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Journal of Bacteriology, October 2008, p. 6660-6667, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation {triangledown} ,{dagger}

Yong-Suk Che,1,2 Shuichi Nakamura,1,2 Seiji Kojima,2,3 Nobunori Kami-ike,2 Keiichi Namba,1,2* and Tohru Minamino1,2*

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,2 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan3

Received 13 April 2008/ Accepted 9 July 2008


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ABSTRACT
 
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.


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INTRODUCTION
 
The bacterial flagellum, a locomotive organelle that allows bacteria to swim in a liquid environment, is a rotary nanomachine composed of a motor and a helical propeller. The energy source for the Salmonella enterica serovar Typhimurium flagellar motor is an inwardly directed electrochemical gradient of protons across the cytoplasmic membrane, the proton motive force (1, 2, 25). The molecular mechanism of energy coupling between proton influx and flagellar motor rotation remains unknown. An increase in the intracellular proton concentration suppresses or even abolishes the flagellar motor function, suggesting that the absolute concentration of protons in the cytoplasm is one of the factors that determine the rotation speed of the motor (28).

Five proteins, MotA, MotB, FliG, FliM, and FliN, are responsible for torque generation in the flagellar motor (1, 2, 25). MotA and MotB are cytoplasmic membrane proteins and form a complex consisting of four copies of MotA and two copies of MotB (8, 13, 24, 35, 36, 37, 39, 40). The MotA/B complex is the stator of the motor and plays an important role in proton conductance (3, 4, 6, 20, 36). Recently, it was estimated that there are at least 11 copies of the MotA/B complex around the flagellar basal body (30). Since MotB has a potential peptidoglycan-binding motif in its C-terminal periplasmic domain, the MotA/B complex is postulated to be anchored to the peptidoglycan layer (14). However, MotB is not always tightly associated with the peptidoglycan layer, because the stators are dynamically replaced even during the motor rotation (5, 7, 26, 34). FliG, FliM, and FliN, which are also responsible for switching the direction of torque (22, 41), form the C ring on the cytoplasmic side of the flagellar MS ring and act as the rotor (15). Recently, it has been observed that there are 26 steps in each revolution of the flagellar motor, which is consistent with the number of FliG subunits in the C ring, the proposed site of torque generation in the rotor (34).

MotA consists of four transmembrane spans (TH1 to TH4), two short periplasmic loops and two extensive cytoplasmic regions. MotB consists of an N-terminal cytoplasmic region, one transmembrane span (TM), and a large periplasmic region containing the potential peptidoglycan-binding motif (Fig. 1A). Cross-linking experiments have shown that the four MotA subunits in the stator complex are positioned with their TM3 and TM4 segments adjacent to the dimer of MotB-TM located at the center and their TM1 and TM2 segments in outer positions (Fig. 1B) (8). Two charged residues in the cytoplasmic loop of MotA (MotAC), Arg-90 and Glu-98, are responsible for the interaction with charged residues of FliG to produce torque (44). Two conserved proline residues of MotA, Pro-173 and Pro-222, are thought to be involved in conformational changes of the stator complex that couple proton influx with torque generation (10). The absolutely conserved and functionally critical aspartic acid residue Asp-33 of Salmonella MotB, which corresponds to Asp-32 of Escherichia coli MotB, is located near the cytoplasmic end of its TM segment and is postulated to be a proton-binding site (32, 38, 45). MotB exists as a dimer in the stator complex, and these aspartic acid residues are positioned on the surface of the MotB-TM dimer facing MotA TMs, suggesting that the stator complex is likely to have two proton-conducting pathways (9). Both protonation and deprotonation of this aspartic acid residue cause conformational changes in MotAC, which may drive flagellar motor rotation (23). Therefore, proton translocation through the proton-conducting pathway and cyclic conformational changes of the stator complex for torque generation are likely to be coupled and mutually regulated with each other, although it remains unknown how.


