Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Temperature-hypersensitive sites of the flagellar switch component FliG in Salmonella enterica serovar typhimurium

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla(-), Mot(-), and Che(-)), depending on the sites and types of mutations. Some of the Mot(-) mutants previously characterized are found to be motile. Reexamination of all Mot(-) mutants in fliGMN genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20 degrees C but nonmotile at 37 degrees C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of fliM and fliN and most of the fliG TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the fliG mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.     

Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium.

Defects in the chemotaxis proteins CheY and CheZ of Salmonella typhimurium can be suppressed by mutations in the flagellar switch, such that swarming of a pseudorevertant on semisolid plates is significantly better than that of its parent. cheY suppressors contribute to a clockwise switch bias, and cheZ suppressors contribute to a counterclockwise bias. Among the three known switch genes, fliM contributes most examples of such suppressor mutations. We have investigated the changes in FliM that are responsible for suppression, as well as the changes in CheY or CheZ that are being compensated for. Ten independently isolated parental cheY mutations represented nine distinct mutations, one an amino acid duplication and the rest missense mutations. Several of the altered amino acids lie on one face of the three-dimensional structure of CheY (A. M. Stock, J. M. Mottonen, J. B. Stock, and C. E. Schutt, Nature (London) 337:745-749, 1989; K. Volz and P. Matsumura, J. Biol. Chem. 266:15511-15519, 1991); this face may constitute the binding site for the switch. All 10 cheZ mutations were distinct, with several of them resulting in premature termination. cheY and cheZ suppressors in FliM occurred in clusters, which in general did not overlap. A few cheZ suppressors and one cheY suppressor involved changes near the N terminus of FliM, but neither cheY nor cheZ suppressors involved changes near the C terminus. Among the strongest cheY suppressors were changes from Arg to a neutral amino acid or from Val to Glu, suggesting that electrostatic interactions may play an important role in switching. A given cheY or cheZ mutation could be suppressed by many different fliM mutations; conversely, a given fliM mutation was often encountered as a suppressor of more than one cheY or cheZ mutation. The data suggest that an important factor in suppression is a balancing of the shift in switch bias introduced by alteration of CheY or CheZ with an appropriate opposing shift introduced by alteration of FliM. For strains with a severe parental mutation, such as the cheZ null mutations, adjustment of switch bias is essentially the only factor in suppression, since the attractant L-aspartate caused at most a slight further enhancement of the swarming rate over that occurring in the absence of a chemotactic stimulus. We discuss a model for switching in which there are distinct interactions for the counterclockwise and clockwise states, with suppression occurring by impairment of one of the states and hence by relative enhancement of the other state. FliM can also undergo amino acid changes that result in a paralyzed (Mot-) phenotype; these changes were confined to a very few residues in the protein.     

Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization of the switch complex

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In Escherichia coli and Salmonella, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an alpha-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ(125-128) and R(160) (the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione S-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.     

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.     

Overproduced Salmonella typhimurium flagellar motor switch complexes

Three Salmonella typhimurium flagellar motor proteins, FliG, FliM and FliN, are required for the switching of rotation sense. The proteins have been localized to the cytoplasmic module of the flagellar base. Structures, which were morphologically indistinguishable from the native transmembrane MS-ring and cytoplasmic C-ring basal body modules, formed in Escherichia coli upon plasmid-encoded synthesis of these proteins together with FliF. The structures localized to the cell membrane and contained all three motor proteins, as determined by immuno-electron microscopy. This result supports the deduction, based on earlier biochemical analysis, that the C-ring is composed entirely of these proteins and, therefore, functions as a dedicated motor component. In addition, it demonstrates that the morphologically correct assembly of the C-ring onto the MS-ring proceeds independently of other structural components of these modules.     

Genetic and behavioral analysis of flagellar switch mutants of Salmonella typhimurium.

At the interface between the sensory transduction system and the flagellar motor system of Salmonella typhimurium, the switch complex plays an important role in both sensory transduction and energy transduction. To examine the function of the switch complex, we isolated from 10 cheY mutants 500 pseudorevertants with a suppressor mutation in one of the three genes (fliG, fliM, and fliN) encoding the switch complex. Detailed mapping revealed that these suppressor mutations were localized to several segments of each switch gene, suggesting localization of functional sites on the switch complex. These switch mutations were introduced into the wild-type background and into a chemotaxis deletion background. Behavior of the pseudorevertants and their derivatives (1,500 strains in all) was observed by light microscopy. In the chemotaxis deletion background, about 70% of the switch mutants showed smooth swimming and the rest showed more or less tumbly swimming. There was some correlation between the mutational sites and the swimming patterns in the chemotaxis deletion background, suggesting that there is segregation of functional sites on the switch complex. The interaction of the switch complex with the chemotaxis protein, CheY, and the stochastic nature of switching in the absence of CheY are discussed.     

Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG

The FliG protein is essential for assembly, rotation and clockwise/counter-clockwise (CW/CCW) switching of the bacterial flagellum. About 25 copies of FliG are present in a large rotor-mounted assembly termed the 'switch complex', which also contains the proteins FliM and FliN. Mutational studies have identified the segments of FliG most crucial for flagellar assembly, rotation and switching. The structure of the C-terminal domain, which functions specifically in rotation, was reported previously. Here, we describe the crystal structure of a larger fragment of the FliG protein from Thermotoga maritima, which encompasses the middle and C-terminal parts of the protein (termed FliG-MC). The FliG-MC molecule consists of two compact globular domains, linked by an alpha-helix and an extended segment that contains a well-conserved Gly-Gly motif. Mutational studies indicate that FliM binds to both of the globular domains, and given the flexibility of the linking segment, FliM is likely to determine the relative orientation of the domains in the flagellum. We propose a model for the organization of FliG-MC molecules in the flagellum, and suggest that CW/CCW switching might occur by movement of the C-terminal domain relative to other parts of FliG, under the control of FliM.     

Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in highly viscous media and blocks its infectivity

The flagellar motor switch complex protein FliG plays an essential role in flagella biosynthesis and motility. In most motile bacteria, only one fliG homologue is present in the genome. However, several spirochete species have two putative fliG genes (referred to as fliG1 and fliG2) and their roles in flagella assembly and motility remain unknown. In this report, the Lyme disease spirochete Borrelia burgdorferi was used as a genetic model to investigate the roles of these two fliG homologues. It was found that fliG2 encodes a typical motor switch complex protein that is required for the flagellation and motility of B. burgdorferi. In contrast, the function of fliG1 is quite unique. Disruption of fliG1 did not affect flagellation and the mutant was still motile but failed to translate in highly viscous media. GFP‐fusion and motion tracking analyses revealed that FliG1 asymmetrically locates at one end of cells and the loss of fliG1 somehow impacted one bundle of flagella rotation. In addition, animal studies demonstrated that the fliG1? mutant was quickly cleared after inoculation into the murine host, which highlights the importance of the ability to swim in highly viscous media in the infectivity of B. burgdorferi and probably other pathogenic spirochetes.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

FliG subunit arrangement in the flagellar rotor probed by targeted cross-linking

FliG is a component of the switch complex on the rotor of the bacterial flagellum. Each flagellar motor contains about 25 FliG molecules. The protein of Escherichia coli has 331 amino acid residues and comprises at least two discrete domains. A C-terminal domain of about 100 residues functions in rotation and includes charged residues that interact with the stator protein MotA. Other parts of the FliG protein are essential for flagellar assembly and interact with the MS ring protein FliF and the switch complex protein FliM. The crystal structure of the middle and C-terminal parts of FliG shows two globular domains joined by an alpha-helix and a short extended segment that contains two well-conserved glycine residues. Here, we describe targeted cross-linking studies of FliG that reveal features of its organization in the flagellum. Cys residues were introduced at various positions, singly or in pairs, and cross-linking by a maleimide or disulfide-inducing oxidant was examined. FliG molecules with pairs of Cys residues at certain positions in the middle domain formed disulfide-linked dimers and larger multimers with a high yield, showing that the middle domains of adjacent subunits are in fairly close proximity and putting constraints on the relative orientation of the domains. Certain proteins with single Cys replacements in the C-terminal domain formed dimers with moderate yields but not larger multimers. On the basis of the cross-linking results and the data available from mutational and electron microscopic studies, we propose a model for the organization of FliG subunits in the flagellum.     

Analysis of a FliM-FliN flagellar switch fusion mutant of Salmonella typhimurium.

