Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching.

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.     

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.     

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.     

Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.     

Rotation and switching of the flagellar motor assembly in Halobacterium halobium.

Halobacterium halobium swims with a polarly inserted motor-driven flagellar bundle. The swimming direction of the cell can be reserved by switching the rotational sense of the bundle. The switch is under the control of photoreceptor and chemoreceptor proteins that act through a branched signal chain. The swimming behavior of the cells and the switching process of the flagellar bundle were investigated with a computer-assisted motion analysis system. The cells were shown to swim faster by clockwise than by counterclockwise rotation of the flagellar bundle. From the small magnitude of speed fluctuations, it is concluded that the majority, if not all, of the individual flagellar motors of a cell rotate in the same direction at any given time. After stimulation with light (blue light pulse or orange light step-down), the cells continued swimming with almost constant speed but then slowed before they reversed direction. The cells passed through a pausing state during the change of the rotational sense of the flagellar bundle and then exhibited a transient acceleration. Both the average length of the pausing period and the transient acceleration were independent of the stimulus size and thus represent intrinsic properties of the flagellar motor assembly. The average length of the pausing period of individual cells, however, was not constant. The time course of the probability for spontaneous motor switching was calculated from frequency distribution and shown to be independent of the rotational sense. The time course further characterizes spontaneous switching as a stochastic rather than an oscillator-triggered event.     

The Agrobacterium tumefaciens motor gene,motA, is in a linked cluster with the flagellar switch protein genes,fliG, fliM and fliN

We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.© 1997 Elsevier Science B.V. All rights reserved.     

flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor.

The flaAII gene of Salmonella typhimurium has also been termed motC and cheV, because defective alleles may give rise to a nonflagellate, paralyzed, or nonchemotactic phenotype. We isolated a temperature-sensitive motility mutant (MY1) and have found that the mutation occurs in the flaAII gene. In temperature-jump experiments, MY1 could be converted from highly motile to paralyzed within 0.5 s, demonstrating that flaAII is a structural gene whose product is immediately essential for motor rotation. The mutant, although chemotactic at permissive temperatures (less than 36 degrees C), had a higher clockwise rotational bias than did the wild type; it can therefore be regarded simultaneously as motC(Ts) and cheV (tumbly). The only previously reported S. typhimurium cheV mutant was smooth-swimming. A shift toward counterclockwise bias accompanied loss of rotational speed in the restrictive temperature range. This result, by analogy with known proton motive force effects on motor switching, further indicates a central role of the flaAII (motC, cheV) protein in the energy transduction and switching process. Since there is no evidence associating it with the isolable entity known as the basal body, it may reside at the cytoplasmic face of the flagellar motor.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium.

Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.     

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium.

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.     

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.     

Temperature-induced switching of the bacterial flagellar motor.

Chemotaxis signaling proteins normally control the direction of rotation of the flagellar motor of Escherichia coli. In their absence, a wild-type motor spins exclusively counterclockwise. Although the signaling pathway is well defined, relatively little is known about switching, the mechanism that enables the motor to change direction. We found that switching occurs in the absence of signaling proteins when cells are cooled to temperatures below about 10 degrees C. The forward rate constant (for counterclockwise to clockwise, CCW to CW, switching) increases and the reverse rate constant (for CW to CCW switching) decreases as the temperature is lowered. At about -2 degrees C, most motors spin exclusively CW. At temperatures for which reversals are frequent enough to generate a sizable data set, both CCW and CW interval distributions appear to be exponential. From the rate constants we computed equilibrium constants and standard free energy changes, and from the temperature dependence of the standard free energy changes we determined standard enthalpy and entropy changes. Using transition-state theory, we also calculated the activation free energy, enthalpy, and entropy. We conclude that the CW state is preferred at very low temperatures and that it is relatively more highly bonded and restricted than the CCW state.     

The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.     

An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB.

Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM. Most of the suppressing mutations lay in flagellar region IIIb of the chromosome. One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II. SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium. Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state. The location of the mutation further underscores the conclusion from a previous study of spontaneous missense mutants (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically important for switching. All of the second-site mutations in motA and motB caused some impairment of motility, both in the pseudorevertants and in a wild-type fliG background. The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed. The mutations in the MotA and MotB sequences were clustered to a considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB. The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed.