Shuichi Nakamura

Structural insight into the rotational switching mechanism of the bacterial flagellar motor.

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.

Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C‐terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP‐MotB and MotA‐mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP‐MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild‐type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA‐mCherry was observed to colocalize with GFP‐FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotAC), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotAC with FliG is required for the efficient assembly of the stators around the rotor.

Effect of intracellular pH on the torque-speed relationship of bacterial proton-driven flagellar motor.

Bacterial flagella responsible for motility are driven by rotary motors powered by the electrochemical potential difference of specific ions across the cytoplasmic membrane. The stator of proton-driven flagellar motor converts proton influx into mechanical work. However, the energy conversion mechanism remains unclear. Here, we show that the motor is sensitive to intracellular proton concentration for high-speed rotation at low load, which was considerably impaired by lowering intracellular pH, while zero-speed torque was not affected. The change in extracellular pH did not show any effect. These results suggest that a high intracellular proton concentration decreases the rate of proton translocation and therefore that of the mechanochemical reaction cycle of the motor but not the actual torque generation step within the cycle by the stator-rotor interactions.

Solution-Processed Single-Walled Carbon Nanotube Transistors with High Mobility and Large On/Off Ratio

We have examined the device characteristics of solution-processed single-walled carbon nanotube (SWNT) transistors. By using an electrical breakdown, SWNT-field-effect transistors (FETs) exhibited an on/off ratio (Ion/Ioff) of 104 and a field-effect mobility of 3.6 cm2 V-1 s-1 in air, which are comparable to those of other organic FETs. We investigated the detailed mechanism of carrier injection from electrode metals into SWNTs. From the temperature dependence of source–drain current, we evaluated the effective Schottky barrier height for holes to be 170 meV.

Flagellin Redundancy in Caulobacter crescentus and Its Implications for Flagellar Filament Assembly

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

Role of a conserved prolyl residue (Pro173) of MotA in the mechanochemical reaction cycle of the proton-driven flagellar motor of Salmonella.

The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator-rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque-speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor-stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque-speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle.

Control of injected carriers in tetracyano-p-quinodimethane encapsulated carbon nanotube transistors

We examined transistor characteristics of tetracyano-p-quinodimethane encapsulated single-walled carbon nanotubes (TCNQ@SWNTs). In device operations, a clear conversion to a p-type character was observed and the stability of carriers, previously doped into SWNTs, were simultaneously clarified. Because of an energy band shift, between the electrodes and the doped SWNTs induced by the doping, electron injection was achieved only by application of a high source-drain voltage, while holes were easily injected because of decrease in hole barrier height.

Band structure modulation by carrier doping in random-network carbon nanotube transistors

We investigated a role of a Schottky barrier (SB) in carrier doped random-network single-walled carbon nanotube field effect transistors (RN-SWNT-FETs) and the precise estimation of the SB height by a suitable combination of the gate and source-drain voltages. The SB heights were 70meV for hole and 100meV for electron in p- and n-type FETs, respectively. Furthermore, the barrier height was able to be modulated by changing the doping level, which indicates the possibility of controlling the characteristics of RN-SWNT-FETs.

Characterization of SWNT‐Thin‐Film Transistors

Characteristics of network SWNT FETs (SWNT‐TFTs) were examined. The SWNTs were dispersed in a solution of dimethylformamide in a narrow bundle structure to form non‐aligned arrays, which became channels of FETs. The network‐SWNT‐FETs produced in this solution process was found to have a mobility of 10.9 cm2/Vs, ≈ 100 times as high as those reported for other solution‐processed organic thin‐film FETs formed by solution processes, although the on/off ratio was 102. To improve the low on/off ratio, so‐called electrical breakdown was introduced. By this procedure, 1.1 cm2/Vs of mobility and 106 of the on/off ratio were simultaneously achieved.