Effect of the MotA(M206I) Mutation on Torque Generation and Stator Assembly in the Salmonella H+-Driven Flagellar Motor.

The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-µm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-µm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

Genetic analysis of revertants isolated from the rod-fragile fliF mutant of Salmonella.

FliF is the protein comprising the MS-ring of the bacterial flagellar basal body, which is the base for the assembly of flagellar axial structures. From a fliF mutant that easily releases the rod-hook-filament in viscous environments, more than 400 revertants that recovered their swarming ability in viscous conditions, were isolated. The second-site mutations were determined for approximately 70% of them. There were three regions where the mutations were localized: two in Region I, 112 in Region II, and 71 in Region III including the true reversion. In Region I, second-site mutations were found in FlgC and FlgF of the proximal rod, suggesting that they affect the interaction between the MS-ring and the rod. In Region II, there were 69 and 42 mutations in MotA and MotB, respectively, suggesting that the second-site mutations in MotA and MotB may decrease the rotational speed of the flagellar motor to reduce the probability of releasing the rod under this condition. One exception is a mutation in FlhC that caused a down regulation of the flagellar proteins production but it may directly affect transcription or translation of motA and motB. In Region III, there were 44, 24, and 3 mutations in FliG, FliM, and FliF, respectively. There were no second-site mutations identified in FliN although it is involved in torque generation as a component of the C-ring. Many of the mutations were involved in the motor rotation, and it is suggested that such reduced speeds result in stabilizing the filament attachment to the motor.

SptP106-136 plays a role in the complex formation with SptP-specific chaperone SicP.

SptP is a virulence effector protein of Salmonella that is involved in bacterial invasion into a host cell. For effective secretion, SptP forms a complex with SptP-specific chaperone SicP through its chaperone-binding domain, residues 35-139. Here, we suggest the possibility that residues 106-136 of SptP are important for complex formation with SicP by in vitro reconstitution experiments.

Key amino acid residues involved in the transitions of L- to R-type protofilaments of the Salmonella flagellar filament.

The flagellar filament enables bacteria to swim by functioning as a helical propeller. The filament is a supercoiled assembly of a single protein, flagellin, and is formed by 11 protofilaments arranged in a circle. Bacterial swimming and tumbling correlate with changes of the various helical structures, called polymorphic transformation, that are determined by the ratios of two distinct forms of protofilaments, the L and R types. The polymorphic transformation is caused by transition of the protofilament between L and R types. Elucidation of this transition mechanism has been addressed by comparing the atomic structures of L- and R-type straight filaments or using massive molecular dynamic simulation. Here, we found amino acid residues required for the transition of the protofilament using fliC-intragenic suppressor analysis. We isolated a number of revertants producing supercoiled filaments from mutants with straight filaments and identified the second-site mutations in all of the revertants. The results suggest that Asp107, Gly426, and Ser448 and Ser106, Ala416, Ala427, and Arg431 are the key residues involved in inducing supercoiled filaments from the R- and the L-type straight filaments, respectively. Considering the structures of the R- and L-type protofilaments and the relationship between the rotation of the flagellar motor and the polymorphic transformation, we propose that Gly426, Ala427, and Arg431 contribute to the first stage of the transition and that Ser106, Asp107, and Ala416 play a role in propagating the transitions along the flagellar filament.

Analysis of tandem repeats found in 44 prokaryotic genomes.

Genomes contain various types of repetitive sequences. They may be used as probes for seeking genome rearrangements because they are rather free from the natural selection if they are located in the intergenic regions. In this study, we searched for tandem repeats (TRs) in 44 prokaryotic genomes by the color-coding method and sought the signs of genome rearrangements by detailed analysis of the detected TRs. We found 13,542 tandem repeats from 44 prokaryotic genomes in total ranging from several tens to one thousand per genome. The results of statistical analysis show that TRs tend to exist on high base composition bias regions in some genomes. Moreover, we recognized the characteristic distribution patterns of equivalent TR-pairs in 12 genomes, which are expected to indicate the occurrence of whole-genome duplication (WGD) on the genomes. It is demonstrated that TRs could indeed be used for seeking genome rearrangements. Although it has not been made clear at this time whether or not WGD had occurred in prokaryotic genomes, the results of the analyses of equivalent TR-pairs in this study are thought to be evidences of WGD in these genomes.

gamma-Mangostin inhibits inhibitor-kappaB kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in C6 rat glioma cells.