Figure 1
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  		FIG. 1. Topology of MotA and MotB. (A) Cartoon representing the domain organization of MotA and MotB and location of the second-site mutations of pseudorevertants isolated from the motB(D33E) mutant. Salmonella MotA and MotB consist of 295 and 309 amino acid residues, respectively. MotA has four transmembrane segments (TM1 to TM4), and MotB has only one (TM). MotB has a peptidoglycan-binding motif (PG), by which the MotA/B complex is thought to be anchored and act as the stator. Asp-33 of MotB (closed circle), which is located near the cytoplasmic end of TM, is postulated to play a role in proton translocation. Open circles indicate mutated amino acid residues that are capable of suppressing the first-site D33E mutation. The number in parentheses indicates the number of revertants isolated and sequenced. N and C indicate the N and C termini of the proteins. CM, cytoplasmic membrane; cyto, cytoplasm; peri, periplasmic space. (B) Model for the arrangement of transmembrane segments of the MotA/B complex, which consists of four copies of MotA and two copies of MotB. The view is from the periplasmic side of the membrane. The complex is postulated to have two proton-conducting pathways, shown by gray circles. The closed circle indicates Asp-33 of MotB, while the open circle, open triangle, and open squire indicate V35 of MotA, V185 of MotA, and F34 of MotB, respectively, at which mutations suppress the D33E defect. Cross-linking experiments have shown that four MotA subunits are positioned with their TM3 (A3) and TM4 (A4) segments adjacent to the MotB dimer and their TM1 (A1) and TM2 (A2) segments on the outside, although the exact positions of TM1 and TM2 remain unknown (8). Because the V35F mutation in TM2 suppresses the MotB(D33E) defect, TM2 is presumably close to TM3 and/or TM4 to maintain the proton pathway formed at the interface between MotA and MotB.

To further characterize the properties of the proton-conducting pathway in the flagellar motor and its relationship with torque generation cycle, we first confirmed that MotB(D33E) forms the complex with MotA in the cytoplasmic membrane. We then isolated suppressor mutants from the motB(D33E) mutant, identified the suppressor mutation sites, and characterized the torque-speed relationship of the flagellar motors of the parent mutant and its suppressors by tethered-cell measurements and bead assays. We show that, while the wild-type motor torques are almost constant over a wide range of rotation rates, the motB(D33E) mutation causes ca. 40% reduction in stall torque and a sharp decline in the torque-speed curve, with an apparent maximal rotation rate of ca. 20 Hz, and that the second-site mutations restore the stall torque to the wild-type level but not the sharp decline in the torque-speed curve. Based on these results, we will discuss the proton translocation rates and dynamic conformational changes of the stator complex coupled with proton translocation of the wild type, the motB(D33E) mutant, and its pseudorevertants.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, transductional crosses, DNA manipulations, and media. The bacterial strains and plasmids used in this study are listed in Table 1. P22-mediated transductional crosses were carried out using p22HTint as described previously (42). DNA manipulations were carried out as described before (31). Site-directed mutagenesis was carried out using the QuikChange site-directed mutagenesis method as described in the manufacturer's instructions (Stratagene). DNA sequencing was performed with an ABI PRISM 377 DNA sequencer (Applied Biosystems). S broth contained 1.2% Bacto tryptone, 2.4% yeast extract, 1.25% KHPO4, 0.38% KH2PO4, and 0.5% (vol/vol) glycerol. L broth, T broth, soft agar plates, and motility medium were prepared as described before (28, 29). A 100-µg/ml ampicillin solution was added to the medium as needed.


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  		TABLE 1. Strains and plasmids used in this study

Motility assays with soft agar plates. Fresh colonies were inoculated onto soft tryptone agar plates and incubated at 30°C.

Preparation of cell extracts and immunoblotting. Fractionation of cell extracts of SJW1103 and MMTB6055 was carried out as described previously (27). Cells were exponentially grown in 40 ml L broth at 30°C with shaking. The cells were harvested, resuspended in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, and sonicated. After the cell debris was removed by low-speed centrifugation, the cell lysate was ultracentrifuged (100,000 x g, 60 min, 4°C). The soluble and membrane fractions were collected separately. Aliquots of the membrane fractions were treated with 1 M NaCl or 1.2% sodium lauryl sarcosinate (Sarkosyl) for 1 h at room temperature and ultracentrifuged. The supernatant and the pellet fractions were collected separately. After the proteins in each fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting with a polyclonal anti-MotB antibody (MBL Co., Ltd.) was carried out as described before (29). Detection was performed with an enhanced chemiluminescence immunoblotting detection kit (GE Healthcare).

