Changes in the expression of mexB, mexY, and oprD in clinical Pseudomonas aeruginosa isolates.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-ß-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-ß-lactamase (MBL) and 6'-N-aminoglycoside acetyltransferase [AAC(6')-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6')-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.

Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments.

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant ßE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

The 3 × 120° rotary mechanism of Paracoccus denitrificans F1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases.

The rotation of Paracoccus denitrificans F1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below Km, PdF1 showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1 executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1 molecules suggested that PdF1 executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1 is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase from Thermus thermophilus This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1 Comprehensive analysis of rotary catalysis of F1 from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1 and Fo domains of F-ATP synthase. F1 motors with more distinctive steps are coupled with proton-conducting Fo rings with fewer proteolipid subunits, giving insight into the design principle the F1Fo of ATP synthase.

Supramolecular Mechanosensitive Potassium Channel Formed by Fluorinated Amphiphilic Cyclophane.

Inspired by mechanosensitive potassium channels found in nature, we developed a fluorinated amphiphilic cyclophane composed of fluorinated rigid aromatic units connected via flexible hydrophilic octa(ethylene glycol) chains. Microscopic and emission spectroscopic studies revealed that the cyclophane could be incorporated into the hydrophobic layer of the lipid bilayer membranes and self-assembled to form a supramolecular transmembrane ion channel. Current recording measurements using cyclophane-containing planer lipid bilayer membranes successfully demonstrated an efficient transmembrane ion transport. We also demonstrated that the ion transport property was sensitive to the mechanical forces applied to the membranes. In addition, ion transport assays using pH-sensitive fluorescence dye revealed that the supramolecular channel possesses potassium ion selectivity. We also performed all-atom hybrid quantum-mechanical/molecular mechanical simulations to assess the channel structures at atomic resolution and the mechanism of selective potassium ion transport. This research demonstrated the first example of a synthetic mechanosensitive potassium channel, which would open a new door to sensing and manipulating biologically important processes and purification of key materials in industries.

Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

Synthetic Ion Channel Formed by Multiblock Amphiphile with Anisotropic Dual-Stimuli-Responsiveness.

Transmembrane proteins within biological membranes exhibit varieties of important functions that are vital for many cellular activities, and the development of their synthetic mimetics allows for deep understanding in related biological events. Inspired by the structures and functions of natural ion channels that can respond to multiple stimuli in an anisotropic manner, we developed multiblock amphiphile VF in this study. When VF was incorporated into the lipid bilayer membranes, VF formed a supramolecular ion channel whose ion transport property was controllable by the polarity and amplitude of the applied voltage. Microscopic emission spectroscopy revealed that VF changed its molecular conformation in response to the applied voltage. Furthermore, the ion transport property of VF could be reversibly switched by the addition of (R)-propranolol, an aromatic amine known as an antiarrhythmic agent, followed by the addition of ß-cyclodextrin for its removal. The highly regulated orientation of VF allowed for an anisotropic dual-stimuli-responsiveness for the first time as a synthetic ion channel.

Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device.

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multiconditional and multiparametric assays, hindering in-depth understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a digital bioassay platform with easy solution exchanges, called multidimensional (MD) digital bioassay. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. With multiconditional assays based on our system, we could quantitatively determine inhibitor sensitivities of single influenza A virus particles and single alkaline phosphatase (ALP) molecules, which has never been achieved with conventional digital bioassays. Further, we demonstrated that ALPs from two origins can be precisely distinguished by a single-molecule multiparametric assay with our system, which was also difficult with conventional digital bioassays. Thus, MD digital bioassay is a versatile platform to gain in-depth insight into biological entities in unprecedented resolution.

Multiparameter single-particle motion analysis for homogeneous digital immunoassay.

Digital homogeneous non-enzymatic immunosorbent assay (digital Ho-Non ELISA) is a new class of digital immunoassay that enables highly sensitive quantification of biomolecules using a simple protocol. In digital Ho-Non ELISA, nanoparticles are tethered onto the surface of femtoliter reactors via captured target molecules. The tethered particles capturing target molecules are identified as those showing a confined Brownian motion with root-mean-square displacement (RMSD) values in a defined range. The present work aims to improve the specificity to discriminate tethered particles via single-target molecules from non-specifically immobilized particles by analyzing two nanoparticle parameters. First, in order to suppress the broadening of RMSD due to the heterogeneity of bead size, we corrected the RMSD with the fluorescence intensity of the beads. Second, focusing on the shape of Brownian motion in the x-y trajectory, we classified motion patterns by aspect ratio of the trajectory. By using multiparameter single-particle motion analysis with corrected RMSD and aspect ratio, a 3.9-fold enhanced sensitivity in PSA assay was achieved compared to the conventional RMSD analysis approach. This new strategy would increase the potential of digital immunoassays.

Single-molecule analysis of phospholipid scrambling by TMEM16F.

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.

Use of Ghost Cytometry to Differentiate Cells with Similar Gross Morphologic Characteristics.

Imaging flow cytometry shows significant potential for increasing our understanding of heterogeneous and complex life systems and is useful for biomedical applications. Ghost cytometry is a recently proposed approach for directly analyzing compressively measured signals of cells, thereby relieving a computational bottleneck for real-time data analysis in high-throughput imaging cytometry. In our previous work, we demonstrated that this image-free approach could distinguish cells from two cell lines prepared with the same fluorescence staining method. However, the demonstration using different cell lines could not exclude the possibility that classification was based on non-morphological factors such as the speed of cells in flow, which could be encoded in the compressed signals. In this study, we show that GC can classify cells from the same cell line but with different fluorescence distributions in space, supporting the strength of our image-free approach for accurate morphological cell analysis. © 2020 International Society for Advancement of Cytometry.

Revealing the Metabolic Activity of Persisters in Mycobacteria by Single-Cell D2O Raman Imaging Spectroscopy.

The metabolic activity of bacterial cells largely differentiates even within a clonal population. Such metabolic divergence among cells is thought to play an important role for phenotypic adaptation to ever-changing environmental conditions, such as antibiotic persistence. It has long been thought that persisters are in a state called dormancy, in which cells are metabolically inactive and do not grow. However, recent studies suggest that some types of persisters are not necessarily dormant, triggering a debate about the mechanisms of persisters. Here, we combined single-cell Raman imaging spectroscopy and D2O labeling to analyze metabolic activities of bacterial persister cells. Metabolically active cells uptake deuterium through metabolic processes and give distinct C-D Raman bands, which are direct indicators of metabolic activity. Using this imaging method, we characterized the metabolic activity of Mycobacterium smegmatis, a fast-growing model for Mycobacterium tuberculosis. We found that persister cells of M. smegmatis show certain metabolic activity and active cell growth in the presence of the antibiotic rifampicin. Interestingly, persistence is not correlated with growth rate prior to antibiotic exposure. These results show that dormancy is not responsible for the persistence of M. smegmatis cells against rifampicin, suggesting that the mechanism of persistence largely varies depending on the type of antibiotics and bacteria. Our results successfully demonstrate the potential of our perfusion-based single-cell D2O Raman imaging system for the analysis of the metabolic activity and growth of bacterial persister cells.

Rotation of artificial rotor axles in rotary molecular motors.

F1- and V1-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F1 or of V1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F1 or V1 These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F1 and V1 The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F1-FliJ chimera generates only 10% of WT F1, the V1-FliJ chimera generates torque comparable to that of V1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F1 This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F1.

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase.

F1-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F1 exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F1-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.

Automatic Quantitative Segmentation of Myotubes Reveals Single-cell Dynamics of S6 Kinase Activation.

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.

Direct observation of intermediate states during the stepping motion of kinesin-1.

The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.

Digital enzyme assay using attoliter droplet array.

Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed 'nanocell'. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using ß-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicability of digital bioassay for enzymes under non-optimum conditions or enzymes of low turnover rate. The sealing of the reactor with air would also expand the applicability, allowing the use of fluorescent dyes that leak into oil.

Effects of non-equilibrium angle fluctuation on F1-ATPase kinetics induced by temperature increase.

F1-ATPase (F1) is an efficient rotary protein motor, whose reactivity is modulated by the rotary angle to utilize thermal fluctuation. In order to elucidate how its kinetics are affected by the change in the fluctuation, we have extended the reaction-diffusion formalism [R. Watanabe et al., Biophys. J., 2013, 105, 2385] applicable to a wider range of temperatures based on experimental data analysis of F1 derived from thermophilic Bacillus under high ATP concentration conditions. Our simulation shows that the rotary angle distribution manifests a stronger non-equilibrium feature as the temperature increases, because ATP hydrolysis and Pi release are more accelerated compared with the timescale of rotary angle relaxation. This effect causes the rate coefficient obtained from dwell time fitting to deviate from the Arrhenius relation in Pi release, which has been assumed in the previous activation thermodynamic quantities estimation using linear Arrhenius fitting. Larger negative correlation is also found between hydrolysis and Pi release waiting time in a catalytic dwell with the increase in temperature. This loss of independence between the two successive reactions at the catalytic dwell sheds doubt on the conventional dwell time fitting to obtain rate coefficients with a double exponential function at temperatures higher than 65 °C, which is close to the physiological temperature of the thermophilic Bacillus.

Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 µm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M µm and 8.1 × 10-10 M µm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.