Hiroyuki Terashima

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.

Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel

Phospholipid scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca2+-gated channels have been shown to be involved in Ca2+-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus, is a dual-function protein: it is a Ca2+-gated channel, with characteristics of other TMEM16 homologues, and a Ca2+-dependent scramblase, with the expected properties of mammalian phospholipid scramblases. Remarkably, we find that a single Ca2+ site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases.

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.

Purified TMEM16A is sufficient to form Ca2+-activated Cl− channels

Significance Calcium-activated chloride channels play key roles in physiology, from mediating sensory transduction and nociception to regulating mucine secretion in airway epithelia and controlling excitability of smooth muscle fibers. Recently, TMEM16A was identified as the pore-forming subunit of these channels. It remains unclear, however, whether this protein is sufficient to form calcium-activated chloride channels, or whether association with other subunits is required for function. Recently, association with calmodulin has been proposed to be required for the calcium-dependent activation and ion selectivity of these channels. Here we show that purified and reconstituted TMEM16A is necessary and sufficient to recapitulate the properties of native and heterologously expressed calcium-activated chloride currents. Thus, association of TMEM16A with other proteins is not required for function. Ca2+-activated Cl− channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca2+ sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca2+-dependent Cl− transport with submicromolar sensitivity to Ca2+, consistent with what is seen in patch–clamp experiments. The channel is synergistically gated by Ca2+ and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca2+ dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl− transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.

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.

The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor

In Vibrio alginolyticus, the flagellar motor can rotate at a remarkably high speed, ca. three to four times faster than the Escherichia coli or Salmonella motor. Here, we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla⁻ and Mot⁻ phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of ΔflgT cells revealed a smaller LP ring structure compared to the wild type, and most of the T ring was lost. His₆-tagged FlgT could be coisolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.

Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from Vibrio alginolyticus.

Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm.

Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT

Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus, is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coli. The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FlgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FlgT at 2.0-Å resolution. FlgT is composed of three domains, the N-terminal domain (FlgT-N), the middle domain (FlgT-M), and the C-terminal domain (FlgT-C). FlgT-M is similar to the N-terminal domain of TolB, and FlgT-C resembles the N-terminal domain of FliI and the α/β subunits of F1-ATPase. To elucidate the role of each domain, we prepared domain deletion mutants of FlgT and analyzed their effects on the basal-body ring formation. The results suggest that FlgT-N contributes to the construction of the H-ring structure, and FlgT-M mediates the T-ring association on the LP ring. FlgT-C is not essential but stabilizes the H-ring structure. On the basis of these results, we propose an assembly mechanism for the basal-body rings and the stator units of the sodium-driven flagellar motor.

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.