We investigated the effect of gamma-mangostin purified from the fruit hull of the medicinal plant Garcinia mangostana on spontaneous prostaglandin E(2) (PGE(2)) genase release and inducible cyclooxy-2 (COX-2) gene expression in C6 rat glioma cells. An 18-h treatment with gamma-mangostin potently inhibited spontaneous PGE(2) release in a concentration-dependent manner with the IC(50) value of approximately 2 microM, without affecting the cell viability even at 30 microM. By immunoblotting and reverse-transcription polymerase chain reaction, we showed that gamma-mangostin concentration-dependently inhibited lipopolysaccharide (LPS)-induced expression of COX-2 protein and its mRNA, but not those of constitutive COX-1 cyclooxygenase. Because LPS is known to stimulate inhibitor kappaB (IkappaB) kinase (IKK)-mediated phosphorylation of IkappaB followed by its degradation, which in turn induces nuclear factor (NF)-kappaB nuclear translocation leading to transcriptional activation of COX-2 gene, the effect of gamma-mangostin on the IKK/IkappaB cascade controlling the NF-kappaB activation was examined. An in vitro IKK assay using IKK protein immunoprecipitated from C6 cell extract showed that this compound inhibited IKK activity in a concentration-dependent manner, with the IC(50) value of approximately 10 microM. Consistently gamma-mangostin was also observed to decrease the LPS-induced IkappaB degradation and phosphorylation in a concentration-dependent manner, as assayed by immunoblotting. Furthermore, luciferase reporter assays showed that gamma-mangostin reduced the LPS-inducible activation of NF-kappaB-and human COX-2 gene promoter region-dependent transcription. gamma-Mangostin also inhibited rat carrageenan-induced paw edema. These results suggest that gamma-mangostin directly inhibits IKK activity and thereby prevents COX-2 gene transcription, an NF-kappaB target gene, probably to decrease the inflammatory agent-stimulated PGE(2) production in vivo, and is a new useful lead compound for anti-inflammatory drug development.

In vitro characterization of FlgB, FlgC, FlgF, FlgG, and FliE, flagellar basal body proteins of Salmonella.

The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.

Effect of intracellular pH on rotational speed of bacterial flagellar motors.

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.

Inhibitions of histamine release and prostaglandin E2 synthesis by mangosteen, a Thai medicinal plant.

The fruit hull of mangosteen, Garcinia mangostana L. has been used as a Thai indigenous medicine for many years. However, its mechanism of action as a medicine has not been elucidated. The present study was undertaken to examine the effects of mangosteen extracts (100% ethanol, 70% ethanol, 40% ethanol and water) on histamine release and prostaglandin E2 synthesis. We found that the 40% ethanol extract of mangosteen inhibited IgE-mediated histamine release from RBL-2H3 cells with greater potency than the water extract of Rubus suavissimus that has been used as an anti-allergy crude drug in Japan. All extracts of mangosteen potently inhibited A23187-induced prostaglandin E2 synthesis in C6 rat glioma cells, while the water extract of Rubus suavissimus had no effect. The 40% ethanol extract of mangosteen inhibited the prostaglandin E2 synthesis in a concentration-dependent manner with relatively lower concentrations than the histamine release. In addition, passive cutaneous anaphylaxis (PCA) reactions in rats were significantly inhibited by this ethanol extract as well as by the water extract of Rubus suavissimus. These results suggest that the 40% ethanol extract of mangosteen has potent inhibitory activities of both histamine release and prostaglandin E2 synthesis.

Structural properties of FliH, an ATPase regulatory component of the Salmonella type III flagellar export apparatus.

FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation.