M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF.

The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings. When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane. Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings. Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body. We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body.     

Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na(+)-driven flagella of Vibrio alginolyticus

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na⁺-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.     

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.     

Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium.

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.     

Roles of charged residues of rotor and stator in flagellar rotation: comparative study using H+-driven and Na+-driven motors in Escherichia coli

In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.     

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.     

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.     

Spirochetes flagellar collar protein FlbB has astounding effects in orientation of periplasmic flagella,bacterial shape,motility, and assembly of motors in Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, is a highly motile spirochete, and motility, which is provided by its periplasmic flagella, is critical for every part of the spirochete's enzootic life cycle. Unlike externally flagellated bacteria, spirochetes possess a unique periplasmic flagellar structure called the collar. This spirochete‐specific novel component is linked to the flagellar basal body; however, nothing is known about the proteins encoding the collar or their function in any spirochete. To identify a collar protein and determine its function, we employed a comprehensive strategy that included genetic, biochemical, and microscopic analyses. We found that BB0286 (FlbB) is a novel flagellar motor protein, which is located around the flagellar basal body. Deletion of bb0286 has a profound effect on collar formation, assembly of other flagellar structures, morphology, and motility of the spirochete. Orientation of the flagella toward the cell body is critical for determination of wild‐type spirochete's wave‐like morphology and motility. Here, we provide the first evidence that FlbB is a key determinant of normal orientation of the flagella and collar assembly.     

Variable symmetry in Salmonella typhimurium flagellar motors

Electron cryomicroscopy of rotor complexes of the Salmonella typhimurium flagellar motor, overproduced in a nonmotile Escherichia coli host, has revealed a variation in subunit symmetry of the cytoplasmic ring (C ring) module. C rings with subunit symmetries ranging from 31 to 38 were found. They formed a Gaussian distribution around a mean between 34 and 35, a similar number to that determined for native C rings. C-ring diameter scaled with the number of subunits, indicating that the elliptical-shaped subunits maintained constant intersubunit spacing. Taken together with evidence that the M ring does not correspondingly increase in size, this finding indicates that rotor assembly does not require strict stoichiometric interactions between the M- and C-ring subunits. Implications for motor function are discussed.     

Substructure of the flagellar basal body of Salmonella typhimurium.

The Salmonella typhimurium basal body, a part of the flagellar rotary motor, consists of four rings (denoted M, S, P and L) and a coaxial rod. Using low-dose electron microscopy and image averaging methods on negatively stained and frozen-hydrated preparations, we examined whole basal body complexes and subcomplexes obtained by dissociation in acid. Dissociation occurs in steps, allowing us to obtain images of substructures lacking the M ring, lacking the M and S rings, and lacking the M and S rings and the proximal portion of the rod. We obtained images of the L and P ring subcomplex. The existence of a subcomplex missing only the M ring suggests either that the S and M rings derive from two different proteins, or that the M ring is a labile domain of a single protein, which makes up both rings. At the 25 to 30 A resolution of our averaged images, the L, P and S rings appear cylindrically symmetric. Images of the M ring show variability that may be due to differences in angular orientation of the grid, but equally could be due to structural variations. Three-dimensional reconstructions of these structures from the averaged images reveal the internal structure and spatial organization of these components.     

Regulation cascade of flagellar expression in Gram-negative bacteria

Flagellar motility helps bacteria to reach the most favourable environments and to successfully compete with other micro-organisms. These complex organelles also play an important role in adhesion to substrates, biofilm formation and virulence process. In addition, because their synthesis and functioning are very expensive for the cell (about 2% of biosynthetic energy expenditure in Escherichia coli) and may induce a strong immune response in the host organism, the expression of flagellar genes is highly regulated by environmental conditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellated bacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largely unknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of the cascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatory regions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similar hierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests an evolutionary conservation of regulatory mechanisms in Gram-negative bacteria.     

Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators.

The FliM protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of controlled low-level expression of FliM in a fliM null strain. Disruption of the fliM gene abolishes flagellation. Underexpression of FliM causes cells to produce comparatively few flagella, and most flagella built are defective, producing subnormal average torque and fluctuating rapidly in speed. The results imply that in a normal flagellar motor, multiple molecules of FliM are present and can function independently to some degree. The speed fluctuations indicate that stable operation requires most, possibly all, of the normal complement of FliM. Thus, the FliM subunits are not as fully independent as the motility proteins MotA and MotB characterized in earlier work, suggesting that FliM occupies a location in the motor distinct from the MotA/MotB torque generators. Several mutations in fliM previously reported to cause flagellar paralysis in Salmonella typhimurium (H. Sockett, S. Yamaguchi, M. Kihara, V.M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992) were made and characterized in E. coli. These mutations did not cause flagellar paralysis in E. coli; their phenotypes were more complex and suggest that FliM is not directly involved in torque generation.     

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.     

Bacterial chemotaxis in an optical trap

An optical trapping technique is implemented to investigate the chemotactic behavior of a marine bacterial strain Vibrio alginolyticus. The technique takes the advantage that the bacterium has only a single polar flagellum, which can rotate either in the counter-clock-wise or clock-wise direction. The two rotation states of the motor can be readily and instantaneously resolved in the optical trap, allowing the flagellar motor switching rate S(t) to be measured under different chemical stimulations. In this paper the focus will be on the bacterial response to an impulsive change of chemoattractant serine. Despite different propulsion apparati and motility patterns, cells of V. alginolyticus apparently use a similar response as Escherichia coli to regulate their chemotactic behavior. Specifically, we found that the switching rate S(t) of the bacterial motor exhibits a biphasic behavior, showing a fast initial response followed by a slow relaxation to the steady-state switching rate S0. The measured S(t) can be mimicked by a model that has been recently proposed for chemotaxis in E. coli. The similarity in the response to the brief chemical stimulation in these two different bacteria is striking, suggesting that the biphasic response may be evolutionarily conserved. This study also demonstrated that optical tweezers can be a useful tool for chemotaxis studies and should be applicable to other polarly flagellated bacteria.     

A Caulobacter gene involved in polar morphogenesis.

At specific times in the cell cycle, the bacterium Caulobacter crescentus assembles two major polar organelles, the flagellum and the stalk. Previous studies have shown that flbT mutants overproduce flagellins and are unable to form chemotaxis swarm rings. In this paper, we report alterations in both the stalk and the flagellar structure that result from a mutation in the flagellar gene flbT. Mutant strains produce some stalks that have a flagellum, produce some stalks that have an extra lobe protruding from their sides, have filaments lacking the 29-kilodalton flagellin, and produce several unusual cell types, including filamentous cells as well as predivisional cells with two stalks and predivisional cells with no stalk at all. We propose that flagellated stalks arise as a consequence of a failure to eject the flagellum at the correct time in the cell cycle and that the extra stalk lobe is due to a second site for the initiation of stalk biogenesis. Thus, a step in the pathway that establishes the characteristic asymmetry of the C. crescentus cell appears to be disrupted in flbT mutants. We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle. The polar particles appear as a cluster of approximately 1 to 10 stain-excluding rings, visible in electron micrographs of negatively stained wild-type cells. This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells.     

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.     

Starvation-Induced Changes in Motility, Chemotaxis, and Flagellation of Rhizobium meliloti

The changes in motility, chemotactic responsiveness, and flagellation of Rhizobium meliloti RMB7201, L5-30, and JJ1c10 were analyzed after transfer of the bacteria to buffer with no available C, N, or phosphate. Cells of these three strains remained viable for weeks after transfer to starvation buffer (SB) but lost all motility within just 8 to 72 h after transfer to SB. The rates of motility loss differed by severalfold among the strains. Each strain showed a transient, two- to sixfold increase in chemotactic responsiveness toward glutamine within a few hours after transfer to SB, even though motility dropped substantially during the same period. Strains L5-30 and JJ1c10 also showed increased responsiveness to the nonmetabolizable chemoattractant cycloleucine. Cycloleucine partially restored the motility of starving cells when added after transfer and prevented the loss of motility when included in the SB used for initial suspension of the cells. Thus, interactions between chemoattractants and their receptors appear to affect the regulation of motility in response to starvation independently of nutrient or energy source availability. Electron microscopic observations revealed that R. meliloti cells lost flagella and flagellar integrity during starvation, but not as fast, nor to such a great extent, as the cells lost motility. Even after prolonged starvation, when none of the cells were actively motile, about one-third to one-half of the initially flagellated cells retained some flagella. Inactivation of flagellar motors therefore appears to be a rapid and important response of R. meliloti to starvation conditions. Flagellar-motor inactivation was at least partially reversible by addition of either cycloleucine or glucose. During starvation, some cells appeared to retain normal flagellation, normal motor activity, or both for relatively long periods while other cells rapidly lost flagella, motor activity, or both, indicating that starvation-induced regulation of motility may proceed differently in various cell subpopulations.     

The ability of Proteus mirabilis to sense surfaces and regulate virulence gene expression involves FliL, a flagellar basal body protein

Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL- mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.