In Vitro Flagellar Type III Protein Transport Assay Using Inverted Membrane Vesicles.

The flagellar axial proteins are transported across the cytoplasmic membrane into the central channel of the growing flagellum via the flagellar protein export apparatus, a member of the type III secretion system (T3SS). To reveal the molecular mechanism of protein transport by the T3SS, accurate measurement of protein transport under various conditions is essential. In this chapter, we describe an in vitro method for flagellar protein transport assay using inverted membrane vesicles (IMVs) prepared from Salmonella cells. This method can easily and precisely control the condition around the T3SS and be applied to other T3SSs.

Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo.

The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin.

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20 mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365 nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.

Chiral separation of catechin and epicatechin by reversed phase high-performance liquid chromatography with ß-cyclodextrin stepwise and linear gradient elution modes.

Catechin and epicatechin were enantioseparated by high-performance liquid chromatography (HPLC) with a phenyl column and aqueous mobile phases containing 0.05% (w/v) and 0.6% (w/v) of ß-cyclodextrin for catechin and epicatechin, respectively. ß-Cyclodextrin was found to be scarcely retained on a phenyl column. Consequently, it was suggested that catechin, which was eluted earlier than epicatechin, formed more stable inclusion complex with ß-cyclodextrin than epicatechin and earlier eluted enantiomers, (-)-catechin and (+)-epicatechin, formed more stable diastereomer complexes with ß-cyclodextrin than the respective enantiomers. This was confirmed by ß-cyclodextrin-modified micellar electrokinetic chromatography and Benesi-Hildebrand plots by fluorescence spectrophotometry. Effect of sugars (D-sucrose, D-glucose, and D-fructose) on the epimerization of (+)-catechin and (+)-epicatechin by heating was investigated by HPLC with a ß-cyclodextrin stepwise elution mode, in which two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used by turns. The epimerization of the two enantiomers was suppressed only when D-fructose was added. Separation of ten kinds of catechins including catechin and epicatechin enantiomers was investigated by a ß-cyclodextrin linear gradient HPLC elution mode without using organic solvents, where two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used with changing their ratio gradually. These catechins in a green tea infusion could be separated successfully by this method.

Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor.

The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction.

Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor.

Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four transmembrane regions and a cytoplasmic domain of approximately 100 residues, which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The 3D structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66 and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

Putative Spanner Function of the Vibrio PomB Plug Region in the Stator Rotation Model for Flagellar Motor.

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Enantioseparation of phenethylamines by using high-performance liquid chromatography column permanently coated with methylated ß-cyclodextrin.

Cyclodextrins and their derivatives have been used for chiral high-performance liquid chromatography selectors, while they are costly to use as mobile phase additives in high-performance liquid chromatography. Here, we report application of phenyl column coated permanently with methylated ß-cyclodextrin for chiral high-performance liquid chromatography. A 0.1% (v/v) phosphoric acid solution containing 1 M NaCl and 0.5% (w/v) methylated ß-cyclodextrin was subjected to a phenyl column at a flow rate of 0.5 mL/min at 30°C for 2 h. Using the precoating phenyl column, all the enantiomers of the four phenethylamines (norepinephrine, epinephrine, octopamine, and synephrine) were successfully separated simultaneously by high-performance liquid chromatography with a mobile phase without methylated ß-cyclodextrin at a flow rate of 0.2 mL/min at 30°C. The enantioseparation ability was retained for successive analyses during 1 week. It is suggested that inclusion complex of methylated ß-cyclodextrin with a phenyl group on the surface of the stationary phase could be formed and that the inclusion complex could form the ternary complex with the injected analytes. The longer retention time of (S)-enantiomers of analytes than corresponding (R)-enantiomers for high-performance liquid chromatography could be explained from the higher stability of the methylated ß-cyclodextrin complexes with (S)-enantiomers, which were confirmed by capillary electrophoresis and 1 H NMR spectroscopy experiments.

Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body.

The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor.

The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses, but have not been demonstrated biochemically. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.ImportanceThe bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo- and disulfide-crosslinking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species.

The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

Regulation of the Single Polar Flagellar Biogenesis.

Some bacterial species, such as the marine bacterium Vibrio alginolyticus, have a single polar flagellum that allows it to swim in liquid environments. Two regulators, FlhF and FlhG, function antagonistically to generate only one flagellum at the cell pole. FlhF, a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase, works as a positive regulator for flagellar biogenesis and determines the location of flagellar assembly at the pole, whereas FlhG, a MinD-type ATPase, works as a negative regulator that inhibits flagellar formation. FlhF intrinsically localizes at the cell pole, and guanosine triphosphate (GTP) binding to FlhF is critical for its polar localization and flagellation. FlhG also localizes at the cell pole via the polar landmark protein HubP to directly inhibit FlhF function at the cell pole, and this localization depends on ATP binding to FlhG. However, the detailed regulatory mechanisms involved, played by FlhF and FlhG as the major factors, remain largely unknown. This article reviews recent studies that highlight the post-translational regulation mechanism that allows the synthesis of only a single flagellum at the cell pole.

In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles.

The bacterial flagellum is a filamentous organelle extending from the cell surface. The axial structure of the flagellum consists of the rod, hook, junction, filament, and cap. The axial structure is formed by axial component proteins exported via a specific protein export apparatus in a well-regulated manner. Although previous studies have revealed the outline of the flagellar construction process, the mechanism of axial structure formation, including axial protein export, is still obscure due to difficulties in direct observation of protein export and assembly in vivo. We recently developed an in vitro flagellar protein transport assay system using inverted membrane vesicles (IMVs) and succeeded in reproducing the early stage of flagellar assembly. However, the late stage of the flagellar formation process remained to be examined in the IMVs. In this study, we showed that the filament-type proteins are transported into the IMVs to produce the filament on the hook inside the IMVs. Furthermore, we provide direct evidence that coordinated flagellar protein export and assembly can occur at the post-translational level. These results indicate that the ordered construction of the entire flagellar structure can be regulated by only the interactions between the protein export apparatus, the export substrate proteins, and their cognate chaperones.

In Situ Structure of the Vibrio Polar Flagellum Reveals a Distinct Outer Membrane Complex and Its Specific Interaction with the Stator.

The bacterial flagellum is a biological nanomachine that rotates to allow bacteria to swim. For flagellar rotation, torque is generated by interactions between a rotor and a stator. The stator, which is composed of MotA and MotB subunit proteins in the membrane, is thought to bind to the peptidoglycan (PG) layer, which anchors the stator around the rotor. Detailed information on the stator and its interactions with the rotor remains unclear. Here, we deployed cryo-electron tomography and genetic analysis to characterize in situ structure of the bacterial flagellar motor in Vibrio alginolyticus, which is best known for its polar sheathed flagellum and high-speed rotation. We determined in situ structure of the motor at unprecedented resolution and revealed the unique protein-protein interactions among Vibrio-specific features, namely the H ring and T ring. Specifically, the H ring is composed of 26 copies of FlgT and FlgO, and the T ring consists of 26 copies of a MotX-MotY heterodimer. We revealed for the first time a specific interaction between the T ring and the stator PomB subunit, providing direct evidence that the stator unit undergoes a large conformational change from a compact form to an extended form. The T ring facilitates the recruitment of the extended stator units for the high-speed motility in Vibrio species.IMPORTANCE The torque of flagellar rotation is generated by interactions between a rotor and a stator; however, detailed structural information is lacking. Here, we utilized cryo-electron tomography and advanced imaging analysis to obtain a high-resolution in situ flagellar basal body structure in Vibrio alginolyticus, which is a Gram-negative marine bacterium. Our high-resolution motor structure not only revealed detailed protein-protein interactions among unique Vibrio-specific features, the T ring and H ring, but also provided the first structural evidence that the T ring interacts directly with the periplasmic domain of the stator. Docking atomic structures of key components into the in situ motor map allowed us to visualize the pseudoatomic architecture of the polar sheathed flagellum in Vibrio spp. and provides novel insight into its assembly and function.

Essential ion binding residues for Na+ flow in stator complex of the Vibrio flagellar motor.

The bacterial flagellar motor is a unique supramolecular complex which converts ion flow into rotational force. Many biological devices mainly use two types of ions, proton and sodium ion. This is probably because of the fact that life originated in seawater, which is rich in protons and sodium ions. The polar flagellar motor in Vibrio is coupled with sodium ion and the energy converting unit of the motor is composed of two membrane proteins, PomA and PomB. It has been shown that the ion binding residue essential for ion transduction is the conserved aspartic acid residue (PomB-D24) in the PomB transmembrane region. To reveal the mechanism of ion selectivity, we identified essential residues, PomA-T158 and PomA-T186, other than PomB-D24, in the Na+-driven flagellar motor. It has been shown that the side chain of threonine contacts Na+ in Na+-coupled transporters. We monitored the Na+-binding specific structural changes using ATR-FTIR spectroscopy. The signals were abolished in PomA-T158A and -T186A, as well as in PomB-D24N. Molecular dynamics simulations further confirmed the strong binding of Na+ to D24 and showed that T158A and T186A hindered the Na+ binding and transportation. The data indicate that two threonine residues (PomA-T158 and PomA-T186), together with PomB-D24, are important for Na+ conduction in the Vibrio flagellar motor. The results contribute to clarify the mechanism of ion recognition and conversion of ion flow into mechanical force.

Structure of Vibrio FliL, a New Stomatin-like Protein That Assists the Bacterial Flagellar Motor Function.

Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.

