Seiji Kojima

The bacterial flagellar motor: structure and function of a complex molecular machine.

The bacterial flagellar motor harnesses ion flow to drive rotary motion, at speeds reaching 100000 rpm and with apparently tight coupling. The functional properties of the motor are quite well understood, but its molecular mechanism remains unknown. Studies of motor physiology, together with mutational and biochemical studies of the components, place significant constraints on the mechanism. Rotation is probably driven by conformational changes in membrane-protein complexes that form the stator. These conformational changes occur as protons move on and off a critical aspartate residue in the stator protein MotB, and the resulting forces are applied to the rotor protein FliG. The bacterial flagellum is a complex structure built from about two dozen proteins. Its construction requires an apparatus at the base that exports many flagellar components to their sites of installation by way of an axial channel through the structure. The sequence of events in assembly is understood in general terms, but not yet at the molecular level. A fuller understanding of motor rotation and flagellar assembly will require more data on the structures and organization of the constituent proteins.

FLAGELLAR MOTILITY IN BACTERIA : STRUCTURE AND FUNCTION OF FLAGELLAR MOTOR

Bacterial flagella are filamentous organelles that drive cell locomotion. They thrust cells in liquids (swimming) or on surfaces (swarming) so that cells can move toward favorable environments. At the base of each flagellum, a reversible rotary motor, which is powered by the proton- or the sodium-motive force, is embedded in the cell envelope. The motor consists of two parts: the rotating part, or rotor, that is connected to the hook and the filament, and the nonrotating part, or stator, that conducts coupling ion and is responsible for energy conversion. Intensive genetic and biochemical studies of the flagellum have been conducted in Salmonella typhimurium and Escherichia coli, and more than 50 gene products are known to be involved in flagellar assembly and function. The energy-coupling mechanism, however, is still not known. In this chapter, we survey our current knowledge of the flagellar system, based mostly on studies from Salmonella, E. coli, and marine species Vibrio alginolyticus, supplemented with distinct aspects of other bacterial species revealed by recent studies.

Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB

Torque generation in the Salmonella flagellar motor is coupled to translocation of H+ ions through the proton‐conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan‐binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C‐terminal periplasmic fragment of MotB (MotBC) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton‐conducting channel. Drastic conformational changes in the N‐terminal portion of MotBC are required both for PG binding and the proton channel activation.

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na+‐driven flagella and required for stator formation

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na+‐driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a ΔmotX or ΔmotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.

Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors.

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H+ or Na+. The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na+ motor of Vibrio alginolyticus, is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein‐fused stator proteins. D24 of PomB is the predicted Na+‐binding site. Furthermore, we demonstrated that the coupling ion, Na+, is required for stator assembly and that phenamil (an inhibitor of the Na+‐driven motor) inhibited the assembly. Carbonyl cyanide m‐chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na+ influx through the channel, including Na+ binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.

The Effects of Cell Sizes, Environmental Conditions, and Growth Phases on the Strength of Individual W303 Yeast Cells Inside ESEM

We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM)-nanomanipulator system. Compression experiments to penetrate the cell walls of single cells of different cell sizes (about 3-6 mu m diameter), environmental conditions (600 Pa and 3 mPa), and growth phases (early log, mid log, late log and saturation) were conducted. The compression experiments were performed inside ESEM, embedded with a 7 DOF nanomanipulator with a sharp pyramidal end effector and a cooling stage, i.e., a temperature controller. ESEM itself can control the chamber pressure. Data clearly show an increment in penetration force, i.e., 96plusmn2, 124 plusmn10, 163plusmn1, and 234plusmn14 nN at 3, 4, 5, and 6 mu m cell diameters, respectively. Whereas, 20-fold increase in penetration forces was recorded at different environmental conditions for 5 mu m cell diameter, i.e., 163plusmn1 nN and 2.95plusmn0.23 mu N at 600 Pa (ESEM mode) and 3 mPa (HV mode), respectively. This was further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e., ESEM mode (3.31plusmn0.11 MPa) and HV mode (26.02plusmn3.66 MPa) for 5 mu m cell diameter. Finally, the penetration forces at different cell growth phases also show the increment pattern from log (early, mid, and late) to saturation phases, i.e., 161plusmn 25, 216plusmn15, 255 plusmn21, and 408plusmn41 nN, respectively.

Sodium‐driven motor of the polar flagellum in marine bacteria Vibrio

The Na+‐driven bacterial flagellar motor is a molecular machine powered by an electrochemical potential gradient of sodium ions across the cytoplasmic membrane. The marine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim in liquid. The flagellar motor contains a basal body and a stator complexes, which are composed of several proteins. PomA, PomB, MotX, and MotY are thought to be essential components of the stator that are required to generate the torque of the rotation. Several mutations have been investigated to understand the characteristics and function of the ion channel in the stator and the mechanism of its assembly around the rotor to complete the motor. In this review, we summarize recent results of the Na+‐driven motor in the polar flagellum of Vibrio.

Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus.

Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number of polar flagella is positively regulated by FlhF and negatively regulated by FlhG. We now show that DeltaflhF cells are non-flagellated as are most DeltaflhFG cells; however, some of the DeltaflhFG cells have several flagella at lateral positions. We found that FlhF-GFP was localized at the flagellated pole, and its polar localization was seen more intensely in DeltaflhFG cells. On the other hand, most of the FlhG-GFP was diffused throughout the cytoplasm, although some was localized at the pole. To investigate the FlhF-FlhG interaction, immunoprecipitation was performed by using an anti-FlhF antibody, and FlhG co-precipitated with FlhF. From these results we propose a model in which FlhF localization at the pole determines polar location and production of a flagellum, FlhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FlhG negatively regulates flagellar number in V. alginolyticus cells.

Insights into the stator assembly of the Vibrio flagellar motor from the crystal structure of MotY

Rotation of the sodium-driven polar flagellum of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX, and MotY. PomA and PomB form a sodium-ion channel in the cytoplasmic membrane that functions as a stator complex to couple sodium-ion flux with torque generation. MotX and MotY are components of the T-ring, which is located beneath the P-ring of the polar flagellar basal body and is involved in incorporation of the PomA/PomB complex into the motor. Here, we describe the determination of the crystal structure of MotY at 2.9 Å resolution. The structure shows two distinct domains: an N-terminal domain (MotY-N) and a C-terminal domain (MotY-C). MotY-N has a unique structure. MotY-C contains a putative peptidoglycan-binding motif that is remarkably similar to those of peptidoglycan-binding proteins, such as Pal and RmpM, but this region is disordered in MotY. Motility assay of cells producing either of the MotY-N and MotY-C fragments and subsequent biochemical analyses indicate that MotY-N is essential for association of the stator units around the rotor, whereas MotY-C stabilizes the association by binding to the peptidoglycan layer. Based on these observations, we propose a model for the mechanism of stator assembly around the rotor.

Nanoindentation Methods to Measure Viscoelastic Properties of Single Cells Using Sharp, Flat, and Buckling Tips Inside ESEM

In this paper, methods to measure viscoelastic properties of time-dependent materials are proposed using sharp, flat, and buckling tips inside an environmental SEM. Single W303 yeast cells were employed in this study. Each of the tips was used to indent single cells in a nanoindentation test. Three loading histories were used: 1) a ramp loading history, in which a sharp indenter was used; 2) a step loading history, in which a flat indenter was implemented; and 3) a fast unloading history, in which a buckling nanoneedle was applied. Analysis of the viscoelastic properties of single cells was performed for each of the loading histories by choosing an appropriate theory between the correspondence principle and the functional equation. Results from each of the tests show good agreement, from which strong conclusion can be drawn.