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.     

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.     

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.     

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.     

Specific inactivator of flagellar reversal in Salmonella typhimurium.

Specific inhibition of flagellar rotation reversal was observed after exposure of chemotactic Salmonella typhimurium to citrate autoclaved at neutral pH. The presence of a rotation reversal inactivator was established in autoclaved citrate-containing media and nutrient broth. Since modulation of flagellar rotation by attractants and repellents is the basis of chemotactic behavior, a specific inhibitor of rotation reversal, which is essential for tumble generation, provides a useful probe into the molecular mechanism of bacterial chemotaxis. The inactivator inhibits clockwise rotation without affecting counterclockwise rotation, speed of rotation, or the capacity of the cells to grow and divide. Inactivation of clockwise rotation is gradual and irreversible, differing from the transient inhibition of clockwise rotation by attractants, which is characterized by an immediate suppression followed by a return to normal rotation patterns. The rotation reversal inactivator is stable to acidification, rotary evaporation, lyophilization, and rehydration.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

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.     

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.     

Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes.     

Conformational switching in the flagellar filament of Salmonella typhimurium.

The flagellar filament of the mutant Salmonella typhimurium strain SJW814 is straight, and has a right-handed twist like the filament of SJW1655. Three-dimensional reconstructions from electron micrographs of ice-embedded filaments reveal a flagellin subunit that has the same domain organization as that of SJW1655. Both show slight changes from the domain organization of the subunits from SJW1660, which possesses a straight, left-handed filament. This points to the possible role of changes in subunit conformation in the left-to-right-handed structural transition in filaments. Comparison of the left and right-handed filaments shows that the subunit's orientation and intersubunit bonding appear to change. The orientation of the subunit in the SJW814 filament is intermediate between that of SJW1655 and SJW1660. Its intermediate orientation may explain why the filaments of SJW1655 and SJW1660 are locked in one conformation, whereas the filament of SJW814 can be induced to switch by, for example, changes in pH and ionic strength.     

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.     

Operon structure of flagellar genes in Salmonella typhimurium

Summary In Salmonella typhimurium, more than 40 genes have been shown to be involved in flagellar formation and function and almost all of them have been assigned to three regions of the chromosome, termed region I, region II, and region III. In the present study, a large number of transposon-insertion mutants in these flagellar genes were isolated using Tn10 and Mud1. The flaV gene was found to be a strong hot spot for Tn10 insertion. Complementation analysis of the polarity effects exerted by the transposon-insertion mutants defined 13 different flagellar operons; 3 in region I, 4 in region II, and 6 in region III. These results are compared with the reported arrangement of the corresponding genes in Escherichia coli.     

Structural genes for flagellar hook-associated proteins in Salmonella typhimurium.

The flaW, flaU, and flaV genes of Salmonella typhimurium LT2 were cloned into pBR322. These genes were mapped on the cloned DNA fragments by restriction endonuclease analysis and construction of the deletion derivatives. Their gene products were identified, by the minicell method, as proteins whose molecular weights were estimated to be 59,000 for the flaW product, 31,000 for the flaU product, and 48,000 for the flaV product. These values are identical to those of three species of hook-associated proteins (HAPs), namely, HAP1, HAP3, and HAP2. Furthermore, antibodies against HAP1, HAP3, and HAP2 specifically reacted with the gene products of flaW, flaU, and flaV, respectively. Therefore, we concluded that they are structural genes for HAPs. The antibodies against HAP1 and HAP3 also specifically reacted with the gene products of flaS and flaT of Escherichia coli, respectively. This indicates that these gene products are HAPs in E. coli. This result is consistent with the demonstration that flaS and flaT of E. coli are functionally homologous with flaW and flaU of S. typhimurium.     

Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium.

Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure.     

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.     

Excretion of flagellin by a short-flagella mutant of Salmonella typhimurium.

A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (less than 0.1 micron long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.