Pulldown assays. Detergent solubilization of membrane fractions and pulldown assays were carried out as described before (24, 37). BL21(DE3) cells were transformed with plasmid pMM650 (encoding MotA and MotB), pNSK2 (encoding MotA-His and MotB), or pYC9 [encoding MotA-His and MotB(D33E)], and the resulting transformants were inoculated into 25 ml S broth containing 100 µg/ml ampicillin. Cultures were incubated at 24°C with shaking until the cell density reached an optical density at 600 nm of ca. 0.2. After addition of 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside), incubation was continued overnight at 24°C. Cells were collected by centrifugation, suspended in a 1-ml solution containing 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, and protease inhibitors (complete tablet; Roche Diagnostics), and then sonicated. Samples were centrifuged at 8,000 x g for 10 min at 4°C to remove undisrupted cells, and then membranes were collected by ultracentrifugation (100,000 x g, 60 min, 4°C). The membrane fractions were resuspended in a 1-ml solution containing 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 0.3% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}. After centrifugation (23,000 x g, 15 min, 4°C), the supernatant fractions were incubated with a Ni+-nitrilotriacetic acid (NTA) resin (Qiagen) for 1 h at 4°C. Beads were washed three times with a solution containing 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole, 10% (vol/vol) glycerol, 0.3% (wt/vol) CHAPS. Proteins were eluted with a solution containing 50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, 10% glycerol, 0.3% CHAPS. Eluted materials were mixed with a sodium dodecyl sulfate-loading buffer and boiled.

Rotation measurement of tethered cells. Salmonella cells were grown at 30°C in T broth until the cell density reached an optical density at 600 nm of ca. 0.8. The flagella were sheared off by passing them through a 19-gauge needle, and cells with a single filament were attached to a glass surface by using a polyclonal antiserum active against FliC purified from SJW1103 (MBL Co., Ltd.). The cells were observed using a phase-contrast microscope (CH40; Olympus) at ca. 25°C, and their rotation was recorded on videotape. The rotation rates of individual tethered cells were analyzed by an image-processing program that we developed on the basis of the Igor Pro 5.03j software package (WaveMetrics). Torque calculation is detailed in the supplemental material.

Bead assay. Cells were exponentially grown in T broth at 30°C with shaking. The cells were passed through a 25-gauge needle to partially shear their sticky flagellar filaments. After centrifugation, the cells were resuspended into motility buffer and incubated at 37°C for 30 min. The cells were allowed to settle on and attach to a MAS (Matsunami adhesive saline)-coated coverslip (Matsunami glass). Then, latex beads with diameters of 2.0 µm (Molecular Probes), 1.5 µm (Molecular Probes), or 0.8 µm (Bangs Laboratory) were attached to the filament. The rotation speed was measured by projecting the phase-contrast image of each bead onto a quadrant photodiode through a 60x oil immersion objective lens as described by Sowa et al. (33). All data were recorded at 1.0-ms intervals by a 16-bit A-D board (Microscience), using the LaBDAQ software package (Matsuyama Advance), and analyzed by programs that we developed on the basis of Igor Pro. All experiments were done at ca. 25°C. Torque calculation is described in detail in the supplemental material.


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RESULTS
 
Protein stability and localization of MotB(D33E). The D33E mutation in MotB was originally isolated as a suppressor of an extremely clockwise-biased fliG mutant (38), indicating that the motB(D33E) mutant retains the ability to rotate its motor to some degree. This motB mutant moved very slowly, suggesting that the impaired motility was a consequence of a lowered proton-conducting activity for the MotA/B complex (45). However, there was another possibility that this mutation might influence the targeting of MotB to the cytoplasmic membrane because Asp-33 is located near the cytoplasmic end of its transmembrane segment. Therefore, we first carried out cell fractionation experiments (Fig. 2A). The cells of Salmonella wild-type strain SJW1103 and the motB(D33E) mutant strain MMTB6055 were disrupted by sonication, and the cell lysates were fractionated into the soluble and membrane fractions by ultracentrifugation. Wild-type MotB and MotB(D33E) were both found in the membrane fraction (lane 2). Then, the membrane fraction was treated with either 1.2% sodium lauryl sarcosinate (Sarkosyl) or 1 M NaCl and separated by ultracentrifugation into the pellet and supernatant fractions. Both wild-type MotB and MotB(D33E) were solubilized by Sarkosyl (lanes 5 and 6) but not by 1 M NaCl (lanes 3 and 4). As integral membrane proteins are usually solubilized by Sarkosyl treatment but not by salt treatment, we conclude that MotB(D33E) was expressed from the chromosome at wild-type levels and properly targeted to the cytoplasmic membrane.


