Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1.
ABSTRACT: The bacterial flagellum is a motility organelle that consists of a rotary motor and a helical propeller. The flagella usually work individually or by forming a loose bundle to produce thrust. However, the flagellar apparatus of marine bacterium MO-1 is a tight bundle of seven flagellar filaments enveloped in a sheath, and it has been a mystery as to how the flagella rotate smoothly in coordination. Here we have used electron cryotomography to visualize the 3D architecture of the sheathed flagella. The seven filaments are enveloped with 24 fibrils in the sheath, and their basal bodies are arranged in an intertwined hexagonal array similar to the thick and thin filaments of vertebrate skeletal muscles. This complex and exquisite architecture strongly suggests that the fibrils counter-rotate between flagella in direct contact to minimize the friction of high-speed rotation of individual flagella in the tight bundle within the sheath to enable MO-1 cells to swim at about 300 µm/s.
Project description:UNLABELLED:Motility is essential for the pathogenesis of many bacterial species. Most bacteria move using flagella, which are multiprotein filaments that rotate propelled by a cell wall-anchored motor using chemical energy. Although some components of the flagellar apparatus are common to many bacterial species, recent studies have shown significant differences in the flagellar structures of different bacterial species. The molecular bases for these differences, however, are not understood. The flagella from epsilonproteobacteria, which include the bacterial pathogens Campylobacter jejuni and Helicobacter pylori, are among the most divergent. Using next-generation sequencing combined with transposon mutagenesis, we have conducted a comprehensive high-throughput genetic screen in Campylobacter jejuni, which identified several novel components of its flagellar system. Biochemical analyses detected interactions between the identified proteins and known components of the flagellar machinery, and in vivo imaging located them to the bacterial poles, where flagella assemble. Most of the identified new components are conserved within but restricted to epsilonproteobacteria. These studies provide insight into the divergent flagella of this group of bacteria and highlight the complexity of this remarkable structure, which has adapted to carry out its conserved functions in the context of widely diverse bacterial species. IMPORTANCE:Motility is essential for the normal physiology and pathogenesis of many bacterial species. Most bacteria move using flagella, which are multiprotein filaments that rotate propelled by a motor that uses chemical energy as fuel. Although some components of the flagellar apparatus are common to many bacterial species, recent studies have shown significant divergence in the flagellar structures across bacterial species. However, the molecular bases for these differences are not understood. The flagella from epsilonproteobacteria, which include the bacterial pathogens Campylobacter jejuni and Helicobacter pylori, are among the most divergent. We conducted a comprehensive genetic screen in Campylobacter jejuni and identified several novel components of the flagellar system. These studies provide important information to understand how flagella have adapted to function in the context of widely diverse sets of bacterial species and bring unique insight into the evolution and function of this remarkable bacterial organelle.
Project description:We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per ?m of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The "dry" colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.
Project description:Actinoplanes missouriensis spores swim with a tuft of flagella. Flagella of newborn spores are wrapped with a membranous sheath. When the sheath is unwrapped, spores start swimming. Flagellar length is kept short, at around 1.9 ?m, which covers half the circumference of the spore.
Project description:Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 ?m and a diameter of 1.4 ?m, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional.R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.
Project description:Cross-linked filament bundles, such as in cilia and flagella, are ubiquitous in biology. They are considered in textbooks as simple filaments with larger stiffness. Recent observations of flagellar counterbend, however, show that induction of curvature in one section of a passive flagellum instigates a compensatory counter-curvature elsewhere, exposing the intricate role of the diminutive cross-linking proteins at large scales. We show that this effect, a material property of the cross-linking mechanics, modifies the bundle dynamics and induces a bimodal L2 - L3 length-dependent material response that departs from the Euler-Bernoulli theory. Hence, the use of simpler theories to analyse experiments can result in paradoxical interpretations. Remarkably, the counterbend dynamics instigates counter-waves in opposition to driven oscillations in distant parts of the bundle, with potential impact on the regulation of flagellar bending waves. These results have a range of physical and biological applications, including the empirical disentanglement of material quantities via counterbend dynamics.
Project description:Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.
Project description:A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.
Project description:The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.
Project description:The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.
Project description:Many species of bacteria swim through viscous environments by rotating multiple helical flagella. The filaments gather behind the cell body and form a close helical bundle, which propels the cell forward during a "run". The filaments inside the bundle cannot be continuously actuated, nor can they easily unbundle, if they are tangled around one another. The fact that bacteria can passively form coherent bundles, i.e. bundles which do not contain tangled pairs of filaments, may appear surprising given that flagella are actuated by uncoordinated motors. In this article, we establish the theoretical conditions under which a pair of rigid helical filaments can form a tangled bundle, and we compare these constraints with experimental data collected from the literature. Our results suggest that bacterial flagella are too straight and too far apart to form tangled bundles based on their intrinsic, undeformed geometry alone. This makes the formation of coherent bundles more robust against the passive nature of the bundling process, where the position of individual filaments cannot be controlled.