Tuning the stator subunit of the flagellar motor with coiled-coil engineering.

Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA5 MotB2 , an heptameric 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 modeling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modeled the central MotB dimer using coiled-coil engineering and modeling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found three 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 modeling.

Microstructure characterization and mechanistic insight into polyether polyols and their associated polyurethanes.

This article reviews the analytical tool chest used for characterizing alkoxylates and their associated copolymer mixtures. Specific emphasis will be placed upon the use of mass spectrometry-based techniques as rapid characterization tools for optimizing reaction processes in an industrial R&D setting. An initial tutorial will cover the use of matrix-assisted laser desorption/ionization-mass spectrometry and tandem mass spectrometry fragmentation for detailed component analysis (e.g., polyol and isocyanate) of a model polyurethane-based foam. Next, this critical feedback information will be used with the guidance of mass spectrometry to initiate the development of a new, more efficient, tris(pentafluorophenyl)borane (FAB) catalyst-based alkoxylation process for generating the next generation of glycerin-initiated poly(propylene oxide)-co-poly(ethylene oxide) copolymers. Examples will be provided for each step in the FAB-based optimization process that were required to generate the final product. Following this example, two-dimensional liquid chromatography, supercritical fluid chromatography, and ion mobility separations, along with their coupling to mass spectrometry, will be reviewed for their efficiency in characterizing and quantitating the components within these complex polyether polyol mixtures.

Ion-Powered Rotary Motors: Where Did They Come from and Where They Are Going?

Molecular motors are found in many living organisms. One such molecular machine, the ion-powered rotary motor (IRM), requires the movement of ions across a membrane against a concentration gradient to drive rotational movement. The bacterial flagellar motor (BFM) is an example of an IRM which relies on ion movement through the stator proteins to generate the rotation of the flagella. There are many ions which can be used by the BFM stators to power motility and different ions can be used by a single bacterium expressing multiple stator variants. The use of ancestral sequence reconstruction (ASR) and functional analysis of reconstructed stators shows promise for understanding how these proteins evolved and when the divergence in ion use may have occurred. In this review, we discuss extant BFM stators and the ions that power them as well as recent examples of the use of ASR to study ion-channel selectivity and how this might be applied to further study of the BFM stator complex.

Microbial stir bars: Light-activated rotation of tethered bacterial cells to enhance mixing in stagnant fluids.

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).

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility.

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.

The rapid evolution of flagellar ion selectivity in experimental populations of E. coli.

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.

Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures.

DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse 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 samples 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.

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers.

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.

Binding of DNA origami to lipids: maximizing yield and switching via strand displacement.

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.

Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics.

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 example 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.

Flagellar export apparatus and ATP synthetase: Homology evidenced by synteny predating the Last Universal Common Ancestor.

We report evidence further supporting homology between proteins in the F1 FO -ATP synthetase and the bacterial flagellar motor (BFM). BFM proteins FliH, FliI, and FliJ have been hypothesized to be homologous to FO -b + F1 -δ, F1 -α/ß, and F1 -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 F1 FO -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).

Corrigendum: Ancestral Sequence Reconstructions of MotB Are Proton-Motile and Require MotA for Motility.

[This corrects the article DOI: 10.3389/fmicb.2020.625837.].

Microfluidic techniques for separation of bacterial cells via taxis.

The microbial environment is typically within a fluid and the key processes happen at the microscopic scale where viscosity dominates over inertial forces. Microfluidic tools are thus well suited to study microbial motility because they offer precise control of spatial structures and are ideal for the generation of laminar fluid flows with low Reynolds numbers at microbial lengthscales. These tools have been used in combination with microscopy platforms to visualise and study various microbial taxes. These include establishing concentration and temperature gradients to influence motility via chemotaxis and thermotaxis, or controlling the surrounding microenvironment to influence rheotaxis, magnetotaxis, and phototaxis. Improvements in microfluidic technology have allowed fine separation of cells based on subtle differences in motility traits and have applications in synthetic biology, directed evolution, and applied medical microbiology.

