Noreen R. Francis

The Three-Dimensional Structure of the Flagellar Rotor from a Clockwise-Locked Mutant of Salmonella enterica Serovar Typhimurium

Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM.

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.

Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate

Angle-layered aggregates of F-actin are net-like structures induced by Mg2+ concentrations below that used to form paracrystals. These aggregates incorporate the angular disorder of subunits, which has been described elsewhere for isolated actin filaments. Because this disorder is incorporated into the aggregates in solution at the time they are formed, the possibility of negative stain preparation being responsible for the disorder is excluded. The simple two-layered geometry of the angle-layered aggregate provides information about the shape of the component actin filaments free from the superposition of large numbers of layers. A model for the filament shape, derived from single filaments and confirmed by the angle-layered aggregate, is different from those that have previously emerged from paracrystal studies. An understanding of the interfilament bond in both the angle-layered aggregate and the paracrystal allows one to reconcile these different models. We have found a bipolar bonding rule, with staggered crossover points in the angle-layered aggregate, which we suggest is also responsible for Mg2+ paracrystals. This bonding rule can explain the apparent alignment of crossover points in adjacent filaments in paracrystals as a consequence of the superposition of staggered filaments.

The bacterial flagellar switch complex is getting more complex

The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane‐bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.

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.

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Escherichia coli chemotaxis is mediated by membrane receptor/histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTarC, that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTarC. Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.

Size of the export channel in the flagellar filament of Salmonella typhimurium

The size of the putative export channel in the bacterial flagellar filament appears small (25 A) in studies done by electron microscopy but large (60 A) in studies done by X-ray diffraction. We have undertaken additional studies by electron microscopy to examine some of the possible causes of the difference. A comparison of three-dimensional image reconstructions of native and reconstituted filaments rules out the presence or absence of flagellin monomers in the export channel as the source of the variation in apparent channel size. The channel seen in reconstructions from both kinds of filaments is 25 A in diameter. The difference in the previous studies is more probably a result of artifacts introduced in either the X-ray or the electron microscopical methodology. Comparisons of three-dimensional reconstructions from images of filaments embedded in various stains (anionic, cationic and neutral) and in ice, taken at a range of defocuses, rule out the two most likely sources of artifact in electron microscopy (i.e., staining artifacts and defocus phase contrast). Based on these studies we suggest that the channel seen in the image reconstructions is free of exported flagellin monomers, that its true diameter is about 25 A, and, therefore, that the flagellin monomer must be unfolded to pass along it.

A polymorphism peculiar to bipolar actin bundles.

Both muscle and nonmuscle actins produced magnesium paracrystals which we found indistinguishable from one another. Contrary to some previous reports, calcium ions caused no change in filament organization for either type of actin. The most ordered paracrystals consisted of hexagonally packed filaments with opposite polarities. We suggest that this mode of packing permits a form of disorder not previously described, which may account for some puzzling aspects of earlier observations and may prove useful in analyzing actin bundles formed, for example, with erythrocyte band 4.9 protein.

α-Ketoglutarate dehydrogenase complex may be heterogeneous in quaternary structure*

The quaternary structure of the alpha-ketoglutarate dehydrogenase complex (KGDC) from Escherichia coli has been investigated by electron microscopy. KGDC consists of an octahedral cube-shaped structural core, lipoyl transsuccinylase (E2), to which 12 polypeptide chains each of alpha-ketoglutarate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) are non-covalently bound. The analysis was greatly simplified by analyzing subcomplexes of KGDC prepared by assembly of the purified component enzymes in vitro; the subcomplexes consisted of the E2 component to which only a few E1 or E3 subunits were attached. We find that both the E1 and E3 bind on the surface of the E2 molecule approximately midway between the 4-fold and 2-fold symmetry axes of E2. There are 24 such positions per E2 molecule but, based upon the observed stoichiometries of the component enzymes, it is clear that at least half of these sites are unoccupied in KGDC. If KGDC possesses symmetry, then a mechanism must exist for selecting a symmetrically distributed subset of the potential binding sites for the E1 and E3. However, analysis of images of subcomplexes in which two E1 or E3 subunits are present suggests that binding to the E2 occurs through random selection of the potential binding sites. If native KGDC is assembled by such a mechanism, then KGDC would not have a unique quaternary structure, but instead would consist of a family of structural isomers having up to approximately 125,000 members. Consideration of independent structural and biochemical data regarding the mechanism of action of the E2 indicates that the kind of structural heterogeneity being proposed is consistent with a functional KGDC.

Role of excess lipoyl dehydrogenase in reconstituted α-ketoglutarate dehydrogenase complex of Escherichiacoli

Abstract The α-ketoglutarate dehydrogenase complex of Escherichia coli can bind up to 12 dimers of dihydrolipoyl dehydrogenase (E3) besides those already present. Maximal activity does not increase, however, when surplus E3 is present. This observation was previously interpreted to mean that the excess enzyme is inactive. We have now determined that if the reactions catalyzed by E3 are made rate-limiting, the excess E3 functions equivalently to that in the native complex.