Periplasmic Region Research Articles

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotBC2) and Vibrio alginolyticus (PomBC5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotBC2, the dimeric conformation of the PomBC5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.

Metal interactions with the transmembrane region of HupE Ni2 + transporter explain its efficiency

Efficient nickel transport is crucial for the survival and virulence of various bacteria and fungi, with Ni2+ being required for the activity of nine enzymes such as [NiFe] hydrogenase, which catalyzes the reversible oxidation of molecular hydrogen for energy production.This work focuses on a region of transmembrane domain I from the HupE nickel transporter, highly conserved in the HupE/UreJ and NiCoT permease families, analyzing its interactions with native Ni2+ and two other metal ions (Cu2+ and Zn2+), which might interfere with nickel binding. Metal coordination sites are pointed out and thermodynamic parameters are discussed in detail. Their comparison to the previously studied periplasmic metal binding region satisfies our chemical curiosity and allows to draw conclusions about HupE metal specificity. The results of this study explain one of the reasons why HupE is a medium-affinity and low-capacity transporter – its periplasmic region, 22HVGLHADGTLAGLN35, binds Ni2+ with much higher affinity than the transmembrane 36HPFSGLDH43 one, which should transport the metal inside the cell. Moreover, the specificity of the transmembrane region is similar to that of the periplasmic one and to that of the full-length HupE – Cu2+ ions are able to outcompete Ni2+.

The SecA protein deeply penetrates into the SecYEG channel during insertion, contacting most channel transmembrane helices and periplasmic regions

The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific in vivo photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to "freeze" them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during in vitro protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.

Structural analysis of variant of Helicobacter pylori MotB in its activated form, engineered as chimera of MotB and leucine zipper

Rotation of the bacterial flagellum is powered by a proton influx through the peptidoglycan (PG)-tethered stator ring MotA/B. MotA and MotB form an inner-membrane complex that does not conduct protons and does not bind to PG until it is inserted into the flagellar motor. The opening of the proton channel involves association of the plug helices in the periplasmic region of the MotB dimer into a parallel coiled coil. Here, we have characterised the structure of a soluble variant of full-length Helicobacter pylori MotB in which the plug helix was engineered to be locked in a parallel coiled coil state, mimicking the open state of the stator. Fluorescence resonance energy transfer measurements, combined with PG-binding assays and fitting of the crystal structures of MotB fragments to the small angle X-ray scattering (SAXS) data revealed that the protein’s C-terminal domain has a PG-binding-competent conformation. Molecular modelling against the SAXS data suggested that the linker in H. pylori MotB forms a subdomain between the plug and the C-terminal domain, that ‘clamps’ the coiled coil of the plug, thus stabilising the activated form of the protein. Based on these results, we present a pseudo-atomic model structure of full-length MotB in its activated form.

Braun’s Lipoprotein Facilitates OmpA Interaction with the Escherichia coli Cell Wall

Gram-negative bacteria such as Escherichia coli are protected by a complex cell envelope. The development of novel therapeutics against these bacteria necessitates a molecular level understanding of the structure-dynamics-function relationships of the various components of the cell envelope. We use atomistic MD simulations to reveal the details of covalent and noncovalent protein interactions that link the outer membrane to the aqueous periplasmic region. We show that the Braun’s lipoprotein tilts and bends, and thereby lifts the cell wall closer to the outer membrane. Both monomers and dimers of the outer membrane porin OmpA can interact with peptidoglycan in the presence of Braun’s lipoprotein, but in the absence of the latter, only dimers of OmpA show a propensity to form contacts with peptidoglycan. Our study provides a glimpse of how the molecular components of the bacterial cell envelope interact with each other to mediate cell wall attachment in E. coli.

Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli.

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three Bacillus licheniformis alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

ABSTRACTThe EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this.IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps E. coli to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.

Sinorhizobium meliloti Chemotaxis to Multiple Amino Acids Is Mediated by the Chemoreceptor McpU.

