Robert M. Macnab

Normal-to-curly flagellar transitions and their role in bacterial tumbling. Stabilization of an alternative quaternary structure by mechanical force*

Tumbling, a characteristic feature of the motility and chemotactic response of peritrichously flagellated bacteria, has been examined by dark-field light microscopy and found to be intimately linked to major changes in the quaternary structure of the flagella. The normal structure of the flagella of Salmonella is a left-handed helix of wavelength 2·3 μm and diameter 0·4 μm. Under right-handed torsion generated when the rotary motor is operating in reverse (i.e. in a clockwise sense), the normal structure undergoes a discrete transition to an alternative structure, designated “curly”, which is a right-handed helix of wavelength 1·1 μm and diameter 0·3 μm. The transition proceeds rapidly from the cell body outward but is, at any instant, confined to a very short region of the flagellum. The heteromorphous structure of a flagellum in transition consists of two portions, normal and curly, with a change in axial direction of 64° occurring at the transition region. The chaotic motion of the cell body, in reaction to a number of flagella which are rotating and in transition, constitutes the tumble. The induction of polymorphic transitions by mechanical force is confirmed by studies with motor and flagellar mutants, and with cells converted to curly flagellar phenotype by p -fluorophenylalanine. The phenomenon of polymorphic transitions was examined most extensively in Salmonella , but is also noted in Bacillus subtilis and Escherichia coli and may therefore be a general feature of bacterial flagellar structure. The right-handedness of the curly structures induced mechanically or by incorporation of p -fluorophenylalanine is also found for the curly structures caused by mutation or pH/ionic strength changes (Shimada et al. , 1975). The reasons for the specific interaction between mechanical and molecular forces are discussed.

Interactions among components of the Salmonella flagellar export apparatus and its substrates

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild‐type cells and inhibition of the export of substrates such as the hook protein FlgE. Co‐overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His‐FLAG‐tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook‐type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament‐type export substrates FlgK and FlgL (hook–filament junction proteins) and FliC (flagellin). We tested for protein–protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook‐type substrates were strong, whereas those with filament‐type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP‐dependent process mediated by FliI.

Domain Structure of Salmonella FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching

We have investigated the properties of the cytoplasmic domain (FlhB(C)) of the 383-amino-acid Salmonella membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB(C) was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB(CN) and FlhB(CC), which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB(delta CC) (i.e., the N-terminal transmembrane domain FlhB(TM) plus FlhB(CN)) and FlhB(CC), resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB(C), existing as a FlhB(CN)-FlhB(CC) complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB(C) (mutant or wild type) or the FlhB(CC) polypeptide alone bound FlgD and FliC to about the same extent. FlhB(CN) by itself did not bind substrates appreciably. We propose that FlhB(C) has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB(CN) and FlhB(CC), is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB(C) resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.

FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase activity

Both FliH and the ATPase FliI are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N‐terminus of FliI interacts with FliH and that this interaction is important for the ATPase function of the C‐terminal domain of FliI. N‐terminally histidine‐tagged, wild‐type FliI retarded untagged FliH in a Ni‐NTA affinity chromatography assay, as did N‐His‐tagged versions of FliI carrying catalytic mutations. In contrast, N‐His‐tagged FliI carrying the double mutation R7C/L12P did not, further indicating that the N‐terminus of FliI is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and FliI form a complex. Analytical gel filtration with in‐line multiangle light scattering indicated that FliH alone forms a dimer, FliI alone remains as a monomer, and FliH and FliI together form a (FliH)2FliI complex. Ni‐NTA affinity chromatography using N‐His‐tagged FliH and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH–FliI complex was about 10‐fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.

ENZYMATIC CHARACTERIZATION OF FLII : AN ATPASE INVOLVED IN FLAGELLAR ASSEMBLY IN SALMONELLA TYPHIMURIUM

FliI is a protein needed for flagellar assembly in Salmonella typhimurium. It shows sequence similarity to the catalytic β subunit of the F0F1-ATPase and is even more closely related to putative ATPases in Type III bacterial secretory pathways. A His-tagged version of FliI, which was fully functional in complementation tests, was purified to homogeneity. It had an ATPase activity of 0.16 s−1 at 25°C and pH 7, and a Km for ATP of 0.3 mM; Mg2+ was required. The activity was not affected by inhibitors of the F-, V- or P-type ATPases, or inhibitors of the Type I or Type II bacterial secretory pathways. Mutations K188I and Y363S decreased the ATPase activity about 100-fold, increased the Km about 10-fold, blocked flagellar assembly, and were dominant. Other FliI mutations that disrupted flagellar protein export were found near the N terminus; they permitted essentially wild-type ATPase activity, were not dominant, and showed a dosage-dependent phenotype. We propose that FliI has a C-terminal ATPase domain and an N-terminal domain that interacts with other components in the flagellum-specific export apparatus.

Stoichiometric analysis of the flagellar hook-(basal-body) complex of Salmonella typhimurium

The stoichiometries of components within the flagellar hook-(basal-body) complex of Salmonella typhimurium have been determined. The hook protein (FlgE), the most abundant protein in the complex, is present at approximately 130 subunits. Hook-associated protein 1 (FlgK) is present at approximately 12 subunits. The distal rod protein (FlgG) is present at approximately 26 subunits, while the proximal rod proteins (FlgB, FlgC and FlgF) are present at only approximately six subunits each. The stoichiometries of the proximal rod proteins and hook-associated protein 1 are, within experimental error, consistent with values of 5 or 6, and 11, respectively. Such values would correspond to either one or two turns of a helical structure with a basic helix of approximately 5.5 subunits per turn, which is the geometry of both the hook and the filament and, one supposes, the rod and hook-associated proteins. These stoichiometries may derive from rules for the heterologous interactions that occur when a helical structure consists of successive segments constructed from different proteins; the stoichiometries within the hook and the distal portion of the rod must, however, be set by different mechanisms. The stoichiometries for the ring proteins are approximately 26 subunits each for the M-ring protein (FliF), the P-ring protein (FlgI), and the L-ring protein (FlgH); the protein responsible for the S-ring feature is not known. The rings presumably have rotational rather than helical symmetry, in which case the stoichiometries would be directly constrained by the intersubunit bonding angle. The ring stoichiometries are discussed in light of other information concerning flagellar structure and function.

FLIK, THE PROTEIN RESPONSIBLE FOR FLAGELLAR HOOK LENGTH CONTROL IN SALMONELLA, IS EXPORTED DURING HOOK ASSEMBLY

In wild‐type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at ≈ 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild‐type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook‐filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N‐terminal region of FliK, but not by C‐terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook‐length control and the switching of export substrate specificity.

Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum

Within the bacterial flagellum the basal-body rod, the hook, the hook-associated proteins (HAPs), and the helical filament constitute an axial substructure whose elements share structural features and a common export pathway. We present here the amino acid sequences of the hook protein and the three HAPs of Salmonella typhimurium, as deduced from the DNA sequences of their structural genes (flgE, flgK, flgL and fliD, respectively). We compared these sequences with each other and with those for the filament protein (flagellin) and four rod proteins, which have been described previously (Joys, 1985; Homma et al., 1990; Smith & Selander, 1990). Hook protein most strongly resembled the distal rod protein (FlgG) and the proximal HAP (HAP1), which are thought to be attached to the proximal and distal ends of the hook, respectively; the similarities were most pronounced near the N and C termini. Hook protein and flagellin, which occupy virtually identical helical lattices, did not resemble each other strongly but showed some limited similarities near their termini. HAP3 and HAP2, which form the proximal and distal boundaries of the filament, showed few similarities to flagellin, each other, or the other axial proteins. With the exceptions of the N-terminal region of HAP2, and the C-terminal region of flagellin, proline residues were absent from the terminal regions of the axial proteins. Moreover, with the exception of the N-terminal region of HAP2, the terminal regions contained hydrophobic residues at intervals of seven residues. Together, these observations suggest that the axial proteins may have amphipathic alpha-helical structure at their N and C termini. In the case of the filament and the hook, the terminal regions are believed to be responsible for the quaternary interactions between subunits. We suggest that this is likely to be true of the other axial structures as well, and specifically that interaction between N-terminal and C-terminal alpha-helices may be important in the formation of the axial structures of the flagellum. Although consensus sequences were noted among some of the proteins, such as the rod, hook and HAP1, no consensus extended to the entire set of axial proteins. Thus the basis for recognition of a protein for export by the flagellum-specific pathway remains to be identified.

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body.

Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum‐specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS‐ring protein FliF and immunoblotting isolated hook basal body complexes with anti‐FLAG monoclonal antibody, we estimate (using the FLAG‐tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti‐FLAG antibody and gold–protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.

Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB

FlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella. It permits export of rod/hook‐type proteins before hook completion, whereupon it switches specificity to recognize filament‐type proteins. The cytoplasmic C‐terminal domain of FlhB (FlhBC) is cleaved between Asn‐269 and Pro‐270, defining two subdomains: FlhBCN and FlhBCC. Here, we show that subdomain interactions and cleavage within FlhB are central to substrate‐specificity switching. We found that deletions between residues 216 and 240 of FlhBCN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn‐269 and Pro‐270 abolished both. The mutation N269A prevented cleavage at the FlhBCN–FlhBCC boundary. Cells producing FlhB(N269A) exported the same amounts of hook‐capping protein as cells producing wild‐type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament‐type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook‐type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.