Ancestral Sequence Reconstructions of MotB Are Proton-Motile and Require MotA for Motility.

The bacterial flagellar motor (BFM) is a nanomachine that rotates the flagellum to propel many known bacteria. The BFM is powered by ion transit across the cell membrane through the stator complex, a membrane protein. Different bacteria use various ions to run their BFM, but the majority of BFMs are powered by either proton (H+) or sodium (Na+) ions. The transmembrane (TM) domain of the B-subunit of the stator complex is crucial for ion selectivity, as it forms the ion channel in complex with TM3 and TM4 of the A-subunit. In this study, we reconstructed and engineered thirteen ancestral sequences of the stator B-subunit to evaluate the functional properties and ionic power source of the stator proteins at reconstruction nodes to evaluate the potential of ancestral sequence reconstruction (ASR) methods for stator engineering and to test specific motifs previously hypothesized to be involved in ion-selectivity. We found that all thirteen of our reconstructed ancient B-subunit proteins could assemble into functional stator complexes in combination with the contemporary Escherichia coli MotA-subunit to restore motility in stator deleted E. coli strains. The flagellar rotation of the thirteen ancestral MotBs was found to be Na+ independent which suggested that the F30/Y30 residue was not significantly correlated with sodium/proton phenotype, in contrast to what we had reported previously. Additionally, four among the thirteen reconstructed B-subunits were compatible with the A-subunit of Aquifex aeolicus and able to function in a sodium-independent manner. Overall, this work demonstrates the use of ancestral reconstruction to generate novel stators and quantify which residues are correlated with which ionic power source.

Evolution of the Stator Elements of Rotary Prokaryote Motors.

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. Here, we review the latest structural and biochemical data for the stator complexes and compare these with other ion transporters and microbial motors to examine possible evolutionary origins of the stator complex.

The Fusion of Lipid and DNA Nanotechnology.

Lipid membranes form the boundary of many biological compartments, including organelles and cells. Consisting of two leaflets of amphipathic molecules, the bilayer membrane forms an impermeable barrier to ions and small molecules. Controlled transport of molecules across lipid membranes is a fundamental biological process that is facilitated by a diverse range of membrane proteins, including ion-channels and pores. However, biological membranes and their associated proteins are challenging to experimentally characterize. These challenges have motivated recent advances in nanotechnology towards building and manipulating synthetic lipid systems. Liposomes-aqueous droplets enclosed by a bilayer membrane-can be synthesised in vitro and used as a synthetic model for the cell membrane. In DNA nanotechnology, DNA is used as programmable building material for self-assembling biocompatible nanostructures. DNA nanostructures can be functionalised with hydrophobic chemical modifications, which bind to or bridge lipid membranes. Here, we review approaches that combine techniques from lipid and DNA nanotechnology to engineer the topography, permeability, and surface interactions of membranes, and to direct the fusion and formation of liposomes. These approaches have been used to study the properties of membrane proteins, to build biosensors, and as a pathway towards assembling synthetic multicellular systems.

Photostable Helical Polyfurans.

This report describes the design and synthesis of a new class of polyfurans bearing ester side chains. The macromolecules can be synthesized using catalyst-transfer polycondensation, providing precise control over molecular weight and molecular weight distribution. Such obtained furan ester polymers are significantly more photostable than their alkyl analogues owing to the electron-withdrawing nature of the attached subunit. Most interestingly, they spontaneously fold into a compact π-stacked helix, yielding a complex multilayer cylindrical nanoparticle with a hollow, rigid, conjugated core composed of the polyfuran backbone and a soft, insulating outer layer formed by the ester side chains. The length of polymer side chains dictates the outer diameter of such nanoparticles, which for the hexyl ester groups used in the present study is equal to ∼2.3 nm. The inner cavity of the conjugated core is lined with oxygen atoms, which set its effective diameter to 0.4 nm. Furthermore, installation of bulkier, branched chiral ester side chains on the repeat unit yields structures that, upon change of solvent, can reversibly transition between an ordered chiral helical folded and disordered unfolded state.