40 results found
Ferreira JL, Gao FZ, Rossmann FM, et al., γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures, PLoS Biology, ISSN: 1544-9173
Nguyen LT, Oikonomou CM, Ding HJ, et al., 2019, Simulations suggest a constrictive force is required for Gram-negative bacterial cell division, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723
Thomson NM, Ferreira JL, Matthews-Palmer TR, et al., 2018, Giant flagellins form thick flagellar filaments in two species of marine gamma-proteobacteria, PLoS ONE, Vol: 13, ISSN: 1932-6203
Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode “giant” flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how “the” bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.
Rossmann F, Beeby M, 2018, Insights into the evolution of the bacterial flagellar motor from highthroughput in situ electron cryotomography and subtomogram averaging, Acta Crystallographica Section D: Biological Crystallography, Vol: D74, Pages: 585-594, ISSN: 1399-0047
In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions.
Henderson LD, Beeby M, 2018, High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly., Methods Mol Biol, Vol: 1764, Pages: 29-44
Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.
Chaban B, Coleman I, Beeby M, 2018, Evolution of higher torque in Campylobacter-type bacterial flagellar motors, Scientific Reports, Vol: 8, ISSN: 2045-2322
Understanding the evolution of molecular machines underpins our understanding of the development of life on earth. A well-studied case are bacterial flagellar motors that spin helical propellers for bacterial motility. Diverse motors produce different torques, but how this diversity evolved remains unknown. To gain insights into evolution of the high-torque ε-proteobacterial motor exemplified by the Campylobacter jejuni motor, we inferred ancestral states by combining phylogenetics, electron cryotomography, and motility assays to characterize motors from Wolinella succinogenes, Arcobacter butzleri and Bdellovibrio bacteriovorus. Observation of ~12 stator complexes in many proteobacteria, yet ~17 in ε-proteobacteria suggest a “quantum leap” evolutionary event. Campylobacter-type motors have high stator occupancy in wider rings of additional stator complexes that are scaffolded by large proteinaceous periplasmic rings. We propose a model for motor evolution wherein independent inner- and outer-membrane structures fused to form a scaffold for additional stator complexes. Significantly, inner- and outer-membrane associated structures have evolved independently multiple times, suggesting that evolution of such structures is facile and poised the ε-proteobacteria to fuse them to form the high-torque Campylobacter-type motor.
Ferreira J, Matthews-Palmer T, Beeby M, 2017, Electron cryo-tomography, Cellular Imaging Electron Tomography and Related Techniques, Editors: Hanssen, Publisher: Springer, ISBN: 9783319689975
This book highlights important techniques for cellular imaging and covers the basics and applications of electron tomography and related techniques.
Asmar AT, Ferreira JL, Cohen EJ, et al., 2017, Communication across the bacterial cell envelope depends on the size of the periplasm, PLOS BIOLOGY, Vol: 15, ISSN: 1545-7885
Thecellenvelopeof gram-negativebacteria,a structurecomprisinganouter(OM)andaninner(IM)membrane,is essentialforlife.TheOMandtheIMareseparatedbytheperi-plasm,a compartmentthatcontainsthepeptidoglycan. TheOMis tetheredto thepeptido-glycanviathelipoprotein,Lpp.However,theimportanceof theenvelope’smultilayeredarchitectureremainsunknown.Here,whenweremovedphysicalcouplingbetweentheOMandthepeptidoglycan,cellslosttheabilityto sensedefectsin envelopeintegrity.Furtherexperimentsrevealedthatthecriticalparameterforthetransmissionof stresssignalsfromtheenvelopeto thecytoplasm, wherecellularbehaviouris controlled,is theIM-to-OMdis-tance.Augmentingthisdistancebyincreasingthelengthof thelipoproteinLppdestroyedsignalling,whereassimultaneouslyincreasingthelengthof thestress-sensinglipoproteinRcsFrestoredsignalling.Ourresultsdemonstratethephysiological importanceof thesizeof theperiplasm.TheyalsorevealthatstrictcontrolovertheIM-to-OMdistanceis requiredforeffectiveenvelopesurveillanceandprotection,suggestingthatcellulararchitectureandthestructureof transenvelopeproteincomplexeshavebeenevolutionarilyco-optimisedforcorrectfunction.Similarstrategiesarelikelyat playin cellularcompartmentssurroundedby2 concentricmembranes,suchaschloroplastsandmitochondria.
