ORCID Profile
0000-0002-5839-6904
Current Organisation
University of New South Wales
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Phylogeny and Comparative Analysis | Molecular Evolution | Other Physical Sciences | Biomedical Instrumentation | Biochemistry and Cell Biology | Optical Properties of Materials | Genetics | Synchrotrons; Accelerators; Instruments and Techniques | Soft Condensed Matter | Synthetic Biology | Bacteriology
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Physical Sciences | Expanding Knowledge in Technology |
Publisher: MDPI AG
Date: 25-06-2023
Abstract: 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 ex le 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 ergence in ion use may have occurred. In this review, we discuss extant BFM stators and the ions that power them as well as recent ex les of the use of ASR to study ion-channel selectivity and how this might be applied to further study of the BFM stator complex.
Publisher: Wiley
Date: 19-08-2014
Abstract: Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in in idual pores.
Publisher: AIP Publishing
Date: 05-2021
DOI: 10.1063/5.0046941
Abstract: Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use of synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied ex le is the use of the sodium-powered PomA-PotB chimeric stator unit in the naturally proton-powered Escherichia coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eightfold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement the selection of highly motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.
Publisher: Springer Science and Business Media LLC
Date: 14-05-2018
DOI: 10.1038/S41467-018-04288-4
Abstract: The bacterial flagellum is a large extracellular protein organelle that extrudes from the cell surface. The flagellar filament is assembled from tens of thousands of flagellin subunits that are exported through the flagellar type III secretion system. Here, we measure the growth of Escherichia coli flagella in real time and find that, although the growth rate displays large variations at similar lengths, it decays on average as flagella lengthen. By tracking single flagella, we show that the large variations in growth rate occur as a result of frequent pauses. Furthermore, different flagella on the same cell show variable growth rates with correlation. Our observations are consistent with an injection-diffusion model, and we propose that an insufficient cytoplasmic flagellin supply is responsible for the pauses in flagellar growth in E. coli .
Publisher: Oxford University Press (OUP)
Date: 2023
Abstract: The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H+-powered motors), or PomA and PomB (in Na+-powered motors). In this study, we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed 10 ancestral sequences of MotA and found four of them were motile in combination with contemporary Escherichia coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing, and MotA–MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.
Publisher: Cold Spring Harbor Laboratory
Date: 10-04-2019
DOI: 10.1101/604462
Abstract: Recently, DNA-PAINT single molecule localisation microscopy (SMLM) has shown great promise for quantitative imaging. However, labelling strategies so far have relied on approaches that are multivalent or affinity-based. Here, we demonstrate tagPAINT - the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA docking strands for single molecule localisation microscopy via DNA-PAINT. We utilised tagPAINT for T-cell receptor signalling proteins at the immune synapse as a proof of principle.
Publisher: AIP Publishing
Date: 03-2023
DOI: 10.1063/5.0144934
Abstract: Microfluidics devices are gaining significant interest in biomedical applications. However, in a micron-scale device, reaction speed is often limited by the slow rate of diffusion of the reagents. Several active and passive micro-mixers have been fabricated to enhance mixing in microfluidic devices. Here, we demonstrate external control of mixing by rotating a rod-shaped bacterial cell. This rotation is driven by ion transit across the bacterial flagellar stator complex. We first measured the flow fields generated by rotating a single bacterial cell rotationally locked to rotate either clockwise (CW) or counterclockwise (CCW). Micro-particle image velocimetry (μPIV) and particle tracking velocimetry results showed that a bacterial cell of ∼ 2.75 μm long, rotating at 5.75 ± 0.39 Hz in a counterclockwise direction could generate distinct micro-vortices with circular flow fields with a mean velocity of 4.72 ± 1.67 μm/s and maximum velocity of 7.90 μm/s in aqueous solution. We verified our experimental data with a numerical simulation at matched flow conditions, which revealed vortices of similar dimensions and speed. We observed that the flow-field diminished with increasing z-height above the plane of the rotating cell. Lastly, we showed that we could activate and tune rotational mixing remotely using strains engineered with proteorhodopsin, where rotation could be activated by controlled external illumination using green laser light (561 nm).
Publisher: Wiley
Date: 25-06-2015
Publisher: Wiley
Date: 17-04-2019
DOI: 10.1111/MMI.14246
Abstract: The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that in idual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.
Publisher: Oxford University Press (OUP)
Date: 06-10-2021
DOI: 10.1093/NAR/GKAB888
Abstract: Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.
Publisher: Elsevier BV
Date: 05-2018
Publisher: Springer Science and Business Media LLC
Date: 04-05-2019
Publisher: Informa UK Limited
Date: 2019
Publisher: American Chemical Society (ACS)
Date: 29-05-2018
Abstract: The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.
Publisher: Portland Press Ltd.
Date: 18-02-2021
DOI: 10.1042/BST20200746
Abstract: Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future.
Publisher: Cold Spring Harbor Laboratory
Date: 31-12-2018
DOI: 10.1101/507533
Abstract: The bacterial flagellar motor (BFM) powers the rotation that propels swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus / Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus , and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that in idual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.
Publisher: Springer Science and Business Media LLC
Date: 08-02-2016
DOI: 10.1038/NSMB.3172
Abstract: Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.
Publisher: Cold Spring Harbor Laboratory
Date: 02-06-2020
DOI: 10.1101/2020.06.01.128686
Abstract: Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol-modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA-lipid binding while allowing controlled switching by strand-displacement are not known. Here we characterised the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA-lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl 2 , was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded. For larger DNA origami tiles, 4 – 8 cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid-binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand-displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.
Publisher: Springer Science and Business Media LLC
Date: 15-07-2022
DOI: 10.1038/S41467-022-31898-W
Abstract: Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins alpha-hemolysin and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials.
Publisher: Elsevier BV
Date: 04-2016
Publisher: Elsevier BV
Date: 12-2013
Publisher: Cold Spring Harbor Laboratory
Date: 05-03-2023
DOI: 10.1101/2023.03.05.530362
Abstract: Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA 5 MotB 2 , a heterodimeric complex which forms an ion channel which couples energy from the ion motive force to torque generation. Recent structural work revealed that stator complex consists of a ring of five MotA subunits which rotate around a central dimer of MotB subunits. Transmembrane (TM) domains TM3 and TM4 from MotA combine with the single TM domain from MotB to form two separate ion channels within this complex. Much is known about the ion binding site and ion specificity however, to date, no modelling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modelled the central MotB dimer using coiled-coil engineering and modelling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found 3 stable coiled-coil states with dimer interface angles of 28°, 56° and 64°. We tested the effect of strategic mutagenesis on the comparative energy of the states and correlated motility with a specific hierarchy of stability between the three states. In general, our results indicate agreement with existing models describing a 36° rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modelling.
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2022
DOI: 10.1101/2022.05.10.491423
Abstract: Lentigo maligna (LM), a form of melanoma in situ that predominantly affects sun-exposed areas such as the face, has an ill-defined clinical border and has a high rate of recurrence. Atypical Intraepidermal Melanocytic Proliferation (AIMP) is a term used to describe the melanocytic proliferation of an uncertain malignant potential. Clinically and histologically, AIMP can be difficult to distinguish from LM, and indeed AIMP may in some cases progress to LM. Reflectance Confocal Microscopy (RCM) is often used to investigate these lesions non-invasively, however, RCM is often not readily available nor is the associated expertise for RCM image interpretation. Here, we demonstrate machine learning architectures that can correctly classify lesions between LM and AIMP on stacks of RCM images. Overall, our methods showcase the potential for computer-aided diagnosis in dermatology, which in conjunction with the remote acquisition, can expand the range of diagnostic tools in the community.
Publisher: Proceedings of the National Academy of Sciences
Date: 03-09-2013
Abstract: The twin-arginine translocation (Tat) pathway transports folded proteins across a membrane without significant ion leakage. The mechanism by which Tat is able to carry out this challenging feat is unclear. We used direct imaging of fluorescent protein-tagged Tat components in bacterial cells to show that the TatA element of the Tat system undergoes substrate- and proton motive force-dependent oligomerization. Thus the Tat transporter element is assembled on demand, avoiding the need to seal the transporter between translocation events.
Publisher: MDPI AG
Date: 11-12-2018
DOI: 10.3390/GENES9120621
Abstract: Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.
Publisher: Cold Spring Harbor Laboratory
Date: 30-12-2018
DOI: 10.1101/508176
Abstract: Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis ( Xerocytosis ) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.
Publisher: Elsevier BV
Date: 04-2014
Publisher: Oxford University Press (OUP)
Date: 17-12-2016
DOI: 10.1093/NAR/GKV1466
Publisher: Springer Science and Business Media LLC
Date: 30-01-2011
DOI: 10.1007/S00249-010-0667-Y
Abstract: Modern single-molecule biophysical experiments require high numerical aperture oil-immersion objectives in close contact with the s le. We introduce two methods of high numerical aperture temperature control which can be implemented on any microscope: objective temperature control using a ring-shaped Peltier device, and stage temperature control using a fluid flow cooling chip in close thermal contact with the s le. We demonstrate the efficacy of each system by showing the change in speed with temperature of two molecular motors, the bacterial flagellar motor and skeletal muscle myosin.
Publisher: eLife Sciences Publications, Ltd
Date: 03-12-2016
DOI: 10.7554/ELIFE.20718
Abstract: The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9NR00472F
Abstract: DNA qPAINT can be used to quantify the stoichiometry of dense arrays of molecules separated by as little as 3 nm.
Publisher: Springer Science and Business Media LLC
Date: 23-01-2019
Publisher: The Royal Society
Date: 12-2019
DOI: 10.1098/RSOS.191268
Abstract: Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.
Publisher: eLife Sciences Publications, Ltd
Date: 18-01-2017
DOI: 10.7554/ELIFE.22140
Abstract: Bacterial flagella are extracellular filaments that drive swimming in bacteria. During motor assembly, flagellins are transported unfolded through the central channel in the flagellum to the growing tip. Here, we applied in vivo fluorescent imaging to monitor in real time the Vibrio alginolyticus polar flagella growth. The flagellar growth rate is found to be highly length-dependent. Initially, the flagellum grows at a constant rate (50 nm/min) when shorter than 1500 nm. The growth rate decays sharply when the flagellum grows longer, which decreases to ~9 nm/min at 7500 nm. We modeled flagellin transport inside the channel as a one-dimensional diffusive process with an injection force at its base. When the flagellum is short, its growth rate is determined by the loading speed at the base. Only when the flagellum grows longer does diffusion of flagellin become the rate-limiting step, dramatically reducing the growth rate. Our results shed new light on the dynamic building process of this complex extracellular structure.
Publisher: Cold Spring Harbor Laboratory
Date: 13-04-2021
DOI: 10.1101/2021.04.13.439105
Abstract: The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H + ) and sodium ions (Na + ). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na + motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H + motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na + and H + stator types, and in pathogenic and non-pathogenic strains. Here we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 μM across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus . Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.
Publisher: Informa UK Limited
Date: 11-2009
Publisher: American Association for the Advancement of Science (AAAS)
Date: 25-11-2022
Abstract: Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants that rescue function. The bacterial flagellar motor (BFM) is an excellent candidate—an ancient and highly conserved molecular complex for bacterial propulsion toward favorable environments. Motor rotation is often powered by H + or Na + ion transit through the torque-generating stator subunit of the motor complex, and ion selectivity has adapted over evolutionary time scales. Here, we used CRISPR engineering to replace the native Escherichia coli H + -powered stator with Na + -powered stator genes and report the spontaneous reversion of our edit in a low-sodium environment. We followed the evolution of the stators during their reversion to H + -powered motility and used both whole-genome and RNA sequencing to identify genes involved in the cell’s adaptation. Our transplant of an unfit protein and the cells’ rapid response to this edit demonstrate the adaptability of the stator subunit and highlight the hierarchical modularity of the flagellar motor.
Publisher: Oxford University Press (OUP)
Date: 12-11-2021
DOI: 10.1093/NAR/GKAB1163
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2AN16261J
Abstract: Carbon black (CB) nanoparticles modified with fluorescein, a highly fluorescent molecule, were prepared using a facile and efficient methodology. Simply stirring CB in aqueous solution containing fluorescein resulted in the strong physisorption of fluorescein onto the CB surface. The resulting Fluorescein/CB was then characterised by means of X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), fluorescence microscopy and fluorescence spectroscopy. The optimum experimental conditions for fluorescence of Fluorescein/CB viz. fluorescence excitation and emission wavelengths, O(2) removal and the amount of Fluorescein/CB used, were investigated. The Fluorescein/CB was used as a fluorescent probe for the sensitive detection of Pd(II) in water, based on fluorescence quenching. The results demonstrated that the fluorescence intensity of Fluorescein/CB decreased with increasing Pd(II) concentration, and the fluorescence quenching process could be described by the Stern-Volmer equation. The limit of detection (LOD) for the fluorescence quenching of Fluorescein/CB by Pd(II) in aqueous solution was found to be 1.07 μM (based on 3σ). Last, approaches were studied for the removal of Fe(III) which interferes with the fluorescence quenching of Fluorescein/CB. Complexation of Fe(III) with salicylic acid was used to enhance and control the selectivity of Fluorescein/CB sensor towards Pd(II) in the presence of Fe(III).
Publisher: MDPI AG
Date: 04-11-2021
DOI: 10.3390/MEMBRANES11110857
Abstract: Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.
Publisher: MDPI AG
Date: 30-11-2021
DOI: 10.3390/MEMBRANES11120950
Abstract: DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with erse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but s les re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics.
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2019
DOI: 10.1101/525345
Abstract: Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.
Publisher: Springer International Publishing
Date: 2016
DOI: 10.1007/978-3-319-32189-9_14
Abstract: Motor proteins are molecules which convert chemical energy to mechanical work and are responsible for motility across all levels: for transport within a cell, for the motion of an in idual cell in its surroundings, and for movement in multicellular aggregates, such as muscles. The bacterial flagellar motor is one of the canonical ex les of a molecular complex made from several motor proteins, which self-assembles on demand and provides the locomotive force for bacteria. This locomotion provides a key aspect of bacteria's prevalence. Here, we outline the biophysics behind the assembly, the energetics, the switching and the rotation of this remarkable nanoscale electric motor that is Nature's first wheel.
Publisher: American Society for Microbiology
Date: 25-10-2021
DOI: 10.1128/JB.00367-21
Abstract: Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 μM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus .
Publisher: Wiley
Date: 16-05-2021
Abstract: We report evidence further supporting homology between proteins in the F 1 F O ‐ATP synthetase and the bacterial flagellar motor (BFM). BFM proteins FliH, FliI, and FliJ have been hypothesized to be homologous to F O ‐b + F 1 ‐δ, F 1 ‐α/β, and F 1 ‐γ, with similar structure and interactions. We conduct a further test by constructing a gene order dataset, examining the order of fliH , fliI , and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar type 3 secretion systems, and comparing this to published surveys of gene order in the F 1 F O ‐ATP synthetase, its N‐ATPase relatives, and the bacterial/archaeal V‐ and A‐type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F‐ATPase gene order atpFHAG , coding for subunits b‐δ‐α‐γ. The V/A‐type ATPases have a similar conserved gene order. Our results confirm homology between these systems, and suggest a rare case of synteny conserved over billions of years, predating the Last Universal Common Ancestor (LUCA).
Publisher: Springer Science and Business Media LLC
Date: 04-09-2019
Publisher: Cold Spring Harbor Laboratory
Date: 27-01-2021
DOI: 10.1101/2021.01.26.427765
Abstract: Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants which rescue function. The Bacterial Flagellar Motor (BFM) is an excellent candidate – an ancient and highly conserved molecular complex for propulsion which navigates bacteria towards favourable environments. In most species, rotation is powered by H + or Na + ion transit through the torque-generating stator subunit of the motor complex. The ion that drives the rotor has changed over evolutionary timescales but the molecular basis of this selectivity remains unknown. Here we used CRISPR engineering to replace the native Escherichia coli H + -powered stator with Na + -powered stator genes and report the rapid and spontaneous reversion of our edit in a low sodium environment. We followed the evolution of the stators during their reversion to H + -powered motility and used whole genome and transcriptome sequencing to identify both flagellar- and non-flagellar-associated genes involved in the cell’s adaptation. Our transplant of an unfit protein and the cells’ rapid response to this edit demonstrates the adaptability of the stator subunit and highlights the hierarchical modularity of the flagellar motor.
Publisher: Wiley
Date: 23-10-2023
DOI: 10.1002/PRO.4811
Publisher: Cold Spring Harbor Laboratory
Date: 14-01-2022
DOI: 10.1101/2022.01.13.476178
Abstract: Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins alpha-hemolysin and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ . In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.
Publisher: Springer Science and Business Media LLC
Date: 27-03-2017
DOI: 10.1038/SREP45180
Abstract: The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli , (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.
Publisher: Elsevier BV
Date: 03-2016
Publisher: MDPI AG
Date: 23-02-2023
Abstract: Lentigo maligna (LM) is an early form of pre-invasive melanoma that predominantly affects sun-exposed areas such as the face. LM is highly treatable when identified early but has an ill-defined clinical border and a high rate of recurrence. Atypical intraepidermal melanocytic proliferation (AIMP), also known as atypical melanocytic hyperplasia (AMH), is a histological description that indicates melanocytic proliferation with uncertain malignant potential. Clinically and histologically, AIMP can be difficult to distinguish from LM, and indeed AIMP may, in some cases, progress to LM. The early diagnosis and distinction of LM from AIMP are important since LM requires a definitive treatment. Reflectance confocal microscopy (RCM) is an imaging technique often used to investigate these lesions non-invasively, without biopsy. However, RCM equipment is often not readily available, nor is the associated expertise for RCM image interpretation easy to find. Here, we implemented a machine learning classifier using popular convolutional neural network (CNN) architectures and demonstrated that it could correctly classify lesions between LM and AIMP on biopsy-confirmed RCM image stacks. We identified local z-projection (LZP) as a recent fast approach for projecting a 3D image into 2D while preserving information and achieved high-accuracy machine classification with minimal computational requirements.
Publisher: American Society for Microbiology
Date: 15-01-2020
DOI: 10.1128/JB.00557-19
Abstract: The bacterial flagellar motor is driven by an ion flux that is converted to torque by motor-attendant complexes known as stators. The dynamics of stator assembly around the motor in response to external stimuli have been the subject of much recent research, but less is known about the evolutionary origins of stator complexes and how they select for specific ions.
Publisher: Cold Spring Harbor Laboratory
Date: 02-01-2021
DOI: 10.1101/2021.01.01.425057
Abstract: Evidence of homology between proteins in the ATP synthetase and the bacterial flagellar motor (BFM) has been accumulating since the 1980s. Specifically, the BFM’s Type 3 Secretion System (T3SS) export apparatus FliH, FliI, and FliJ are considered homologous to F O -b + F 1 -δ, F 1 -α/β, and F 1 -γ, and have similar structure and interactions. We review the discoveries that advanced the homology hypothesis and then conduct a further test by examining gene order in the two systems and their relatives. Conservation of gene order, or synteny, is often observed between closely related prokaryote species, but usually degrades with phylogenetic distance. As a result, observed conservation of synteny over vast phylogenetic distances can be evidence of shared ancestral coexpression, interaction, and function. We constructed a gene order dataset by examining the order of fliH , fliI , and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar T3SS. We compared this to published surveys of gene order in the F 1 F O -ATP synthetase, its N-ATPase relatives, and the bacterial/archaeal V- and A-type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F-ATPase gene order atpFHAG , coding for subunits b - δ - α - γ . The V/A-type ATPases have a similar conserved gene order shared for homologous components. Our results further strengthen the argument for homology between these systems, and suggest a rare case of synteny conserved over billions of years, dating back to well before the Last Universal Common Ancestor (LUCA).
Publisher: Cold Spring Harbor Laboratory
Date: 18-10-2022
DOI: 10.1101/2022.10.17.512626
Abstract: The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H + powered motors), or PomA and PomB (in Na + powered motors). In this study we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed ten ancestral sequences of MotA and found four of them were motile in combination with contemporary E. coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing and MotA-MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.
Publisher: Cold Spring Harbor Laboratory
Date: 24-11-2020
DOI: 10.1101/2020.11.24.395772
Abstract: Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied ex le is the use of the sodium powered PomA-PotB chimeric stator unit in the naturally proton-powered E. coli . Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive-force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eight-fold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement selection of highly-motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.
Publisher: eLife Sciences Publications, Ltd
Date: 2016
Publisher: Cold Spring Harbor Laboratory
Date: 26-01-2023
DOI: 10.1101/2023.01.26.525760
Abstract: Microfluidics devices are gaining significant interest in biomedical applications. However, in a micron-scale device, reaction speed is often limited by the slow rate of diffusion of the reagents. Several active and passive micro-mixers have been fabricated to enhance mixing in microfluidic devices. Here, we demonstrate external control of mixing by rotating a rodshaped bacterial cell. This rotation is driven by ion transit across the bacterial flagellar stator complex. We first measured the flow fields generated by rotating a single bacterial cell rotationally locked to rotate either clockwise (CW) or counterclockwise (CCW). Micro-Particle Image Velocimetry (μPIV) and Particle Tracking Velocimetry results showed that a bacterial cell of ~ 2.75 μm long, rotating at 5.75 ± 0.39 Hz in a counterclockwise direction could generate distinct micro-vortices with circular flow fields with a mean velocity of 4.72 ± 1.67 μm/s and maximum velocity of 7.90 μm/s in aqueous solution. We verified our experimental data with a numerical simulation at matched flow conditions which revealed vortices of similar dimensions and speed. We observed that the flow-field diminished with increasing z-height above the plane of the rotating cell. Lastly, we showed we could activate and tune rotational mixing remotely using strains engineered with Proteorhodopsin (PR), where rotation could be activated by controlled external illumination using green laser light (561 nm).
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2019
End Date: 07-2022
Amount: $405,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2024
Amount: $488,914.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2019
End Date: 12-2019
Amount: $1,480,000.00
Funder: Australian Research Council
View Funded Activity