Figure 2
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  		FIG. 2. Biochemical properties of MotB(D33E). (A) Subcellular location of MotB. The whole-cell extract of SJW1103 (WT) or MMTB6055 [MotB(D33E)] (lane 1) was separated by ultracentrifugation to isolate the membrane fraction (lane 2). Then, the membrane fraction was treated with either 1 M NaCl (lanes 3 and 4) or 1.2% Sarkosyl (lanes 5 and 6), separated by ultracentrifugation into the pellet (ppt) and supernatant (sup) fractions, and immunoblotted with the anti-MotB antibody. The positions of wild-type MotB and MotB(D33E) are shown by arrowheads. The positions of molecular-mass markers are shown on the left. (B) Interaction of MotB(D33E) with MotA. The membrane fractions from BL21(DE3) cells transformed with pMM650 (encoding MotA and MotB) (lanes 1 and 2), pNSK2 (encoding MotA-His and MotB) (lanes 3 and 4), and pYC9 [encoding MotA-His and MotB(D33E)] (lanes 5 and 6) were solubilized by 0.3% CHAPS and loaded onto Ni+-NTA agarose columns. After being washed, proteins were eluted with 300 mM imidazole. The solubilized fraction loaded on the column (L) and the eluted proteins (E) were analyzed by immunoblotting with the anti-MotB antibody.

Interaction of MotB(D33E) with MotA. It has been shown that several combinations of Cys residues introduced by site-directed mutagenesis into the transmembrane segments MotA-TM3 and MotB-TM give a high yield of disulfide-linked MotA/B heterodimers upon oxidation with iodine (8). In fact, the E. coli motA(P173C) motB(D32C) double mutant strain gave a significant yield of a MotA/B cross-linking product. To test if the D33E mutation of Salmonella affects the MotA-MotB interaction, we carried out pulldown assays. If MotB(D33E) strongly associates with MotA, it should be copurified with a His-tagged variant of MotA by Ni+-NTA affinity chromatography. A His tag was attached to the C terminus of MotA. Membrane fractions were prepared from BL21(DE3) transformed with pYC9, which encodes both C-terminally His-tagged MotA (MotA-His) and untagged MotB(D33E) on pET22b; solubilized by 0.3% CHAPS; and centrifuged. Then, the supernatant fractions were loaded onto a Ni+-NTA column. After being washed, the bound materials were eluted at high imidazole concentrations and analyzed by immunoblotting with the polyclonal anti-MotB antibody (Fig. 2B).

MotB(D33E) was expressed at a level similar to that of wild-type MotB derived from both pMM650 and pNSK2 (Fig. 2B, lanes 1, 3, and 5). In the elution fractions, the amounts of MotB(D33E) were essentially the same as those of MotB obtained from the wild-type MotB-plus-MotA-His sample (lanes 4 and 6), indicating that untagged MotB(D33E) is copurified with MotA-His almost as efficiently as wild-type MotB by Ni+-NTA affinity chromatography. The negative control (lane 2) shows a faint band of MotB, but this is probably due to unavoidable nonspecific protein binding of untagged MotB to Ni+-NTA beads. Therefore, we conclude that the D33E mutation does not affect the interaction with MotA.

Isolation of pseudorevertants from the motB(D33E) mutant. To understand how the D33E mutation considerably interferes with flagellar motor rotation, pseudorevertants were isolated from MMTB6055 [motB(D33E)] by streaking an overnight culture out on soft tryptone agar plates, incubating it at 30°C for 2 days, and looking for motility halos. Seventeen motile colonies were purified from such halos. The motility of these pseudorevertants was better than that of the parent strain although not as good as that of the wild-type strain (Fig. 3A and B).