The legume symbiont Sinorhizobium meliloti is chemoattracted to compounds exuded by germinating seeds of its host alfalfa. This response is mainly mediated by the S. meliloti chemoreceptor McpU. McpU also has a prominent contribution in sensing a synthetic amino acid (aa) mixture mimicking the amounts and composition observed in seed exudate. Here, we used the hydrogel capillary assay to quantify chemotactic responses of S. meliloti to individual aa exuded by germinating alfalfa seeds and to define the role of McpU in this behavior. S. meliloti exhibited positive chemotaxis responses to all proteinogenic aa, except for aspartate, and to citrulline, cystine, gamma-aminobutyric acid, and ornithine. Wild-type responses were diverse in intensity, while a strain lacking mcpU displayed strongly diminished responses. Differential scanning fluorimetry demonstrated interaction of the purified periplasmic region of McpU (McpU-PR) with the aa, except glutamate and aspartate. We additionally tested organic acids and sugars, but there were no significant interactions with the McpU ligand-binding domain, except for citrate. Using ligand displacement, we confirmed the interaction of McpU-PR with aa representing strong and weak attractants. Our results show that S. meliloti McpU is a broad-range aa receptor mediating differential responses to individual attractants, which does not bind negatively charged aa.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

New Insights into the Regulation of Cell-Surface Signaling Activity Acquired from a Mutagenesis Screen of the Pseudomonas putida IutY Sigma/Anti-Sigma Factor.

Cell-surface signaling (CSS) is a signal transfer system that allows Gram-negative bacteria to detect environmental signals and generate a cytosolic response. These systems are composed of an outer membrane receptor that senses the inducing signal, an extracytoplasmic function sigma factor (σECF) that targets the cytosolic response by modifying gene expression and a cytoplasmic membrane anti-sigma factor that keeps the σECF in an inactive state in the absence of the signal and transduces its presence from the outer membrane to the cytosol. Although CSS systems regulate bacterial processes as crucial as stress response, iron scavenging and virulence, the exact mechanisms that drive CSS are still not completely understood. Binding of the signal to the CSS receptor is known to trigger a signaling cascade that results in the regulated proteolysis of the anti-sigma factor and the activation of the σECF in the cytosol. This study was carried out to generate new insights in the proteolytic activation of CSS σECF. We performed a random mutagenesis screen of the unique IutY protein of Pseudomonas putida, a protein that combines a cytosolic σECF domain and a periplasmic anti-sigma factor domain in a single polypeptide. In response to the presence of an iron carrier, the siderophore aerobactin, in the extracellular medium, IutY is processed by two different proteases, Prc and RseP, which results in the release and activation of the σIutY domain. Our experiments show that all IutY mutant proteins that contain periplasmic residues depend on RseP for activation. In contrast, Prc is only required for mutant variants with a periplasmic domain longer than 50 amino acids, which indicates that the periplasmic region of IutY is trimmed down to ~50 amino acids creating the RseP substrate. Moreover, we have identified several conserved residues in the CSS anti-sigma factor family of which mutation leads to constitutive activation of their cognate σECF. These findings advance our knowledge on how CSS activity is regulated by the consecutive action of two proteases. Elucidation of the exact mechanism behind CSS activation will enable the development of strategies to block CSS in pathogenic bacteria.

Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS) is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR), detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular iron to modulate bacterial iron homeostasis.

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus.

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.

Sinorhizobium meliloti chemotaxis to quaternary ammonium compounds is mediated by the chemoreceptorMcpX.

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpXPR and McpX34-306 ) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpXPR with dissociation constants (Kd ) in the nanomolar range for choline and glycine betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant-derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding.

Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts.

The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by escaping predators. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory”plug”. Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell “ghosts” persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as “bacterial skeletons” for housing artificial chromosomes.

Computer simulations suggest direct and stable tip to tip interaction between the outer membrane channel TolC and the isolated docking domain of the multidrug RND efflux transporter AcrB

One way by which bacteria achieve antibiotics resistance is preventing drug access to its target molecule for example through an overproduction of multi-drug efflux pumps of the resistance nodulation division (RND) protein super family of which AcrAB–TolC in Escherichia coli is a prominent example. Although representing one of the best studied efflux systems, the question of how AcrB and TolC interact is still unclear as the available experimental data suggest that either both proteins interact in a tip to tip manner or do not interact at all but are instead connected by a hexamer of AcrA molecules. Addressing the question of TolC–AcrB interaction, we performed a series of 100 ns - 1 µs–molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain (AcrBDD). In 5/6 simulations we observe direct TolC–AcrBDD interaction that is only stable on the simulated time scale when both proteins engage in a tip to tip manner. At the same time we find TolC opening and closing freely on extracellular side while remaining closed at the inner periplasmic bottleneck region, suggesting that either the simulated time is too short or additional components are required to unlock TolC.

Serine suppresses the motor function of a periplasmic PomB mutation in the Vibrio flagella stator.

The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross-linking experiments of double-Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double-Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)-biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che(-) mutants, in either a CCW-locked or clockwise (CW)-locked strain. In contrast, in a ΔcheY (CCW-locked) strain, Vibrio (L160C/I186C) required serine to be rescued. We speculate that CheY affects stator conformation and motility restoration by serine is independent on the chemotaxis signaling pathway.

Structural modeling of the flagellum MS ring protein FliF reveals similarities to the type III secretion system and sporulation complex.

The flagellum is a large proteinaceous organelle found at the surface of many bacteria, whose primary role is to allow motility through the rotation of a long extracellular filament. It is an essential virulence factor in many pathogenic species, and is also a priming component in the formation of antibiotic-resistant biofilms. The flagellum consists of the export apparatus on the cytosolic side; the basal body and rotor, spanning the bacterial membrane(s) and periplasm; and the hook-filament, that protrudes away from the bacterial surface. Formation of the basal body MS ring region, constituted of multiple copies of the protein FliF, is one of the initial steps of flagellum assembly. However, the precise architecture of FliF is poorly understood. Here, I report a bioinformatics analysis of the FliF sequence from various bacterial species, suggesting that its periplasmic region is composed of three globular domains. The first two are homologous to that of the type III secretion system injectisome proteins SctJ, and the third possesses a similar fold to that of the sporulation complex component SpoIIIAG. I also describe that Chlamydia possesses an unusual FliF protein, lacking part of the SctJ homology domain and the SpoIIIAG-like domain, and fused to the rotor component FliG at its C-terminus. Finally, I have combined the sequence analysis of FliF with the EM map of the MS ring, to propose the first atomic model for the FliF oligomer, suggesting that FliF is structurally akin to a fusion of the two injectisome components SctJ and SctD. These results further define the relationship between the flagellum, injectisome and sporulation complex, and will facilitate future structural characterization of the flagellum basal body.

Sodium-Driven Energy Conversion for Flagellar Rotation of the Earliest Divergent Hyperthermophilic Bacterium

Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here, we cultured this hyperthermophilic bacterium and observed motile behavior with a variable–temperature chamber for optical microscopy [1, 2]. The swimming of the cells was about 90 µm s−1 at 85 °C, and decreased with decreasing temperature of the chamber [3]. Next, we expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.[1] Nishiyama, M. and Sowa Y. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J. 102, 1872-1880.[2] Nishiyama, M. et al. 2013. High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor. J. Bactetiol. 195, 1809-1814.[3] Takekawa, N. et al. 2015. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium. Sci. Rep. 5, 12711.

Studies on the mechanism of bacterial flagellar rotation and the flagellar number regulation.

Many motile bacteria have the motility organ, the flagellum. It rotates by the rotary motor driven by the ion-motive force and is embedded in the cell surface at the base of each flagellar filament. Many researchers have been studying its rotary mechanism for years, but most of the energy conversion processes have been remained in mystery. We focused on the flagellar stator, which works at the core process of energy conversion, and found that the periplasmic region of the stator changes its conformation to be activated only when the stator units are incorporated into the motor and anchored at the cell wall. Meanwhile, the physiologically important supramolecular complex is localized in the cell at the right place and the right time with a proper amount. How the cell achieves such a proper localization is the fundamental question for life science, and we undertake this problem by analyzing the mechanism for biogenesis of a single polar flagellum of Vibrio alginolyticus. Here I describe the molecular mechanism of how the flagellum is generated at the specific place with a proper number, and also how the flagellar stator is incorporated into the motor to complete the functional motor assembly, based on our studies.

Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.