Publications
78 results found
Ahmadi Y, Umrekar TR, Mutter N, et al., 2024, DNA origami-enhanced binding of aptamers to Staphylococcus aureus cells, Biosensors and Bioelectronics: X, Vol: 16
The combination of DNA origami nanostructures and aptamers provides a powerful technology for diagnostic assays. Here, we functionalized a DNA origami nanostructure with a Protein-A binding aptamer to target Staphylococcus aureus bacterial cells. Using an enzyme-linked oligonucleotide assay (ELONA), we semi-quantitatively analyzed and compared the interaction of the aptamer and aptamer-modified DNA origamis with Staphylococcus aureus bacterial isolates. The results showed that aptamer-functionalized DNA nanostructures bind with five times higher affinity (KD: 34 ± 5 nM) compared to the aptamer alone (KD: 160 ± 9 nM). Visualising the interaction of bacterial cells and nanostructures with electron cryotomography further confirmed the aptamer-mediated specific interaction of DNA nanostructures with bacterial cells.
Huang X, Torre I, Chiappi M, et al., 2023, Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments, JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, Vol: 44, Pages: 165-178, ISSN: 0142-4319
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- Citations: 2
Wilkinson MD, Ferreira JL, Beeby M, et al., 2022, The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 9
Kaplan M, Oikonomou CM, Wood CR, et al., 2022, Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System, JOURNAL OF BACTERIOLOGY, Vol: 204, ISSN: 0021-9193
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- Citations: 1
Kaplan M, Oikonomou CM, Wood CR, et al., 2022, Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors, EMBO JOURNAL, Vol: 41, ISSN: 0261-4189
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- Citations: 6
Ortega D, Beeby M, 2022, How did the archaellum get its rotation?, Frontiers in Microbiology, Vol: 12, Pages: 1-6, ISSN: 1664-302X
How changes in function evolve fascinates many evolutionary biologists. Particularly captivating is the evolution of rotation in molecular machines, as it evokes familiar machines that we have made ourselves. The archaellum, an archaeal analog of the bacterial flagellum, is one of the simplest rotary motors. It features a long helical propeller attached to a cell envelope-embedded rotary motor. Satisfyingly, the archaellum is one of many members of the large type IV filament superfamily, which includes pili, secretion systems, and adhesins, relationships that promise clues as to how the rotating archaellum evolved from a non-rotary ancestor. Nevertheless,determining exactly how the archaellum got its rotation remains frustratingly elusive. Here we review what is known about how the archaellum got its rotation, what clues exist, and what more is needed to address this question.
Mandela E, Stubenrauch CJ, Ryoo D, et al., 2022, Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability, ELIFE, Vol: 11, ISSN: 2050-084X
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- Citations: 9
Umrekar T, Winterborn Y, Sivabalasarma S, et al., 2021, Evolution of archaellum rotation involved invention of a stator complex by duplicating and modifying a core component, Frontiers in Microbiology, Vol: 12, Pages: 1-10, ISSN: 1664-302X
Novelty in biology can arise from opportunistic repurposing of nascent characteristics ofexisting features. Understanding how this process happens at the molecular scale, however,suffers from a lack of case studies. The evolutionary emergence of rotary motors is aparticularly clear example of evolution of a new function. The simplest of rotary motors is thearchaellum, a molecular motor that spins a helical propeller for archaeal motility analogous tothe bacterial flagellum. Curiously, emergence of archaellar rotation may have pivoted on thesimple duplication and repurposing of a pre-existing component to produce a stator complexthat anchors to the cell superstructure to enable productive rotation of the rotor component.This putative stator complex is composed of ArlF and ArlG, gene duplications of the filamentcomponent ArlB, providing an opportunity to study how gene duplication andneofunctionalization contributed to the radical innovation of rotary function. Towardunderstanding how this happened, we used electron cryomicroscopy to determine thestructure of isolated ArlG filaments, the major component of the stator complex. Using ahybrid modeling approach incorporating structure prediction and validation, we show thatArlG filaments are open helices distinct to the closed helical filaments of ArlB. Curiously,further analysis reveals that ArlG retains a subset of the inter-protomer interactions ofhomologous ArlB, resulting in a superficially different assembly that nevertheless reflects thecommon ancestry of the two structures. This relatively simple mechanism to changequaternary structure was likely associated with the evolutionary neofunctionalization of thearchaellar stator complex, and we speculate that the relative deformable elasticity of an openhelix may facilitate elastic energy storage during the transmission of the discrete bursts ofenergy released by ATP hydrolysis to continuous archaellar rotation, allowing the inherentproperties of a duplicated ArlB to be
Ferreira JL, Coleman I, Addison ML, et al., 2021, The "Jack-of-all-Trades" Flagellum From Salmonella and E. coli Was Horizontally Acquired From an Ancestral B-Proteobacterium (vol 12, 643180, 2021), FRONTIERS IN MICROBIOLOGY, Vol: 12
Gumbart JC, Ferreira JL, Hwang H, et al., 2021, Lpp positions peptidoglycan at the AcrA-ToIC interface in the AcrAB-ToIC multidrug efflux pump, BIOPHYSICAL JOURNAL, Vol: 120, Pages: 3973-3982, ISSN: 0006-3495
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- Citations: 8
Kaplan M, Oikonomou CM, Wood CR, et al., 2021, A novel widespread bacterial structure related to the flagellar type III secretion system
<jats:title>Abstract</jats:title><jats:p>The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.</jats:p>
Kaplan M, Chreifi G, Metskas LA, et al., 2021, In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryotomograpy, ELIFE, Vol: 10, ISSN: 2050-084X
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- Citations: 5
Kaplan M, Tocheva EI, Briegel A, et al., 2021, Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis, MBIO, Vol: 12, ISSN: 2150-7511
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- Citations: 4
Matthews-Palmer T, Gonzalez-Rodriguez N, Calcraft T, et al., 2021, Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism, Journal of Structural Biology, Vol: 213, ISSN: 1047-8477
Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Ferreira J, coleman I, Addison M, et al., 2021, The 'jack-of-all-trades' flagellum from Salmonella and E. coli was horizontally acquired from an ancestral beta-proteobacterium, Frontiers in Microbiology, Vol: 12, ISSN: 1664-302X
The γ-proteobacteria are a group of diverse bacteria including pathogenic Escherichia, Salmonella, Vibrio, and Pseudomonas species. The majority swim in liquids with polar, sodium-driven flagella and swarm on surfaces with lateral, non-chemotactic flagella. Notable exceptions are the enteric Enterobacteriaceae such as Salmonella and E. coli. Many of the well-studied Enterobacteriaceae are gut bacteria that both swim and swarm with the same proton-driven peritrichous flagella. How different flagella evolved in closely related lineages, however, has remained unclear. Here, we describe our phylogenetic finding that Enterobacteriaceae flagella are not native polar or lateral γ-proteobacterial flagella but were horizontally acquired from an ancestral β-proteobacterium. Using electron cryo-tomography and subtomogram averaging, we confirmed that Enterobacteriaceae flagellar motors resemble contemporary β-proteobacterial motors and are distinct to the polar and lateral motors of other γ-proteobacteria. Structural comparisons support a model in which γ-proteobacterial motors have specialized, suggesting that acquisition of a β-proteobacterial flagellum may have been beneficial as a general-purpose motor suitable for adjusting to diverse conditions. This acquisition may have played a role in the development of the enteric lifestyle.
Umrekar TR, Cohen E, Drobnic T, et al., 2021, CryoEM of bacterial secretion systems: A primer for microbiologists, MOLECULAR MICROBIOLOGY, Vol: 115, Pages: 366-382, ISSN: 0950-382X
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- Citations: 8
Sivabalasarma S, Wetzel H, Nussbaum P, et al., 2021, Analysis of Cell-Cell Bridges in <i>Haloferax volcanii</i> Using Electron Cryo-Tomography Reveal a Continuous Cytoplasm and S-Layer, FRONTIERS IN MICROBIOLOGY, Vol: 11, ISSN: 1664-302X
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- Citations: 7
Alvira S, Watkins DW, Troman L, et al., 2020, Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis, ELIFE, Vol: 9, ISSN: 2050-084X
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- Citations: 21
Sivabalasarma S, Wetzel H, Nußbaum P, et al., 2020, Analysis of cell-cell bridges in <i>Haloferax volcanii</i> using Electron cryo-tomography reveal a continuous cytoplasm and S-layer
<jats:p>Halophilic archaea exchange DNA and proteins using a fusion-based mating mechanism. Scanning electron microscopy previously suggested that mating involves an intermediate state, where cells are connected by an intercellular bridge. To better understand this process, we used electron cryotomography and fluorescence microscopy to visualize cells forming these intercellular bridges. Electron cryo-tomography showed that the observed bridges were enveloped by an S-layer and connected mating cells via a continuous cytoplasm. Macromolecular complexes like ribosomes and unknown thin filamentous helical structures were visualized in the cytoplasm inside the bridges, demonstrating that these bridges can facilitate exchange of cellular components. We followed formation of a cell-cell bridge by fluorescence time-lapse microscopy between cells at a distance of 1.5 µm. These results shed light on the process of haloarchaeal mating and highlight further mechanistic questions.</jats:p>
de Llano E, Miao H, Ahmadi Y, et al., 2020, Adenita: interactive 3D modelling and visualization of DNA nanostructures, NUCLEIC ACIDS RESEARCH, Vol: 48, Pages: 8269-8275, ISSN: 0305-1048
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- Citations: 26
Blagotinsek V, Schwan M, Steinchen W, et al., 2020, An ATP-dependent partner switch links flagellar C-ring assembly with gene expression, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 117, Pages: 20826-20835, ISSN: 0027-8424
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- Citations: 7
Taylor PJ, Hagen J, Faruqu FN, et al., 2020, Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression, International Journal for Parasitology, Vol: 50, Pages: 697-705, ISSN: 0020-7519
Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection.