Figure 3
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  		FIG. 3. Characterization of pseudorevertants isolated from the motB(D33E) mutant. (A) Swarming motility assay of SJW1103 (WT), MMTB6055 [motB(D33E)], MSK001 [motA(V35F) motB(D33E)], MSK002 [motA(V185I) motB(D33E)], MSK003 [motB(D33E/F34L)], MSK004 [motB(D33E/L149V)], and MSK005 [motB(D33E/N278K)] with soft agar plates. The plates were incubated at 30°C for 7 h. (B) Relative swarming rates. The diameter of each swarm ring was normalized to that formed by wild-type cells. Vertical bars are standard errors. (C) Relative torques of tethered cells. Each torque was normalized to that produced by wild-type tethered cells. (D) Motilities of the second-site mutants. Results are shown for a swarming motility assay with cells of an E. coli motA motB double-null strain RP6894 transformed with pTrc99A (V), pNSK9 (encoding wild-type MotA and wild-type MotB), pYC3 [encoding wild-type MotA and MotB(D33E)], pYC4 [encoding MotA(V35F) and wild-type MotB], pYC5 [encoding MotA(V185I) and wild-type MotB], pYC6 [encoding wild-type MotA and MotB(F34L)], pYC7 [encoding wild-type MotA and MotB(L149V)], and pYC8 [encoding wild-type MotA and MotB(N278K)] on soft agar plates. The plates were incubated at 30°C for 14 h.

P22-mediated genetic mapping showed that all suppressor mutations were cotransduced with the D33E mutation (data not shown), indicating that they are located near the first-site mutation. DNA sequence analysis revealed that the mutations are all in motA or motB (Fig. 1A). They are all missense mutations: V35F (isolated nine times) and V185I in MotA and F34L, L149V (isolated five times), and N278K in MotB. The V35F and V185I mutations lie in MotA-TM2 and MotA-TM3, respectively. F34L is located in MotB-TM, while the other two are in the periplasmic domain of MotB.

Rotation rate of tethered cells. To investigate how torque generation is affected by the D33E mutation and the second-site mutations at low speed near stall, cells of the motB(D33E) mutant and its pseudorevertants were tethered to glass slides by a single flagellar filament and their rotation rates were measured (Fig. 3C; also see Table S1 in the supplemental material). The rotation speed of wild-type cells was 8.3 ± 1.0 Hz (calculated torque, 1,490 ± 160 pN nm). In agreement with a previous report (45), the rotation speed of the parent motB(D33E) strain was 5.0 ± 1.1 Hz (calculated torque, 790 ± 230 pN nm). In contrast, for the pseudorevertant cells, the rotation rates were significantly improved (ca. 6.8 to 7.8 Hz), and hence, the torque at low speed near stall was restored nearly to the wild-type level or to halfway between the mutant and wild-type levels.

Free-swimming motility of the pseudorevertants was also observed by high-intensity dark-field microscopy. Unlike wild-type cells, which swam with speeds of 20 ± 8 µm/s (n = 50), only a small number of slowly tumbling cells were observed for the motB(D33E) mutant among the population of the cells. Tumbling cells were seen much more frequently for the suppressor mutants than those of the parent mutant. We also observed very slowly motile cells with average swimming speeds approximately five times slower than that of wild-type cells for all suppressor mutants except for MSK003 [motB(D33E/F34L)], albeit the frequency of such observation was very limited.

As all of the pseudorevertants had as many flagellar filaments as wild-type cells (data not shown) and the tumbling is not due to a high clockwise-counterclockwise switching frequency of the motor, as confirmed by the tethered-cell experiment (data not shown), these data indicate that the high-speed motor rotation of the pseudorevertants under the low-load condition is still severely impaired.

Rotation measurement of single motors by bead assays. To further characterize the effect of the MotB(D33E) mutation and the second-site mutations on the torque generation mechanism, we measured the torque-speed relationship of the wild-type and mutant motors. To measure the torque produced by the motors under a wide range of external-load conditions, we carried out bead assays. To efficiently attach polystyrene beads to flagellar filaments, we introduced a fliC{Delta}(205-293) allele, which lacks residues 205 through 293 in the outer domain D3 of FliC (flagellin) and forms sticky filaments (43), into the motB(D33E) mutant and its pseudorevertants by P22-mediated transduction. Their flagellar filaments were partially sheared off, the filaments were labeled with 0.8-, 1.5-, or 2.0-µm beads, and then rotation measurements were done by projecting the phase-contrast image of each bead onto a quadrant photodiode (Fig. 4; also see Table S2 in the supplemental material).


Figure 4
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  		FIG. 4. Torque-speed relationship of the flagellar motors. Rotation measurements of single flagellar motors were carried out under three different load conditions by tracking the position of a 0.8-µm, 1.5-µm, or 2.0-µm bead attached to the sticky flagellar filament. All the measurements were done at ca. 25°C. Small symbols represent data sets derived from tethered-cell experiments for corresponding strains. WT, wild type.