In the course of an analysis of the three genes encoding the flagellar motor switch, we isolated a paralyzed mutant whose defect proved to be a 4-bp deletion of the ribosome binding sequence of the fliN switch gene (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810,1993). This sequence lies just before the 3' end of the coding sequence of the upstream fliM switch gene, in the same operon. This mutant readily gave rise to pseudorevertants which, though much less motile than the wild type, did exhibit significant swarming. One such pseudorevertant was found to contain a compensating frameshift such that the fliM and fliN genes were placed in frame, coding for an essentially complete FliM-FliN protein fusion. Minicell analysis demonstrated that, as expected, the parental mutant synthesized an essentially full-length FliM protein but no detectable FliN. The pseudorevertant, in contrast, synthesized a protein with the predicted size for the FliM-FliN fusion protein and no detectable FliM or FliN. Immunoblotting of minicells with antibodies against FliM and FliN confirmed the identities of these various proteins. Immunoblotting of book-basal-body complexes from the wild-type strain gave a strong signal for the three switch proteins FliG, FliM, and FliN. Complexes from the FliM-FliN fusion mutant gave a strong signal for FliG but no signal for either FIiM or FliN; a moderately strong signal for the FliM-FliN fusion protein was seen with the anti-FliM antibody, and a weaker signal was seen with the anti-FliN antibody. The cytoplasmic C ring of the structure, which is seen consistently in electron microscopy of wild-type complexes and which is known to contain the FliM and FliN proteins, was much more labile in the FliM-FliN fusion mutant, giving a fragmented and variable appearance or being completely absent. Complementation data indicated that wild-type FliM had a mild dominant negative effect over the fusion protein, that wild-type FliN and the fusion protein work much better than the fusion protein alone, and that wild-type FliM and FliN together have no major positive or negative effect on the function of the fusion protein. We interpret these data to mean that the FliM-FliN fusion protein incorporates into structure but less stably than do the FliM and FliN proteins separately, that wild-type FliM tends to displace the fusion protein, and that wild-type FliN can supplement the FliN domain of the fusion protein without displacing the FliM domain. The data support, but do not prove, a model in which FliM and FliN in the wild-type switch complex are stationary with respect to each other.     

Domain organization of flagellar hook protein from Salmonella typhimurium

Hook forms a universal joint, which mediates the torque of the flagellar motor to the outer helical filaments. Domain organization of hook protein from Salmonella typhimurium was investigated by exploring thermal denaturation properties of its proteolytic fragments. The most stable part of hook protein involves residues 148 to 355 and consists of two domains, as revealed by deconvolution analysis of the calorimetric melting profiles. Residues 72-147 and 356-370 form another domain, while the terminal regions of the molecule, residues 1-71 and 371-403, avoid a compact tertiary structure in the monomeric state. These folding domains were assigned to the morphological domains of hook subunits known from EM image reconstructions, revealing the overall folding of hook protein in its filamentous state.     

Role of FliJ in flagellar protein export in Salmonella

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid Salmonella protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30 degrees C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30 degrees C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42 degrees C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.     

Multiple conformations of the FliG C-terminal domain provide insight into flagellar motor switching

Bacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliG are closely associated with this switching mechanism. We present two crystal structures of FliG(MC) from Helicobacter pylori, each showing distinct domain orientations from previously solved structures. A 180° rotation of the charged ridge-containing C-terminal subdomain FliG(Cα1-6) that is prompted by the rotational freedom of Met245 psi and Phe246 phi at the MFXF motif was revealed. Studies on the swarming and swimming behavior of Escherichia coli mutants further identified the importance of the ???MFXF??? motif and a highly conserved residue, Asn216, in motor switching. Additionally, multiple conformations of FliG(Cα1-6) were demonstrated by intramolecular cysteine crosslinking. The conformational flexibility of FliGc leads us to propose a model that accounts for the symmetrical torque generation process and for the dynamics of the motor.     

Structural insights into the interaction between the bacterial flagellar motor proteins FliF and FliG

The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliG(N)) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ~40 amino acids of FliF (FliF(C)) interact strongly (upper bound K(d) in the low nanomolar range) with FliG(N). The formation of this complex causes extensive conformational changes in FliG(N). We find that T. maritima FliG(N) is homodimeric in the absence of the FliF(C) peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliF(C) mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliF(C) is located on a conserved hydrophobic patch centered along helix 1 of FliG(N). These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring's precise molecular structure and mechanism of rotational switching.     

Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB

Salmonella FliR and FlhB are membrane proteins necessary for flagellar export. In Clostridium a fliR-flhB fusion gene exists. We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio.