Assembly and Dynamics of the Bacterial Flagellum.

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45-50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

Time for bacteria to slow down.

The speed of the bacterial flagellar motor is thought to be regulated by structural changes in the motor. Two new studies, Boehm et al. (2010) in this issue and Paul et al. (2010) in Molecular Cell, now show that cyclic di-GMP also regulates flagellar motor speed through interactions between the cyclic di-GMP binding protein YcgR and the motor proteins.

Motile ghosts of the halophilic archaeon, Haloferax volcanii.

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

Effect of Reaction Media on Grafting Hydrophobic Polymers from Cellulose Nanocrystals via Surface-Initiated Atom-Transfer Radical Polymerization.

Hydrophobic polymer-grafted cellulose nanocrystals (CNCs) were produced via surface-initiated atom-transfer radical polymerization (SI-ATRP) in two different solvents to examine the role of reaction media on the extent of surface modification. Poly(butyl acrylate)-grafted CNCs were synthesized in either dimethylformamide (DMF) (D-PBA-g-CNCs) or toluene (T-PBA-g-CNCs) alongside a free polymer from a sacrificial initiator. The colloidal stability of unmodified CNCs, initiator-modified CNCs, and PBA-g-CNCs in water, DMF, and toluene was evaluated by optical transmittance. The enhanced colloidal stability of initiator-modified CNCs in DMF led to improved accessibility to initiator groups during polymer grafting; D-PBA-g-CNCs had 30 times more grafted chains than T-PBA-g-CNCs, determined by thermogravimetric and elemental analysis. D-PBA-g-CNCs dispersed well in toluene and were hydrophobic with a water contact angle of 124° (for polymer grafts > 13 kDa) compared to 25° for T-PBA-g-CNCs. The cellulose crystal structure was preserved, and individual nanoparticles were retained when grafting was carried out in either solvent. This work highlights that optimizing CNC colloidal stability prior to grafting is more crucial than solvent-polymer compatibility to obtain high graft densities and highly hydrophobic CNCs via SI-ATRP.

Cellulose Nanocrystal (CNC)-Latex Nanocomposites: Effect of CNC Hydrophilicity and Charge on Rheological, Mechanical, and Adhesive Properties.

Cellulose nanocrystals (CNCs), a sustainable nanomaterial, are in situ incorporated into emulsion-based pressure-sensitive adhesives (PSAs). Commercially available CNCs with different surface hydrophilicity and surface charge (CNC101 and CNC103 from CelluForce) are used to explore their role in PSA property modification. Viscosity measurements and atomic force microscopy reveal differences in degree of association between the CNCs and the latex particles depending on the surface properties of the CNCs. The more hydrophilic and higher surface charge CNCs (CNC101) show less association with the latex particles. Dynamic strain sweep tests are used to analyze the strain-softening of the nanocomposites based on CNC type and loading. The CNC101 nanocomposites soften at lower strains than their CNC103 counterparts. This behavior is confirmed via dynamic frequency tests and modeling of the nanocomposites' storage moduli, which suggest the formation of CNC aggregates of, on average, 3.8 CNC101 and 1.3 CNC103 nanoparticles. Finally, PSA properties, i.e., tack, peel strength, and shear strength, simultaneously increase upon addition of both CNC types, although to different extents. The relationship between the PSA properties and CNC surface properties confirms that the less hydrophilic CNCs lead to improved CNC dispersion in the PSA films and therefore, enhance PSA properties.

How latex film formation and adhesion at the nanoscale correlate to performance of pressure sensitive adhesives with cellulose nanocrystals.

Emulsion polymerized latex-based pressure-sensitive adhesives (PSAs) are more environmentally benign because they are synthesized in water but often underperform compared to their solution polymerized counterparts. Studies have shown a simultaneous improvement in the tack, and peel and shear strength of various acrylic PSAs upon the addition of cellulose nanocrystals (CNCs). This work uses atomic force microscopy (AFM) to examine the role of CNCs in (i) the coalescence of hydrophobic 2-ethyl hexyl acrylate/n-butyl acrylate/methyl methacrylate (EHA/BA/MMA) latex films and (ii) as adhesion modifiers over multiple length scales. Thin films with varying solids content and CNC loading were prepared by spin coating. AFM revealed that CNCs lowered the solids content threshold for latex particle coalescence during film formation. This improved the cohesive strength of the films, which was directly reflected in the increased shear strength of the EHA/BA/MMA PSAs with increasing CNC loading. Colloidal probe AFM indicated that the nano-adhesion of thicker continuous latex films increased with CNC loading when measured over small contact areas where the effect of surface roughness was negligible. Conversely, the beneficial effects of the CNCs on macroscopic PSA tack and peel strength were outweighed by the effects of increased surface roughness with increasing CNC loading over larger surface areas. This highlights that CNCs can improve both cohesive and adhesive PSA properties; however, the effects are most pronounced when the CNCs interact favourably with the latex polymer and are uniformly dispersed throughout the adhesive film. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Speed of the bacterial flagellar motor near zero load depends on the number of stator units.

The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque-speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+-driven motors. We measure the full torque-speed relationship of one and two H+ units in Escherichia coli by selecting the number of H+ units and controlling the number of Na+ units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque-speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque-speed curves to be collapsed onto a single curve by simple rescaling.

Catch bond drives stator mechanosensitivity in the bacterial flagellar motor.

The bacterial flagellar motor (BFM) is the rotary motor that rotates each bacterial flagellum, powering the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force-powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechanosensitive, with the number of engaged units dependent on the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechanosensitivity is unknown. Here, we directly measure the kinetics of arrival and departure of the stator units in individual motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. The kinetic rates obtained, robust with respect to the details of the applied adsorption model, indicate that the lifetime of an assembled stator unit increases when a higher force is applied to its anchoring point in the cell wall. This provides strong evidence that a catch bond (a bond strengthened instead of weakened by force) drives mechanosensitivity of the flagellar motor complex. These results add the BFM to a short, but growing, list of systems demonstrating catch bonds, suggesting that this "molecular strategy" is a widespread mechanism to sense and respond to mechanical stress. We propose that force-enhanced stator adhesion allows the cell to adapt to a heterogeneous environmental viscosity and may ultimately play a role in surface-sensing during swarming and biofilm formation.

Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

Mechanics of torque generation in the bacterial flagellar motor.

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Mutations targeting the plug-domain of the Shewanella oneidensis proton-driven stator allow swimming at increased viscosity and under anaerobic conditions.

Shewanella oneidensis MR-1 possesses two different stator units to drive flagellar rotation, the Na+ -dependent PomAB stator and the H+ -driven MotAB stator, the latter possibly acquired by lateral gene transfer. Although either stator can independently drive swimming through liquid, MotAB-driven motors cannot support efficient motility in structured environments or swimming under anaerobic conditions. Using ΔpomAB cells we isolated spontaneous mutants able to move through soft agar. We show that a mutation that alters the structure of the plug domain in MotB affects motor functions and allows cells to swim through media of increased viscosity and under anaerobic conditions. The number and exchange rates of the mutant stator around the rotor were not significantly different from wild-type stators, suggesting that the number of stators engaged is not the cause of increased swimming efficiency. The swimming speeds of planktonic mutant MotAB-driven cells was reduced, and overexpression of some of these stators caused reduced growth rates, implying that mutant stators not engaged with the rotor allow some proton leakage. The results suggest that the mutations in the MotB plug domain alter the proton interactions with the stator ion channel in a way that both increases torque output and allows swimming at decreased pmf values.

Hybrid-fuel bacterial flagellar motors in Escherichia coli.

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H(+) or Na(+) ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H(+)- and Na(+)-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na(+) concentration ([Na(+)]), using speed changes of single motors driving 1-µm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na(+) and H(+) stator components expressed at high and medium levels, respectively, Na(+) stator units dominate at high [Na(+)] and are replaced by H(+) units when Na(+) is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H(+) and Na(+) stator components sharing a common rotor.

The Limiting Speed of the Bacterial Flagellar Motor.

Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating complexes (stators) was shown to vary across applied loads. This finding brings under scrutiny the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. In this study, we propose that, contrary to previous assumptions, the maximum speed of the motor increases as additional stators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence in chimeric motors, as well as with the requirement that a processive motor driving a large load via an elastic linkage must have a high duty ratio.

Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels.

Structural and Energetic Studies on the Interaction of Cationic Surfactants and Cellulose Nanocrystals.

We report a comprehensive study on the interactions between cationic surfactant homologues CnTAB (n = 12, 14, and 16) with negatively charged cellulose nanocrystals (CNCs). By combining different techniques, such as isothermal titration calorimetry (ITC), surface tension, light scattering, electrophoretic mobility, and fluorescence anisotropy measurements, we identified two different driving forces for the formation of surface induced micellar aggregates. For the C12TAB surfactant, a surfactant monolayer with the alkyl chains exposed to the water is formed via electrostatic interactions at low concentration. At a higher surfactant concentration, micellar aggregates are formed at the CNC surface. For the C14TAB and C16TAB systems, micellar aggregates are formed at the CNC surface at a much lower surfactant concentration via electrostatic interactions, followed by hydrophobic interactions between the alkyl chains. At higher surfactant concentration, charge neutralization and association of the surfactant decorated CNC aggregates led to flocculation.

Stimuli-Responsive Cellulose Nanocrystals for Surfactant-Free Oil Harvesting.

Cellulose nanocrystals with grafted binary polymer brushes (CNC-BPB), poly(oligoethylene glycol) methacrylate (POEGMA) and poly(methacrylic acid) (PMAA), were prepared by cerium-mediated polymerization in aqueous solution. The physical properties of CNC-BPB can be controlled by external triggers, such as temperature and pH, which can be utilized to stabilize and destabilize oil-water emulsions. By virtue of the modifications, these bifunctionalized CNCs diffused to the oil-water interface and stabilized the oil droplets at high pHs. When the pH was lowered to 2, strong hydrogen bonding between POEGMA and PMAA chains grafted on the CNC induced the coalescence of the emulsion droplets, resulting in the phase separation of oil and water. For emulsions stabilized by CNC-POEGMA and free PMAA mixtures, instantaneous coalescence was not observed at low pHs. Successive stabilization-destabilization over 5 cycles was demonstrated by modulating the pH with the addition of acid or base without any loss in efficiency. This work demonstrates that functional sustainable nanomaterials can be used for small scale oil-water separations, particularly for oil droplet transportation and harvesting of lipophilic compounds.

Mechanism and kinetics of a sodium-driven bacterial flagellar motor.

The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration.

Modified cellulose nanocrystal for vitamin C delivery.

Cellulose nanocrystal grafted with chitosan oligosaccharide (CNC-CSOS) was used to encapsulate vitamin C and prepare CNCS/VC complexes using tripolyphosphte via ionic complexation. The stability of vitamin C and the antioxidant activity of the CNCS/VC complexes were elucidated. The formation of the complex was confirmed using DSC and UV-vis spectrophotometry, and TEM was used to study the morphology of the complexes. The encapsulation efficiency of vitamin C at pH 3 and 5 was 71.6% ± 6.8 and 91.0 ± 1.0, respectively. Strong exothermic peaks observed in isothermal titration calorimetric (ITC) studies at pH 5 could be attributed to additional electrostatic interactions between CNC-CSOS and vitamin C at pH 5. The in vitro release of vitamin C from CNCS/VC complexes showed a sustained release of up to 20 days. The vitamin C released from CNCS/VC complex displayed higher stability compared with the control vitamin C solution, and this was also confirmed from the ITC thermograms. CNC-CSOS possessed a higher scavenging activity and faster antioxidant activity compared with its precursors, i.e., oxidized CNC and CSOS and their physical mixtures. Complexing vitamin C into CNC-CSOS particles yielded a dynamic antioxidant agent, where the vitamin C is released over time and displayed sustained antioxidant properties. Therefore, CNCS/VC can potentially be used in cosmeceutical applications as topical formulations.

Quantification of flagellar motor stator dynamics through in vivo proton-motive force control.

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).

Dual responsive pickering emulsion stabilized by poly[2-(dimethylamino)ethyl methacrylate] grafted cellulose nanocrystals.

A weak polyelectrolyte, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), was grafted onto the surface of cellulose nanocrystals via free radical polymerization. The resultant suspension of PDMAEMA-grafted-cellulose nanocrystals (PDMAEMA-g-CNC) possessed pH-responsive properties. The grafting was confirmed by FTIR, potentiometric titration, elementary analysis, and thermogravimetric analysis (TGA); the surface and interfacial properties of the modified particles were characterized by surface tensiometer. Compared to pristine cellulose nanocrystals, modified CNC significantly reduced the surface and interfacial tensions. Stable heptane-in-water and toluene-in-water emulsions were prepared with PDMAEMA-g-CNC. Various factors, such as polarity of solvents, concentration of particles, electrolytes, and pH, on the properties of the emulsions were investigated. Using Nile Red as a florescence probe, the stability of the emulsions as a function of pH and temperature was elucidated. It was deduced that PDMAEMA chains promoted the stability of emulsion droplets and their chain conformation varied with pH and temperature to trigger the emulsification and demulsification of oil droplets. Interestingly, for heptane system, the macroscopic colors varied depending on the pH condition, while the color of the toluene system remained the same. Reversible emulsion systems that responded to pH were observed and a thermoresponsive Pickering emulsion system was demonstrated.