bactérias/genética , Biofísica , Íons , Proteínas Motores Moleculares/genética , SódioRESUMO
Based on genome analyses, it has been estimated that more than half of the bacteria have made an important investment into motility since they possess genes encoding the flagellar motor, the flagellum, chemosensory pathways and chemoreceptors. The metabolic burden associated with gene maintenance, protein synthesis and operating these systems is very important. A central question is thus to establish the physiological benefits that compensate such an important investment. In this chapter, we illustrate that benefits are multiple and diverse, including access to nutrients and preferred niches, biofilm formation and bacterial dispersal. There is also evidence that the complete range of advantages still remains to be defined. In these research efforts, Pseudomonas aeruginosa (PA) has played a central role and is among the central model species. Research conducted on PA had a significant impact in the field and has motivated many experiments in the study of other model bacterial species.