Dennis R. Thomas

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.

Assembly of the inner rod determines needle length in the type III secretion injectisome

Assembly of multi-component supramolecular machines is fundamental to biology, yet in most cases, assembly pathways and their control are poorly understood. An example is the type III secretion machine, which mediates the transfer of bacterial virulence proteins into host cells. A central component of this nanomachine is the needle complex or injectisome, an organelle associated with the bacterial envelope that is composed of a multi-ring base, an inner rod, and a protruding needle. Assembly of this organelle proceeds in sequential steps that require the reprogramming of the secretion machine. Here we provide evidence that, in Salmonella typhimurium, completion of the assembly of the inner rod determines the size of the needle substructure. Assembly of the inner rod, which is regulated by the InvJ protein, triggers conformational changes on the cytoplasmic side of the injectisome, reprogramming the secretion apparatus to stop secretion of the needle protein.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Structures of Bacterial Flagellar Motors from Two FliF-FliG Gene Fusion Mutants

Flagella purified from Salmonella enterica serovar Typhimurium contain FliG, FliM, and FliN, cytoplasmic proteins that are important in torque generation and switching, and FliF, a transmembrane structural protein. The motor portion of the flagellum (the basal body complex) has a cytoplasmic C ring and a transmembrane M ring. Incubation of purified basal bodies at pH 4.5 removed FliM and FliN but not FliG or FliF. These basal bodies lacked C rings but had intact M rings, suggesting that FliM and FliN are part of the C ring but not a detectable part of the M ring. Incubation of basal bodies at pH 2.5 removed FliG, FliM, and FliN but not FliF. These basal bodies lacked the C ring, and the cytoplasmic face of the M ring was altered, suggesting that FliG makes up at least part of the cytoplasmic face of the M ring. Further insights into FliG were obtained from cells expressing a fusion protein of FliF and FliG. Flagella from these mutants still rotated but cells were not chemotactic. One mutant is a full-length fusion of FliF and FliG; the second mutant has a deletion lacking the last 56 residues of FliF and the first 94 residues of FliG. In the former, C rings appeared complete, but a portion of the M ring was shifted to higher radius. The C-ring-M-ring interaction appeared to be altered. In basal bodies with the fusion-deletion protein, the C ring was smaller in diameter, and one of its domains occupied space vacated by missing portions of FliF and FliG.

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.

1369.1-Pos Board # B623.1 – The Bacterial Flagellar Hook Structure

A 10-micron long complex of nine proteins makes up the sturdy, segmented, extracellular rod, hook and filament (or axial component) of the flagellum of Salmonella typhimurium. The sequences of the nine proteins except the cap protein (FliD) have at their N and C termini, heptad repeats characteristic of an alpha-helical bundle. Moreover, the segments characterized have a common helical symmetry. The hypothesis that these alpha-helical folds form an interlocking alpha-domain within and between the contiguous segments of the axial structure has received support from structural studies of the filament. We used electron cryomicroscopy to generate a high-resolution map of the hook. We docked atomic models for the two outer domains of the hook subunit into the corresponding features of the map. The innermost domains are interdigitated ∼1 nm rods, which form a tube having a 3 nm axial lumen, a feature seen in maps of the filament. The rods are somewhat shorter than those in the filament consistent with the shorter sequences thought to generate the fold. The N and C termini of the atomic model, which lie in the middle domain, point towards the spoke of density that connects to the inner rods. Our results further support the hypothesis of a common, interlocked alpha domain for the axial proteins.