The rotation rates of the 2.0-, 1.5-, and 0.8-µm beads attached to the sticky filament of the wild-type flagellar motor were 9.3 ± 1.8 Hz (calculated torque, 1,734 ± 333 pN nm), 20.8 ± 3.5 Hz (1,502 ± 248 pN nm), and 79.9 ± 20.6 Hz (1,421 ± 350 pN nm), respectively. In agreement with previous data (11, 12), the torques produced by the wild-type motor are approximately constant over a wide range of motor speeds (Fig. 4). In contrast, the motor speeds of the motB(D33E) mutant were much lower than those of wild-type cells under all of these external-load conditions. The torques produced by the mutant motors labeled with 2.0-µm beads were approximately half of the wild-type level, which is consistent with the data derived from tethered cells (Fig. 3C). When the rotation speeds were increased by lowering the external load, the mutant torques declined steeply and almost linearly (Fig. 4).

The motor speeds of the pseudorevertants were significantly higher than those of the parent strain, although not as high as those of the wild-type strain, under all the external-load conditions that we examined (Fig. 4; also see Table S2 in the supplemental material). However, the torque-speed relationship of the motors of these pseudorevertants showed declines almost the same as or steeper than that of the parent strain (Fig. 4), suggesting that the maximal rate of proton translocation is still limited, although the torques of the motors at a low speed near stall are recovered significantly by the second-site mutations.

Motility of the second-site motA and motB mutants. To examine the effects of the second mutations for motility, we constructed plasmids containing only the second-site motA or motB mutations by site-directed mutagenesis and then analyzed the motility of the E. coli motAB deletion strain RP6894 with these plasmids by using soft tryptone agar plates at 30°C (Fig. 3D). All of the second-site mutants complemented the motAB-null strain for motility at or close to wild-type levels, indicating that no phenotype is associated with them.


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DISCUSSION
 
MotA and MotB form the stator complex of the proton-driven flagellar motor. The MotA/B complex is responsible for proton translocation across the cytoplasmic membrane. A highly conserved and protonatable residue, Asp-33 of MotB, is believed to be involved in the proton relay mechanism (32, 38, 45). An increase in the intracellular proton concentration slows down the flagellar motor rotation, suggesting that a high intracellular proton concentration interferes with proton dissociation from Asp-33 to the cytoplasm (28). Therefore, it seems likely that both protonation and deprotonation of this Asp play key roles in the torque generation cycle. However, it is poorly understood how this process is coupled with torque generation. In this study, we have analyzed the torque-speed relationship of the motors of a motB(D33E) mutant and its pseudorevertants and obtained evidence suggesting that the D33E mutation not only reduces the proton conductivity significantly but also interferes with an actual torque generation step involving stator-rotor interactions considerably.

The MotB(D33E) mutation reduces proton conductivity and the rate of conformational change. Previous studies of motor rotation have given us several important insights into the motor mechanism. At low speed near stall, the motor operates close to thermodynamic equilibrium and hence the torque is affected neither by solvent isotopes nor by temperature (11, 12). In contrast, at high speed, the motor runs far from the equilibrium and hence the torque generated under low-load conditions is dependent on both temperature and solvent isotopes (11, 12). Thus, the intrinsic proton conductivity through the channel limits the rate of torque generation only when the motor operates at high speed under a low-load regime.

In this study, we have analyzed the torque-speed relationship of the motor of a slowly motile motB(D33E) mutant and found that the low-speed, near-stall torque was approximately half of that of the wild-type motor and that high-speed rotation under low-load conditions was severely limited, showing a steep decline in the torque-speed relationship (Fig. 3 and 4). As the D33E mutation reduced the zero-torque speed to ca. 20 Hz, this mutation significantly reduced the rate of the mechanochemical cycle for torque generation. The replacement of Asp-32 with Glu in a MotB-TetA chimera protein, in which the first 60 residues of MotB are fused to a 50-residue sequence encoded by an open reading frame in the tetA gene, has been shown to suppress proton leakage caused by cooverproduction of MotA with the MotB-TetA chimera protein (3, 36, 45). As we confirmed that MotB(D33E) forms a complex with MotA within the cytoplasmic membrane (Fig. 2), the D33E mutation seems to reduce the proton conductivity of the MotA/B complex significantly. Therefore, the reduced zero-torque speed of the mutant motor is likely due to its reduced rate of proton translocation.