Thomson NM, Ferreira JL, Matthews-Palmer TR, et al., 2017, Giant flagellins form thick flagellar filaments in two species of marine γ-proteobacteria, Publisher: Cold Spring Harbor Laboratory
<jats:p>Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode `giant' flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how `the' bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.</jats:p>
Yao Q, Jewett AI, Chang Y-W, et al., 2017, Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis, EMBO JOURNAL, Vol: 36, Pages: 1577-1589, ISSN: 0261-4189
Cohen EJ, Ferreira JL, Ladinsky MS, et al., 2017, Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane, SCIENCE, Vol: 356, Pages: 197-200, ISSN: 0036-8075
The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.
Taylor WR, Matthews-Palmer TR, Beeby M, 2016, Molecular models for the core components of the flagellar type-III secretion complex, PLOS One, Vol: 11, ISSN: 1932-6203
We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments.
Hoffmann L, Schummer A, Reimann J, et al., 2016, Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius, MICROBIOLOGYOPEN, Vol: 6, ISSN: 2045-8827
Haurat MF, Figueiredo AS, Hoffmann L, et al., 2016, ArnS, a kinase involved in starvation-induced archaellum expression, Molecular Microbiology, Vol: 103, Pages: 181-194, ISSN: 1365-2958
Organisms have evolved motility organelles that allow them to move to favorable habitats. Cells integrate environmental stimuli into intracellular signals to motility machineries to direct this migration. Many motility organelles are complex surface appendages that have evolved a tight, hierarchical regulation of expression. In the crenearchaeon Sulfolobus acidocaldarius, biosynthesis of the archaellum is regulated by regulatory network proteins that control expression of archaellum components in a phosphorylation-dependent manner. A major trigger for archaellum expression is nutrient starvation, but although some components are known, the regulatory cascade triggered by starvation is poorly understood. In this work, we identify the starvation-induced Ser/Thr protein kinase ArnS (Saci_1181) which is located proximally to the archaellum operon. Deletion of arnS results in reduced motility, though the archaellum is properly assembled. Therefore, our experimental and modelling results indicate that ArnS plays an essential role in the precisely controlled expression of archaellum components during starvation-induced motility in Sulfolobus acidocaldarius. Furthermore we combine in vivo experiments and mathematical models to describe for the first time in archaea the dynamics of key regulators of archaellum expression.
Beeby M, Ribardo DA, Brennan CA, et al., 2016, Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E1917-E1926, ISSN: 1091-6490
Nguyen LT, Swulius M, Gumbart JC, et al., 2016, Coarse-Grained Simulations Reveal Mechanisms of Bacterial Morphogenesis, 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 468A-468A, ISSN: 0006-3495
Oikonomou CM, Swulius MT, Briegel A, et al., 2016, Electron cryotomography, IMAGING BACTERIAL MOLECULES, STRUCTURES AND CELLS, Editors: Harwood, Jensen, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 115-139
Beeby M, 2015, Motility in the epsilon-proteobacteria, Current Opinion in Microbiology, Vol: 28, Pages: 115-121, ISSN: 1879-0364
The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility.