Cohen EJ, Nakane D, Kabata Y, et al., 2020, Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella, PLoS Pathogens, Vol: 16, Pages: 1-24, ISSN: 1553-7366
Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts’ gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni’s flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni’s opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.
Cohen EJ, Nakane D, Kabata Y, et al., 2020, Campylobacter jejunimotility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella, PLoS Pathogens, Vol: 16, Pages: 1-24, ISSN: 1553-7366
Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts’ gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni’s flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni’s opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.
Beeby M, 2020, Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS, TRENDS IN BIOCHEMICAL SCIENCES, Vol: 45, Pages: 549-551, ISSN: 0968-0004
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- Citations: 3
Ahmadi Y, Nord AL, Wilson AJ, et al., 2020, The Brownian and Flow-Driven Rotational Dynamics of a Multicomponent DNA Origami-Based Rotor, SMALL, Vol: 16, ISSN: 1613-6810
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- Citations: 16
Rossmann F, Hug I, Sangermani M, et al., 2020, In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog, Molecular Microbiology, ISSN: 0950-382X
Beeby M, Ferreira J, Tripp P, et al., 2020, Propulsive nanomachines: the convergent evolution of archaella, flagella, and cilia, FEMS Microbiology Reviews, Vol: 44, Pages: 253-304, ISSN: 0168-6445
Echoing the repeated convergent evolution of flight and vision in large eukaryotes, propulsive swimming motility has evolved independently in microbes in each of the three domains of life. Filamentous appendages—archaella in Archaea, flagella in Bacteria, and cilia in Eukaryotes—wave, whip, or rotate to propel microbes, overcoming diffusion and enabling colonization of new environments. The implementations of the three propulsive nanomachines are distinct, however: archaella and flagella rotate, while cilia beat or wave; flagella and cilia assemble at their tips, while archaella assemble at their base; archaella and cilia use ATP for motility, while flagella use ion-motive force. These underlying differences reflect the tinkering required to evolve a propulsive molecular machine, in which pre-existing machines in the appropriate contexts were iteratively co-opted for new functions, and whose origins are reflected in the resultant mechanisms. Contemporary homologies suggest that archaella evolved from a non-rotary pilus, flagella from a non-rotary appendage or secretion system, and cilia from a passive sensory structure. Here we review the structure, assembly, mechanism, and homologies of the three distinct solutions as a foundation to better understand how propulsive nanomachines evolved three times independently and to highlight principles of molecular evolution.
Kaplan M, Sweredoski MJ, Rodrigues JPGLM, et al., 2020, Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 117, Pages: 8941-8947, ISSN: 0027-8424
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- Citations: 14
Taylor P, Hagen J, Faruqu F, et al., 2020, Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression, Publisher: bioRxiv
Abstract Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment largely encapsulated within small vesicles. Parasite secreted material often contains microRNAs (miRNAs), raising the possibility that they might contribute to pathology by regulating host genes in target cells. Here we characterise material from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis , which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly however, T. spiralis muscle stage larvae (MSL), which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests a new potential mechanism of RNA secretion with implications for the pathology of T. spiralis infection.
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