MotX and MotY Are Required for Flagellar Rotation in Shewanella oneidensis MR-1

The single polar flagellum of Shewanella oneidensis MR-1 is powered by two different stator complexes, the sodium-dependent PomAB and the proton-driven MotAB. In addition, Shewanella harbors two genes with homology to motX and motY of Vibrio species. In Vibrio, the products of these genes are crucial for sodium-dependent flagellar rotation. Resequencing of S. oneidensis MR-1 motY revealed that the gene does not harbor an authentic frameshift as was originally reported. Mutational analysis demonstrated that both MotX and MotY are critical for flagellar rotation of S. oneidensis MR-1 for both sodium- and proton-dependent stator systems but do not affect assembly of the flagellar filament. Fluorescence tagging of MotX and MotY to mCherry revealed that both proteins localize to the flagellated cell pole depending on the presence of the basal flagellar structure. Functional localization of MotX requires MotY, whereas MotY localizes independently of MotX. In contrast to the case in Vibrio, neither protein is crucial for the recruitment of the PomAB or MotAB stator complexes to the flagellated cell pole, nor do they play a major role in the stator selection process. Thus, MotX and MotY are not exclusive features of sodium-dependent flagellar systems. Furthermore, MotX and MotY in Shewanella, and possibly also in other genera, must have functions beyond the recruitment of the stator complexes.Flagellum-mediated swimming motility is a widespread means of locomotion among bacteria. Flagella consist of protein filaments that are rotated at the filament''s base by a membrane-embedded motor (3, 39). Rotation is powered by electrochemical gradients across the cytoplasmic membrane. Thus far, two coupling ions, sodium ions and protons, have been described as energy sources for bacterial flagellar motors (4, 24, 48). Two major components confer the conversion of the ion flux into rotary motion. The first component forms a rotor-mounted ring-like structure at the base of the flagellar basal body and is referred to as the switch complex or the C ring; it is composed of the proteins FliG, FliM, and FliN. The second major component is the stator system, consisting of membrane-embedded stator complexes that surround the C ring (3). Each stator complex is composed of two subunits in a 4:2 stoichiometry. In Escherichia coli, MotA and MotB constitute the stator complex by forming a proton-specific ion channel; the Na⁺-dependent counterpart in Vibrio species consists of the orthologs PomA and PomB (1, 5, 49). MotA and PomA both have four transmembrane domains and are thought to interact with FliG via a cytoplasmic segment to generate torque (2, 50). Stator function is presumably made possible by a peptidoglycan-binding motif located at the C-terminal portion of MotB and PomB that anchors the stator complex to the cell wall (1, 8). In E. coli, at least 11 stator complexes can be synchronously involved in driving flagellar rotation (35). However, a single complex is sufficient for rotation of the filament (36, 40). Despite its tight attachment to the peptidoglycan, the stator ring system was found to form a surprisingly dynamic complex. It has been suggested that inactive precomplexes of the stators form a membrane-located pool before being activated upon incorporation into the stator ring system around the motor (13, 45). In E. coli, the turnover time of stator complexes can be as short as 30 s (21).In Vibrio species, two auxiliary proteins, designated MotX and MotY, are required for motor function of the Na⁺-driven polar flagellar system (22, 23, 28, 31). Recently, it was shown that the proteins associate with the flagellar basal body in Vibrio alginolyticus to form an additional structure, the T ring (42). MotX interacts with MotY and the PomAB stator complexes, and both proteins are thought to be crucial for the acquisition of the stators to the motor of the polar flagellum. (29, 30, 42). A MotY homolog is also associated with the proton-dependent motor system of the lateral flagella of V. alginolyticus that is induced under conditions of elevated viscosity (41).We recently showed that Shewanella oneidensis MR-1 uses two different stator systems to drive the rotation of its single polar flagellum, the Na⁺-dependent PomAB stator and the proton-driven MotAB stator. As suggested by genetic data, the MotAB stator has been acquired by lateral gene transfer, presumably in the process of adaptation from a marine to a freshwater environment (32). The two different stators are recruited to the motor in a way that depends on the sodium ion concentration in the medium. The Na⁺-dependent PomAB stator is present at the flagellated cell pole regardless of the sodium ion concentration, whereas the proton-dependent MotAB stator functionally localizes only under conditions of low sodium or in the absence of PomAB. It is still unclear how stator selection is achieved and whether additional proteins play a role in this process.Orthologs of motX and motY have been annotated in S. oneidensis MR-1. We thus hypothesized that MotX and MotY might play a role in stator selection in S. oneidensis MR-1. However, the originally published sequence of motY harbors a frameshift that would result in a drastically truncated protein lacking a functionally relevant putative peptidoglycan-binding domain at its C terminus (16, 18). This situation seemed inconsistent with a role for MotY in S. oneidensis MR-1.Here we describe a functional analysis of the MotX and MotY orthologs in S. oneidensis MR-1. We found that motY does not, in fact, contain a frameshift mutation, so that MotY is translated in its full-length form. Both MotX and MotY were essential for Na⁺-dependent and proton-dependent motility. Therefore, these proteins have a role in S. oneidensis MR-1 that differs from their function in Vibrio species. We also used fusions to the fluorescent protein mCherry for functional localization studies of MotX and MotY.     

Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.     

Two different stator systems drive a single polar flagellum in Shewanella oneidensis MR-1

The Gram-negative metal ion-reducing bacterium Shewanella oneidensis MR-1 is motile by means of a single polar flagellum. We identified two potential stator systems, PomAB and MotAB, each individually sufficient as a force generator to drive flagellar rotation. Physiological studies indicate that PomAB is sodium-dependent while MotAB is powered by the proton motive force. Flagellar function mainly depends on the PomAB stator; however, the presence of both stator systems under low-sodium conditions results in a faster swimming phenotype. Based on stator homology analysis we speculate that MotAB has been acquired by lateral gene transfer as a consequence of adaptation to a low-sodium environment. Expression analysis at the single cell level showed that both stator systems are expressed simultaneously. An active PomB–mCherry fusion protein effectively localized to the flagellated cell pole in 70–80% of the population independent of sodium concentrations. In contrast, polar localization of MotB–mCherry increased with decreasing sodium concentrations. In the absence of the Pom stator, MotB–mCherry localized to the flagellated cell pole independently of the sodium concentration but was rapidly displaced upon expression of PomAB. We propose that selection of the stator occurs at the level of protein localization in response to sodium concentrations.     

Specificity of motor components in the dual flagellar system of Shewanella putrefaciens CN-32

Bacterial flagellar motors are intricate nanomachines in which the stator units and rotor component FliM may be dynamically exchanged during function. Similar to other bacterial species, the gammaproteobacterium Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system along with a corresponding stator unit. Expression of the secondary system occurs during planktonic growth in complex media and leads to the formation of a subpopulation with one or more additional flagella at random positions in addition to the primary polar system. We used physiological and phenotypic characterizations of defined mutants in concert with fluorescent microscopy on labelled components of the two different systems, the stator proteins PomB and MotB, the rotor components FliM(1) and FliM(2), and the auxiliary motor components MotX and MotY, to determine localization, function and dynamics of the proteins in the flagellar motors. The results demonstrate that the polar flagellum is driven by a Na(+)-dependent FliM(1)/PomAB/MotX/MotY flagellar motor while the secondary system is rotated by a H(+)-dependent FliM(2)/MotAB motor. The components were highly specific for their corresponding motor and are unlikely to be extensively swapped or shared between the two flagellar systems under planktonic conditions. The results have implications for both specificity and dynamics of flagellar motor components.     

Differential effects of sodium ions on motility in the homoacetogenic bacteriaAcetobacterium woodii andSporomusa sphaeroides

The strictly anaerobic homoacetogenic bacteria Acetobacterium woodii and Sporomusa sphaeroides differ with respect to their energy metabolism. Since growth as well as acetate and ATP formation of A. woodii is strictly dependent on Na⁺, but that of S. sphaeroides is not, the question arose whether these organisms also use different coupling ions for mechanical work, i.e. flagellar rotation. During growth on fructose in the presence of Na⁺ (50 mM), cells of A. woodii were vigorously motile, as judged by light microscopy. At low Na⁺ concentrations (0.3 mM), the growth rate decreased by only 15%, but the cells were completely non-motile. Addition of Na⁺ to such cultures restored motility instantaneously. Motility, as determined in swarm agar tubes, was strictly dependent on Na⁺; Li⁺, but not K⁺ partly substituted for Na⁺. Of the amilorides tested, phenamil proved to be a specific inhibitor of the flagellar motor of A. woodii. Growth and motility of S. sphaeroides was neither dependent on Na⁺ nor inhibited by amiloride derivatives. These results indicate that flagellar rotation is driven by Δμ_Na ⁺ in A. woodii, but by Δμ_H ⁺ in S. sphaeroides. Received: 30 May 1995 / Accepted: 31 August 1995     

Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane‐bound stator complexes. We used the light‐driven proton pump proteorhodopsin (pR) to control the proton‐motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s^?1. Using GFP‐tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s^?1.     

Na+-driven bacterial flagellar motors

Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H⁺-driven type found in neutrophiles such asBacillus subtilis andEscherichia coli and the other is the Na⁺-driven type found in alkalophilicBacillus and marineVibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na⁺-driven flagellar motors, which introduces several new aspects in the analysis.     

Carbofuran Induced Oxidative Stress Mediated Alterations in Na+‐K+‐ATPase Activity in Rat Brain: Amelioration by Vitamin E

Pesticides cause oxidative stress and adversely influence Na⁺‐K⁺‐ATPase activity in animals. Since impact of carbofuran has not been properly studied in the mammalian brain, the ability of carbofuran to induce oxidative stress and modulation in Na⁺‐K⁺‐ATPase activity and its amelioration by vitamin E was performed. The rats divided into six groups received two different doses of carbofuran (15% and 30% LD₅₀) for 15 days. The results suggested that the carbofuran treatment caused a significant elevation in levels of malonaldehyde and reduced glutathione and sharp inhibition in the activities of super oxide dismutase, catalase, and glutathione‐S‐transferase; the effect being dose dependent. Carbofuran at different doses also caused sharp reduction in the activity of Na⁺‐K⁺‐ATPase. The pretreatment of vitamin E, however, showed a significant recovery in these indices. The pretreatment of rats with vitamin E offered protection from carbofuran‐induced oxidative stress.     

Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.     

The Function of the Na+-Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

Vibrio cholerae is motile by its polar flagellum, which is driven by a Na⁺-conducting motor. The stators of the motor, composed of four PomA and two PomB subunits, provide access for Na⁺ to the torque-generating unit of the motor. To characterize the Na⁺ pathway formed by the PomAB complex, we studied the influence of chloride salts (chaotropic, Na⁺, and K⁺) and pH on the motility of V. cholerae. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which rules out a direct Na⁺ and H⁺ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na⁺ concentrations but regained motility in the presence of 170 mM chloride. Both PomA and PomB were modified by N,N′-dicyclohexylcarbodiimide (DCCD), indicating the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na⁺ did not protect PomA and PomB from this modification. Our study shows that both osmolality and pH have an influence on the function of the flagellum from V. cholerae. We propose that D23, S26, and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error‐prone PCR to introduce single point mutations into the sodium‐powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium‐channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C‐terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion‐channel blockers.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

Workshop 7: 2

Glutamine, the preferred precursor for neurotransmitter glutamate, is likely to be the principal substrate for the neuronal System A transporter SAT1 in vivo. By measuring currents associated with SAT1 expression in Xenopus oocytes, we found that SAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine, alanine and the System A‐specific analogue 2‐(methylamino) isobutyrate, each with K_0.5 of 0.3–0.5 mm . Amino acid transport is driven by the Na⁺ electrochemical gradient. Kinetic data indicates that Na⁺/cotransport comprises the ordered binding first of Na⁺ (a voltage‐dependent step), then alanine, then simultaneous translocation. Li⁺ (but not H⁺) can substitute for Na⁺ but results in reduced V_max. In the absence of amino acid, SAT1 mediates a cation leak with selectivity Na⁺, Li⁺, H⁺, K⁺. The temperature‐dependence of the leak current (E_a = 17 ± 3 kcal/mol) is consistent with carrier‐mediated Na⁺ uniport activity (cf 13 ± 2 kcal/mol for Na⁺/alanine cotransport) but the leak does not saturate at physiological [Na⁺], suggesting channel activity. Despite a Na⁺ Hill coefficient of 1, we obtained Na⁺/amino acid coupling coefficients greater than 1 from simultaneous measurement of charge and [³H]alanine or [³H]glutamine uptake. Interpretation of these data is model‐dependent and consistent with either (1) an all‐carrier model in which Na⁺/amino acid cotransport is thermodynamically coupled 2 : 1, cotransport is preferred over Na⁺ uniport, and in which there is little cooperativity between Na⁺ binding events, or (2) 1 : 1 coupling in parallel with an always‐on Na⁺ channel activity. In either scenario, the presence of SAT1 at the plasma membrane and resultant Na⁺ fluxes will place a significant energy burden on the cell.     

Determination of the driving force of the Na+ pump in toad bladder by means of vasopressin

Summary Vasopressin stimulates Na⁺ transport across toad bladder largely or entirely by decreasing the resistance to Na⁺ entry into the transporting epithelial cells. Therefore, the hormone should induce proportional changes in short circuit current (I _S ) and tissue conductance; the ratio of these changes should equal the driving force (E _Na) of the Na⁺ pump.Administration of vasopressin provided a rapid, reversible and reproducible technique for the measurement ofE _Na. Values calculated forE _Na ranged from 74 to 186 mV, in agreement with previously published estimates. The results were not dependent on the vasopressin concentration over a wide range of concentrations.Ouabain, an agent thought to inhibit specifically the Na⁺ pump, decreased bothI _S andE _Na. On the other hand, amiloride, a diuretic thought to block specifically Na⁺ entry, markedly reducedI _S , without reducingE _Na.It is concluded that vasopressin constitutes a probe for the rapid reproducible determination ofE _Na under a wide variety of physiological conditions.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Unveiling the Unique Phase Transformation Behavior and Sodiation Kinetics of 1D van der Waals Sb2S3 Anodes for Sodium Ion Batteries

