Load-sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor.

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their 'unplugged' forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.

Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body.

The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane-embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C-terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C-termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF-YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA-CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA-YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co-ordinated manner.

Populational heterogeneity vs. temporal fluctuation in Escherichia coli flagellar motor switching.

The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations.

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor.

The bacterial flagellar motor can rotate in both counterclockwise (CCW) and clockwise (CW) directions. It has been shown that the sodium ion-driven chimeric flagellar motor rotates with 26 steps per revolution, which corresponds to the number of FliG subunits that form part of the rotor ring, but the size of the backward step is smaller than the forward one. Here we report that the proton-driven flagellar motor of Salmonella also rotates with 26 steps per revolution but symmetrical in both CCW and CW directions with occasional smaller backward steps in both directions. Occasional shift in the stepping positions is also observed, suggesting the frequent exchange of stators in one of the 11-12 possible anchoring positions around the rotor. These observations indicate that the elementary process of torque generation by the cyclic association/dissociation of the stator with every FliG subunit along the circumference of the rotor is symmetric in CCW and CW rotation even though the structure of FliG is highly asymmetric and suggests a 180° rotation of a FliG domain for the rotor-stator interaction to reverse the direction of rotation.

Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor.

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA(C) ), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotA(C) with FliG is required for the efficient assembly of the stators around the rotor.

Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus.

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliHDelta1 (missing residues 2-10) but the complex did not show any localization. FliHDelta1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate.

Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation.

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R).

The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex ( Minamino et al., 2011 ). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex ( Minamino et al., 2016 ). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have developed an in vivo pH imaging system with high spatial and pH resolution ( Morimoto et al., 2016 ). Here, we describe how we measure the local pH near the export apparatus in living Salmonella cells ( Morimoto et al., 2016 ). Our approach can be applied to a wide range of living cells. Because local pH is one of the most important parameters to monitor cellular activities of living cells, our protocol would be widely used for diverse areas of life sciences.

Load- and polysaccharide-dependent activation of the Na+-type MotPS stator in the Bacillus subtilis flagellar motor.

The flagellar motor of Bacillus subtilis possesses two distinct H+-type MotAB and Na+-type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner.

High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences.

Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+ flow to drive protein export. IMPORTANCE: The flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed an in vivo pH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.

Effect of the MotB(D33N) mutation on stator assembly and rotation of the proton-driven bacterial flagellar motor.

The bacterial flagellar motor generates torque by converting the energy of proton translocation through the transmembrane proton channel of the stator complex formed by MotA and MotB. The MotA/B complex is thought to be anchored to the peptidoglycan (PG) layer through the PG-binding domain of MotB to act as the stator. The stator units dynamically associate with and dissociate from the motor during flagellar motor rotation, and an electrostatic interaction between MotA and a rotor protein FliG is required for efficient stator assembly. However, the association and dissociation mechanism of the stator units still remains unclear. In this study, we analyzed the speed fluctuation of the flagellar motor of Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional stator complex, MotA/B(D33N), which lost the proton conductivity. The wild-type motor rotated stably but the motor speed fluctuated considerably when the expression level of MotA/B(D33N) was relatively high compared to MotA/B. Rapid accelerations and decelerations were frequently observed. A quantitative analysis of the speed fluctuation and a model simulation suggested that the MotA/B(D33N) stator retains the ability to associate with the motor at a low affinity but dissociates more rapidly than the MotA/B stator. We propose that the stator dissociation process depends on proton translocation through the proton channel.

Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus.

For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assembly-disassembly cycle of FliI during flagellar assembly. We propose that the FliH2FliI complex and FliI6 ring function as a dynamic substrate carrier and a static substrate loader, respectively.

The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor.

The bacterial flagellar motor is made of a rotor and stators. In Salmonella it is thought that about a dozen MotA/B complexes are anchored to the peptidoglycan layer around the motor through the C-terminal peptidoglycan-binding domain of MotB to become active stators as well as proton channels. MotB consists of 309 residues, forming a single transmembrane helix (30-50), a stalk (51-100) and a C-terminal peptidoglycan-binding domain (101-309). Although the stalk is dispensable for torque generation by the motor, it is required for efficient motor performance. Residues 51 to 72 prevent premature proton leakage through the proton channel prior to stator assembly into the motor. However, the role of residues 72-100 remains unknown. Here, we analyzed the torque-speed relationship of the MotB(Δ72-100) motor. At a low speed near stall, this mutant motor produced torque at the wild-type level. Unlike the wild-type motor, however, torque dropped off drastically by slight decrease in external load and then showed a slow exponential decay over a wide range of load by its further reduction. Since it is known that the stator is a mechano-sensor and that the number of active stators changes in a load-dependent manner, we interpreted this unusual torque-speed relationship as anomaly in load-dependent control of the number of active stators. The results suggest that residues 72-100 of MotB is required for proper load-dependent control of the number of active stators around the rotor.

Role of a conserved prolyl residue (Pro173) of MotA in the mechanochemical reaction cycle of the proton-driven flagellar motor of Salmonella.

The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator-rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque-speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor-stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque-speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle.

Effect of intracellular pH on the torque-speed relationship of bacterial proton-driven flagellar motor.

Bacterial flagella responsible for motility are driven by rotary motors powered by the electrochemical potential difference of specific ions across the cytoplasmic membrane. The stator of proton-driven flagellar motor converts proton influx into mechanical work. However, the energy conversion mechanism remains unclear. Here, we show that the motor is sensitive to intracellular proton concentration for high-speed rotation at low load, which was considerably impaired by lowering intracellular pH, while zero-speed torque was not affected. The change in extracellular pH did not show any effect. These results suggest that a high intracellular proton concentration decreases the rate of proton translocation and therefore that of the mechanochemical reaction cycle of the motor but not the actual torque generation step within the cycle by the stator-rotor interactions.

Large-scale screening of Arabidopsis circadian clock mutants by a high-throughput real-time bioluminescence monitoring system.

Using a high-throughput real-time bioluminescence monitoring system, we screened large numbers of Arabidopsis thaliana mutants for extensively altered circadian rhythms. We constructed reporter genes by fusing a promoter of an Arabidopsis flowering-time gene - either GIGANTEA (GI) or FLOWERING LOCUS T (FT) - to a modified firefly luciferase gene (LUC(+)), and we transferred the fusion gene (P(GI)::LUC(+) or P(FT)::LUC(+)) into the Arabidopsis genome. After mutagenesis with ethyl methanesulfonate, 50 000 M(2) seedlings carrying the P(GI)::LUC(+) and 50 000 carrying P(FT)::LUC(+) were screened their bioluminescence rhythms. We isolated six arrhythmic (AR) mutants and 29 other mutants that showed more than 3 h difference in the period length or phase of rhythms compared with the wild-type strains. The shortest period length was 16 h, the longest 27 h. Five of the six AR mutants carrying P(GI)::LUC(+) showed arrhythmia in bioluminescence rhythms in both constant light and constant dark. These five AR mutants also showed arrhythmia in leaf movement rhythms in constant light. Genetic analysis revealed that each of the five AR mutants carried a recessive mutation in a nuclear gene and the mutations belonged to three complementation groups, and at least one of which was mapped on a novel locus. Our results suggest that the three loci identified here may contain central clock or clock-related genes, at least one of which may be a novel.