On the other hand, the approximately 40% reduction in the zero-speed (stall) torque by the D33E mutation, as obtained by zero-speed extrapolation of the torque-speed curve (Fig. 4), cannot be explained by the reduced proton conductivity, because the rate of the mechanochemical cycle is not limited by the proton conductivity in the low-speed regime near stall (11, 12). This suggests that an actual torque generation step involving stator-rotor interactions is also affected by the D33E mutation. Recently, Inoue et al. (21) observed a similar reduction in the near-stall torque of a Na+-driven chimeric mutant motor by the pomA(R232E) mutation and suggested possible explanations for the reduced stall torque: (i) reduced number of stators, (ii) reduction in sodium motive force, (iii) altered energy-coupling ratio (fewer ions per revolution), and (iv) lower energy-coupling efficiency (ion flow without torque generation) (21). However, our preliminary experiments showed that the D33E mutation affected neither the number of stators installed into the motor nor intracellular pH (data not shown), suggesting that the reduced torque near stall is due to neither a reduced number of stators nor reduced proton motive force.

Proton translocation through the channel of the MotA/B complex is believed to induce conformational changes in MotAC (23), which contains conserved, charged residues responsible for electrostatic interactions with the rotor protein FliG (44). The D32E substitution in E. coli MotB has little effect on the proteolysis pattern of the MotA/B complex in the MotAC portion (23), suggesting that the amplitude of the conformational changes of MotAC coupled with proton translocation is likely to be at wild-type levels. Assuming that the number of protons coupled with each revolution of the motor is not affected by the D33E mutation, the significantly reduced zero-speed torque of the motB(D33E) mutant motor (extrapolated values in Fig. 4) is presumably due to a reduced energy-coupling efficiency, for instance, caused by a reduction in the rate of conformational change for power stroke, which would result in a partial loss of coupling between power stroke and rotor-stator interaction. The reduced zero-speed torque cannot be explained by reduced proton conductivity caused by the D33E mutation, because it does not limit the rate of mechanochemical transitions at extreme low speed near stall (11, 12). The torque-speed curve of the flagellar motor shows relatively constant torques up to a characteristic knee point, beyond which the torque declines sharply as speed increases, and reduction in proton conductivity shifts the knee point toward lower speeds (11, 12) but never reduces the stall torque as long as the energy-coupling efficiency is unchanged.

The occurrence of the motB(L149V) and motB(N278K) suppressor mutations in the periplasmic region of MotB of the pseudorevertants, which are located rather away from the proton pathway, implies that these mutations restore the motor function by acting indirectly rather than by altering the proton-conducting pathway directly. As proper positioning of the MotA/B complex relative to the rotor would require a defined conformation of the C-terminal periplasmic domain of MotB (17-19), the reduced energy-coupling efficiency caused by the D33E mutation may be restored by modifications in the geometrical coupling of the rotor-stator interaction.

The second-site suppressor mutations recover a torque generation step but not proton conductivity. The D33E replacement in MotB reduced motor torque, although both Asp and Glu are acidic residues. Because the side chain of Glu is longer than that of Asp, the shift in the position of the carboxyl group must be interfering with proton binding and/or release at position 33 in MotB, and that is probably why the proton conductivity of the MotA/B complex is significantly reduced by the D33E mutation. Suppressor analysis of the motB(D33E) mutant identified two suppressor mutations in MotA (one in TM2 and one in TM3) and three in MotB (one in MotB-TM and two in the periplasmic domain) (Fig. 1). The improved motility of these suppressor mutants on soft agar plates can be attributed to partial restoration of torque under high-load conditions (Fig. 3 and 4). The motor torque at low speeds near stall appears to be high enough to allow the suppressor mutants to crawl through soft agar for swarming. As the zero-speed torque produced by the pseudorevertants' motors was recovered close to the wild-type level (Fig. 4), the second-site mutations seem to recover a torque generation step involving stator-rotor interactions coupled with proton translocation.

The suppressor mutants failed to swim in liquid media, because the high-speed rotation of their motors is still severely impaired, just as for the parent motB(D33E) mutant (Fig. 4), and the rotation rates under low-load conditions, just for driving the filament rotation, are probably below the critical level required for filament bundle formation. The torques of the wild-type flagellar motor are approximately constant up to a certain speed and then fall approximately linearly to zero (12). The steepness of the linear decline in the torque-speed relationship depends on the proton conductivity of the MotA/B complex (11, 12). Therefore, our present observations regarding the flagellar motor rotations of the pseudorevertants, indicating that their zero-speed torques are recovered almost to wild-type levels while their high-speed rotation rates under low-load conditions are still severely limited (Fig. 4), suggest that the impedance of the proton pathway of the pseudorevertants is still much higher than that of the wild type and hence that the maximal proton conductivities of the motors of the pseudorevertants are limited below the critical level required for high-speed motor rotation for swimming in liquid environments.

Effect of the suppressor mutations. Mutants having only one of the second-site suppressor mutations swarmed at essentially wild-type levels (Fig. 3D); thus, there is no phenotype associated with the suppressor mutations alone. Also, these residues (Val-35 and Val-185 in MotA and Phe-34, Leu-149, and Asn-278 in MotB) are neither acidic nor basic. These results imply that they are not directly involved in the proton-conducting pathway. However, we cannot rule out the possibility that they may function to organize water molecules that contribute to the proton pathway and that Phe-34 could contribute to stabilizing of protons more directly through pi-cation interactions.

The proton conductivity of the MotA/B complex is suppressed by a putative plug segment in the C-terminal periplasmic domain of MotB until the complex is installed into the motor (20). Suppressor hunts similar to what we reported here were done for the E. coli motB(D32E) mutant, and four suppressor mutations identified, A59T, Y61N, F62L, and R63Q, were all in the plug segment of MotB (David Blair, personal communication). Therefore, it is also possible that the D33E mutation may prevent the plug segment from being taken away from the proton pathway, thereby reducing the proton conductivity through the stator complex.

The V35F suppressor mutation in MotA-TM2 affects the physical interface between MotA-TM3, MotA-TM4, and MotB-TM. Braun et al. (8) have shown that MotA-TM3 and MotA-TM4 are close to MotB-TM while MotA-TM1 and MotA-TM2 are in the outer positions of the stator complex (Fig. 1B). Tryptophan-scanning mutageneses have shown that MotA-TM1 and MotA-TM2 are in the outer, lipid-exposed locations of the stator complex. In both MotA-TM3 and MotA-TM4, which are facing the proton pathway, the number of residues that tolerate Trp replacement, which are presumably on the helix surfaces pointing away from the proton pathway, is less than those found in MotA-TM1 and MotA-TM2 (32). These results suggest that the non-pathway-facing sides of MotA-TM3 and MotA-TM4 are surrounded by other protein segments, most likely by MotA-TM1 or MotA-TM2. Here, the V35F mutation, which lies in MotA-TM2 (Fig. 1B), was isolated nine times as a suppressor of the motB(D33E) mutant (Fig. 1A and 3). Since it is impossible for the side chain of Val-35 in MotA-TM2 to directly access the proton-conducting pathway because it is likely to be located within the hydrophobic core of the four-transmembrane {alpha}-helix bundle of MotA, the V35F mutation probably affects the conformational changes of the stator complex for torque generation allosterically.


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ACKNOWLEDGMENTS
 
We thank David Blair and May Macnab for critical reading of the manuscript and helpful comments. We acknowledge David Blair for the gift of RP6894 and provision of unpublished data, the late Robert M. Macnab for the gift of MY6055, Fumiaki Makino and Takayuki Kato for measurement of bead diameters, Yoshiyuki Sowa and Akihiko Ishijima for advice in setting up the bead rotation assay system, and Katsumi Imada and Yumiko Saijo-Hamano for stimulating discussion. We are also grateful to Fumio Oosawa and Kelly T. Hughes for encouragement. Y.-S.C. and S.N. are research fellows of the Japan Society for the Promotion of Science.

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.-S.C., S.K., K.N., and T.M.


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FOOTNOTES
 
* Corresponding author. Mailing address: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-4625. Fax: 81-6-6879-4652. E-mail for T. Minamino: tohru{at}fbs.osaka-u.ac.jp. E-mail for K. Namba: keiichi{at}fbs.osaka-u.ac.jp Back

{triangledown} Published ahead of print on 22 August 2008. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, October 2008, p. 6660-6667, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00503-08
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