Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus

The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop(3-4). None of the loop(1-2) residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.     

The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA

PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.     

Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium.

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.     

Multimeric structure of PomA, a component of the Na+-driven polar flagellar motor of vibrio alginolyticus

Four integral membrane proteins, PomA, PomB, MotX, and MotY, are thought to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. Our previous study showed that PomA and PomB form a complex, which catalyzes sodium influx in response to a potassium diffusion potential. PomA forms a stable dimer when expressed in a PomB null mutant. To explore the possible functional dependence of PomA domains in adjacent subunits, we prepared a series of PomA dimer fusions containing different combinations of wild-type or mutant subunits. Introduction of the mutation P199L, which completely inactivates flagellar rotation, into either the first or the second half of the dimer abolished motility. The P199L mutation in monomeric PomA also altered the PomA-PomB interaction. PomA dimer with the P199L mutation even in one subunit also had no ability to interact with PomB, indicating that the both subunits in the dimer are required for the functional interaction between PomA and PomB. Flagellar rotation by wild-type PomA dimer was completely inactivated by phenamil, a sodium channel blocker. However, activity was retained in the presence of phenamil when either half of the dimer was replaced with a phenamil-resistant subunit, indicating that both subunits must bind phenamil for motility to be fully inhibited. These observations demonstrate that both halves of the PomA dimer function together to generate the torque for flagellar rotation.     

Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na(+)-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na(+)-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na(+)-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na(+)-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.     

Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus.

The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motY) encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus. Nucleotide sequence analysis revealed that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is very similar (94.5% identity) to the recently described MotY of V. parahaemolyticus. Their C-terminal domains were similar to the C-terminal domains of many peptidoglycan-interacting proteins, e.g., Escherichia coli MotB and OmpA, suggesting that MotY may interact with peptidoglycan for anchoring the motor. By using the lac promoter-repressor system, motY expression was controlled in V. alginolyticus cells. Swimming ability increased with increasing concentrations of the inducer isopropyl-beta-D-thiogalactopyranoside, and the swimming fraction increased after induction. These results are consistent with the notion that MotY is a component of the force-generating unit. V. alginolyticus motY complemented the motY mutation of V. parahaemolyticus. However, motY appeared to lack a region corresponding to the proposed motY promoter of V. parahaemolyticus. Instead, sequences similar to the sigma54 consensus were found in the upstream regions of both species. We propose that they are transcribed from the sigma54 -specific promoters.     

Multimeric structure of the PomA/PomB channel complex in the Na+-driven flagellar motor of Vibrio alginolyticus

It is known that PomA and PomB form a complex that functions as a Na(+) channel and generates the torque of the Na(+)-driven flagellar motor of Vibrio alginolyticus. It has been suggested that PomA works as a dimer and that the PomA/PomB complex is composed of four PomA and two PomB molecules. PomA does not have any Cys residues and PomB has three Cys residues. Therefore, a mutant PomB (PomB(cl)) whose three Cys residues were replaced by Ala was constructed and found to be motile as well. We carried out gel filtration analysis and examined the effect of cross-linking between the Cys residues of PomB on the formation of the PomA/PomB complex. In the presence of dithiothreitol (DTT), the elution profile of the PomA/PomB complex was shifted to a lower apparent molecular mass fraction similar to that of the complex of the wild-type PomA and PomB(cl) mutant. Next, to analyze the arrangement of PomA molecules in the complex, we introduced the mutation P172C, which has been shown to cross-link PomA molecules, into tandem PomA dimers (PomA approximately PomA). These mutant dimers showed a dominant-negative effect. DTT could restore the function of PomA approximately P172C and P172C approximately P172C, but not P172C approximately PomA. Interdimer and intradimer cross-linked products were observed; the interdimer cross-linked products could be assembled with PomB. The formation of the interdimer cross-link suggests that the channel complex of the Na(+)-driven flagellar motor is composed of two units of a complex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB.     

MotX, the channel component of the sodium-type flagellar motor.

Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.     

Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor gene.

Four motor proteins, MotX, MotY, PomA, and PomB, have been identified as constituents of the Na(+)-driven flagellum of Vibrio species. In this study, the complete motX gene was cloned from Vibrio alginolyticus and shown to complement three mot mutations, motX94, motX115, and motX119, as well as a V. parahaemolyticus motX mutant. The motX94 mutant contains a frameshift at Val86 of MotX, while the motX115 and motX119 mutations comprise substitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX was overexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased, and vice versa, suggesting that MotX and MotY mutually stabilize each other by interacting at the membrane level. When a plasmid containing the motX gene was introduced into motY mutants NMB117 (motY117) and VIO542 (motY542), the mutations were suppressed. In contrast, motY could not cause the recovery of any swarm-defective motX mutants studied. Considering the above evidence, we propose that MotX is more directly involved than MotY in the mechanical functioning of the Na(+)-type flagellar motor, and that MotY may stabilize MotX to support its interaction with other Mot proteins.     

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.     

Intermolecular cross-linking between the periplasmic Loop3-4 regions of PomA, a component of the Na+-driven flagellar motor of Vibrio alginolyticus

PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. PomA has four transmembrane segments. One periplasmic loop (loop(1-2)) connects segments 1 and 2, and another (loop(3-4)), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4. When PomA with an introduced Cys residue (Cys-PomA) in the C-terminal periplasmic loop (loop(3-4)) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl(2). The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility. In the presence of DTT, the ability to swim was partially restored, and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified with N-ethylmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops(3-4) of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.     

MotY, a component of the sodium-type flagellar motor.

Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential. Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined. A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain. Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped. Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This size correlated well with the predicted molecular mass of 33,385 Da. Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility. Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E. coli rnt locus. Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins. To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein. This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.     

Racemization of the amyloidal beta Asp1 residue blocks the acceleration of fibril formation caused by racemization of the Asp23 residue

Amyloid β proteins extracted from the amyloid cores of neuritic plaques are considerably racemized at their Asp residues. To assess the impact of d-Asp on amyloid β_1-42 conformation and on initiation of amyloid fibril formation, we used wild-type amyloid β_1-42 and analogs in which d-Asp was substituted for l-Asp at residues 1, 7, 23, and all combinations of these residues. Amyloid fibril formation was enhanced by d-Asp²³; modulation of Asp chirality at N-terminal position 1 blocked this enhancement and modulation at position 7 augmented it. Knowledge of such chirality modifications may help to develop potent inhibitors of amyloid fibril formation.     

Characterization of PomA mutants defective in the functional assembly of the Na(+)-driven flagellar motor in Vibrio alginolyticus

The polar flagellar motor of Vibrio alginolyticus rotates using Na(+) influx through the stator, which is composed of 2 subunits, PomA and PomB. About a dozen stators dynamically assemble around the rotor, depending on the Na(+) concentration in the surrounding environment. The motor torque is generated by the interaction between the cytoplasmic domain of PomA and the C-terminal region of FliG, a component of the rotor. We had shown previously that mutations of FliG affected the stator assembly around the rotor, which suggested that the PomA-FliG interaction is required for the assembly. In this study, we examined the effects of various mutations mainly in the cytoplasmic domain of PomA on that assembly. All mutant stators examined, which resulted in the loss of motor function, assembled at a lower level than did the wild-type PomA. A His tag pulldown assay showed that some mutations in PomA reduced the PomA-PomB interaction, but other mutations did not. Next, we examined the ion conductivity of the mutants using a mutant stator that lacks the plug domain, PomA/PomB(ΔL)(Δ41-120), which impairs cell growth by overproduction, presumably because a large amount of Na(+) is conducted into the cells. Some PomA mutations suppressed this growth inhibition, suggesting that such mutations reduce Na(+) conductivity, so that the stators could not assemble around the rotor. Only the mutation H136Y did not impair the stator formation and ion conductivity through the stator. We speculate that this particular mutation may affect the PomA-FliG interaction and prevent activation of the stator assembly around the rotor.     

Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.     

MotX and MotY,specific components of the sodium-driven flagellar motor,colocalize to the outer membrane in Vibrio alginolyticus

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.     

Cell-free synthesis of the torque-generating membrane proteins, PomA and PomB, of the Na+-driven flagellar motor in Vibrio alginolyticus

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na(+)-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na(+) channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.     

The MotA protein of E. coli is a proton-conducting component of the flagellar motor

A number of mutants of motA, a gene necessary for flagellar rotation in E. coli, were isolated and characterized. Many mutations were dominant, owing to competition between functional and nonfunctional MotA for a limited number of sites on the flagellar motor. A new class of mutant was discovered in which flagellar torque is normal at low speeds but reduced at high speeds. Hydrogen isotope effects on these mutants indicate that MotA catalyzes proton transfer. We confirmed an earlier observation that overproduction of MotA leads to accumulation of the protein in the cytoplasmic membrane and to significant decreases in growth rate. When nonfunctional mutant variants of MotA were overproduced instead, they accumulated in the cytoplasmic membrane, but growth was not impaired. These results also suggest that MotA conducts protons. This was confirmed by measuring the proton permeabilities of vesicles containing wild-type or mutant MotA proteins.