Carbon‐coated van der Waals stacked Sb₂S₃ nanorods (SSNR/C) are synthesized by facile hydrothermal growth as anodes for sodium ion batteries (SIBs). The sodiation kinetics and phase evolution behavior of the SSNR/C anode during the first and subsequent cycles are unraveled by coupling in situ transmission electron microscopy analysis with first‐principles calculations. During the first sodiation process, Na⁺ ions intercalate into the Sb₂S₃ crystals with an ultrafast speed of 146 nm s^?1. The resulting amorphous Na_x Sb₂S₃ intermediate phases undergo sequential conversion and alloying reactions to form crystalline Na₂S, Na₃Sb, and minor metallic Sb. Upon desodiation, Na⁺ ions extract from the nanocrystalline phases to leave behind the fully desodiated Sb₂S₃ in an amorphous state. Such unique phase evolution behavior gives rise to superb electrochemical performance and leads to an unexpectedly small volume expansion of ≈54%. The first‐principles calculations reveal distinctive phase evolution arising from the synergy between the extremely low Na⁺ ion diffusion barrier of 190 meV and the sharply increased electronic conductivity upon the formation of amorphous Na_x Sb₂S₃ intermediate phases. These findings highlight an anomalous Na⁺ ion storage mechanism and shed new light on the development of high performance SIB anodes based on van der Waals crystals.     

Different neuronal Na+/K+‐ATPase isoforms are involved in diverse signaling pathways

Inhibition of rat neuronal Na⁺/K⁺‐ATPase α3 isoform at low (100 nM) ouabain concentration led to activation of MAP kinase cascade via PKC and PIP₃ kinase. In contrast to ouabain‐sensitive α3 isoform of Na⁺/K⁺‐ATPase, an ouabain‐resistant α1 isoform (inhibition with 1 mM of ouabain) of Na⁺/K⁺‐ATPase regulates MAP kinase via Src kinase dependent reactions. Using of Annexin V‐FITC apoptotic test to determine the cells with early apoptotic features allows to conclude that α3 isoform stimulates and α1 suppresses apoptotic process in cerebellum neurons. These data are the first demonstration showing participation of ouabain‐resistant (α1) and ouabain‐sensitive (α3) Na⁺/K⁺‐ATPase isoforms in diverse signaling pathways in neuronal cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Hydrogen peroxide as a mediator of 5‐aminolevulinic acid‐induced Na+ retention in roots for improving salt tolerance of strawberries

To explore the mechanisms of 5‐aminolevulinic acid (ALA)‐improved plant salt tolerance, strawberries (Fragaria × ananassa Duch. cv. ‘Benihoppe’) were treated with 10 mg l^?1 ALA under 100 mmol l^?1 NaCl stress. We found that the amount of Na⁺ increased in the roots but decreased in the leaves. Laser scanning confocal microscopy (LSCM) observations showed that ALA‐induced roots had more Na⁺ accumulation than NaCl alone. Measurement of the xylem sap revealed that ALA repressed Na⁺ concentrations to a large extent. The electron microprobe X‐ray assay also confirmed ALA‐induced Na⁺ retention in roots. qRT‐PCR showed that ALA upregulated the gene expressions of SOS1 (encoding a plasma membrane Na⁺/H⁺ antiporter), NHX1 (encoding a vacuolar Na⁺/H⁺ antiporter) and HKT1 (encoding a protein of high‐affinity K⁺ uptake), which are associated with Na⁺ exclusion in the roots, Na⁺ sequestration in vacuoles and Na⁺ unloading from the xylem vessels to the parenchyma cells, respectively. Furthermore, we found that ALA treatment reduced the H₂O₂ content in the leaves but increased it in the roots. The exogenous H₂O₂ promoted plant growth, increased root Na⁺ retention and stimulated the gene expressions of NHX1, SOS1 and HKT1. Diphenyleneiodonium (DPI), an inhibitor of H₂O₂ generation, suppressed the effects of ALA or H₂O₂ on Na⁺ retention, gene expressions and salt tolerance. Therefore, we propose that ALA induces H₂O₂ accumulation in roots, which mediates Na⁺ transporter gene expression and more Na⁺ retention in roots, thereby improving plant salt tolerance.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.