Separation of Synephrine Enantiomers in Citrus Fruits by a Reversed Phase HPLC after Chiral Precolumn Derivatization.

Racemic synephrine, which was transformed into diastereomers by derivatization with 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosil isothiocyanate, was resolved by a reversed phase HPLC with UV detection at 254 nm. The total contents of synephrine enantiomers in citrus fruit samples were exocarp > mesocarp > endocarp > sarcocarp, suggesting that synephrine content of outer side of citrus fruits was higher than that of the inner side. (R)-Synephrine was detected in exocarp of eleven fresh citrus fruits, except for lemon, lime, and grapefruit samples. (S)-Synephrine was determined in the exocarp of four citrus fruits (mikan, orange, bitter orange, and ponkan samples) and the ratio of (S)-synephrine to total synephrine was 0.5 - 0.9%. The racemization of (R)-synephrine in aqueous solution during heating at 100°C was also examined. An increase in the heating time brought about an increase in the (S)-synephrine content in a linear fashion. The racemization was found to be significantly reduced by the addition of D-fructose, D-maltose, D-glucose, D-mannose or D-galactose, but not D-sucrose or D-mannitol. It is suggested that the reducibility of sugars may result in the inhibition of racemization.

Novel insight into an energy transduction mechanism of the bacterial flagellar type III protein export.

Type III secretion system (T3SS) is a protein translocator complex family including pathogenic injectisome or bacterial flagellum. The inejectisomal T3SS serves to deliver virulence proteins into host cell and the flagellar T3SS constructs the flagellar axial structure. Although earlier studies have provided many findings on the molecular mechanism of the Type III protein export, they were not sufficient to reveal energy transduction mechanism due to difficulties in controlling measurement conditions in vivo. Recently, we developed an in vitro flagellar Type III protein transport assay system using inverted membrane vesicles (IMVs), and analyzed protein export by using the in vitro method. We reproduced protein export of the flagellar T3SS, hook assembly and substrate specificity switch in IMV to a similar extent to what is seen in living cell. Furthermore, we demonstrated that ATP-hydrolysis energy can drive protein transport even in the absence of proton-motive force (PMF). In this mini-review, we will summarize our new in vitro Type III transport assay method and our findings on the molecular mechanism of Type III protein export.

In Vitro Reconstitution of Functional Type III Protein Export and Insights into Flagellar Assembly.

The type III secretion system (T3SS) forms the functional core of injectisomes, protein transporters that allow bacteria to deliver virulence factors into their hosts for infection, and flagella, which are critical for many pathogens to reach the site of infection. In spite of intensive genetic and biochemical studies, the T3SS protein export mechanism remains unclear due to the difficulty of accurate measurement of protein export in vivo Here, we developed an in vitro flagellar T3S protein transport assay system using an inverted cytoplasmic membrane vesicle (IMV) for accurate and controlled measurements of flagellar protein export. We show that the flagellar T3SS in the IMV fully retains export activity. The flagellar hook was constructed inside the lumen of the IMV by adding purified component proteins externally to the IMV solution. We reproduced the hook length control and export specificity switch in the IMV consistent with that seen in the native cell. Previous in vivo analyses showed that flagellar protein export is driven by proton motive force (PMF) and facilitated by ATP hydrolysis by FliI, a T3SS-specific ATPase. Our in vitro assay recapitulated these previous in vivo observations but furthermore clearly demonstrated that even ATP hydrolysis by FliI alone can drive flagellar protein export. Moreover, this assay showed that addition of the FliH2/FliI complex to the assay solution at a concentration similar to that in the cell dramatically enhanced protein export, confirming that the FliH2/FliI complex in the cytoplasm is important for effective protein transport.IMPORTANCE The type III secretion system (T3SS) is the functional core of the injectisome, a bacterial protein transporter used to deliver virulence proteins into host cells, and bacterial flagella, critical for many pathogens. The molecular mechanism of protein transport is still unclear due to difficulties in accurate measurements of protein transport under well-controlled conditions in vivo We succeeded in developing an in vitro transport assay system of the flagellar T3SS using inverted membrane vesicles (IMVs). Flagellar hook formation was reproduced in the IMV, suggesting that the export apparatus in the IMV retains a protein transport activity similar to that in the cell. Using this system, we revealed that ATP hydrolysis by the T3SS ATPase can drive protein export without PMF.

Chiral separation of isoxanthohumol and 8-prenylnaringenin in beer, hop pellets and hops by HPLC with chiral columns.

Xanthohumol, isoxanthohumol, and 8-prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2-propanol. Fractions of isoxanthohumol and 8-prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8-prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD-H column with a mobile phase composed of hexane-ethanol (90:10, v/v) and a Chiralpak AD-RH column with a mobile phase composed of methanol-2-propanol-water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8-prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2-10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5-5.5) during boiling.