Chaban B, Hughes HV, Beeby M, 2015, The flagellum in bacterial pathogens: For motility and a whole lot more., Seminars in Cell & Developmental Biology, ISSN: 1096-3634
The bacterial flagellum is an amazingly complex molecular machine with a diversity of roles in pathogenesis including reaching the optimal host site, colonization or invasion, maintenance at the infection site, and post-infection dispersal. Multi-megadalton flagellar motors self-assemble across the cell wall to form a reversible rotary motor that spins a helical propeller - the flagellum itself - to drive the motility of diverse bacterial pathogens. The flagellar motor responds to the chemoreceptor system to redirect swimming toward beneficial environments, thus enabling flagellated pathogens to seek out their site of infection. At their target site, additional roles of surface swimming and mechanosensing are mediated by flagella to trigger pathogenesis. Yet while these motility-related functions have long been recognized as virulence factors in bacteria, many bacteria have capitalized upon flagellar structure and function by adapting it to roles in other stages of the infection process. Once at their target site, the flagellum can assist adherence to surfaces, differentiation into biofilms, secretion of effector molecules, further penetration through tissue structures, or in activating phagocytosis to gain entry into eukaryotic cells. Next, upon onset of infection, flagellar expression must be adapted to deal with the host's immune system defenses, either by reduced or altered expression or by flagellar structural modification. Finally, after a successful growth phase on or inside a host, dispersal to new infection sites is often flagellar motility-mediated. Examining examples of all these processes from different bacterial pathogens, it quickly becomes clear that the flagellum is involved in bacterial pathogenesis for motility and a whole lot more.
Nguyen LT, Gumbart JC, Beeby M, et al., 2015, Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 112, Pages: E3689-E3698, ISSN: 0027-8424
Mueller A, Beeby M, McDowall AW, et al., 2014, Ultrastructure and complex polar architecture of the human pathogen Campylobacter jejuni, MicrobiologyOpen, Vol: 3, Pages: 702-710, ISSN: 2045-8827
Campylobacter jejuni is one of the most successful food-borne humanpathogens. Here we use electron cryotomography to explore the ultrastructureof C. jejuni cells in logarithmically growing cultures. This provides the first lookat this pathogen in a near-native state at macromolecular resolution (~5 nm).We find a surprisingly complex polar architecture that includes ribosomeexclusion zones, polyphosphate storage granules, extensive collar-shapedchemoreceptor arrays, and elaborate flagellar motors.
Beeby M, 2014, Evolution of Novel Components of the Bacterial Flagellar Motor, 28th Annual Symposium of the Protein-Society, Publisher: WILEY-BLACKWELL, Pages: 61-61, ISSN: 0961-8368
Gumbart JC, Beeby M, Jensen GJ, et al., 2014, Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale aimulations, PLOS Computational Biology, Vol: 10, ISSN: 1553-734X
Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgorpressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterialcell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid,yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by themolecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is notknown. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in adisordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as knownexperimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other featuressuch as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting thedisordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure andbehavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of allatomsimulations to reproduce a range of macroscopic observables for extended polymer meshes.
Beeby M, Gumbart JC, Roux B, et al., 2013, Architecture and assembly of the Gram-positive cell wall, Molecular Microbiology, Vol: 88, Pages: 664-672, ISSN: 0950-382X
Jensen G, Briegel A, Beeby M, 2013, Visualizing large macromolecular assemblies in vivo with electron cryotomography, 245th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Abrusci P, Vergara-Irigaray M, Johnson S, et al., 2013, Architecture of the major component of the type III secretion system export apparatus, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 20, Pages: 99-U126, ISSN: 1545-9993
Beeby M, Cho M, Stubbe J, et al., 2012, Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha, J. Bacteriol., Vol: 194, Pages: 1092-1099
Briegel A, Beeby M, Thanbichler M, et al., 2011, Activated chemoreceptor arrays remain intact and hexagonally packed, Mol. Microbiol., Vol: 82, Pages: 748-757
Chen S, Beeby M, Murphy GE, et al., 2011, Structural diversity of bacterial flagellar motors, EMBO J., Vol: 30, Pages: 2972-2981
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