Cryoelectron tomography reveals the sequential assembly of bacterial flagella in
            <i>Borrelia burgdorferi</i>

Zhao, Xiaowei; Zhang, Kai; Boquoi, Tristan; Hu, Bo; Motaleb, M. A.; Miller, Kelly A.; James, Milinda E.; Charon, Nyles W.; Manson, Michael D.; Norris, Steven J.; Li, Chunhao; Li, Jun

doi:10.1073/pnas.1308306110

Cited by 105 publications

(169 citation statements)

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“…The secretion channel appeared to be closed at the base, presumably to prevent leakage ( Fig. 2 C and H), reminiscent of findings for the flagellar motor of Borrelia burgdorferi (28).…”

Section: The Presence Of Mxin or Spa33 Has A Dramatic Impact On T3ss supporting

confidence: 68%

“…Two different procedures were used to collect and analyze the tomographic data. Initially, low-dose tilt series were collected at 15,500× magnification and 2 × 2 binning, similar to our previous procedure (28). Subsequently, we collected tilt series in dose fractionation mode, which enabled us to correct the motion-induced image blurring (29) and effectively improve the quality of the final reconstructions.…”

Section: Cryoelectron Tomography Of Shigella Minicells Reveals Intactmentioning

confidence: 99%

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Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.nanomachine | injectisome | pathogen-host interaction | cryo-electron tomography | protein secretion

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“…The secretion channel appeared to be closed at the base, presumably to prevent leakage ( Fig. 2 C and H), reminiscent of findings for the flagellar motor of Borrelia burgdorferi (28).…”

Section: The Presence Of Mxin or Spa33 Has A Dramatic Impact On T3ss supporting

confidence: 68%

Section: Cryoelectron Tomography Of Shigella Minicells Reveals Intactmentioning

confidence: 99%

Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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204

300

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“…It has been suggested that the assembly of the rod is a cooperative process (15). Furthermore, a recent study showed, by cryo-electron microscopy, that the rod structure undergoes rearrangement and acquires the typical rod shape only after the addition of FlgF (38). It is known that the rod components belong to the same export specificity class (23).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

et al. 2016

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The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N-and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCEDespite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled. The bacterial flagellum is a highly efficient nanomachine that propels the cell in liquid and semisolid media and over surfaces. It is composed of approximately 30 different proteins, whose numbers range from a few to several thousand copies (1). Its structure includes three major components, namely, the basal body, the hook, and the filament.The basal body contains the flagellum-specific type III secretion system (T3SS) housed in a bell-like structure, named the C ring, which is also the input for chemotactic signals that control the direction of rotation and consequently cell movement (2). The basal body also contains an inner membrane ring (MS ring) and a periplasmic ring (P ring); in addition, Gram-negative bacteria possess an outer membrane ring (L ring). This structure includes the rod that expands from the MS ring through the L and P rings, as well as the rotary motor driven by proton or sodium ions, which is surrounded by stator subunits that are located around the MS ring and harness energy from the electrochemical gradient.The hook is composed of about 120 copies of FlgE and is connected proximally to the rod and distally to the filament, by means of two hook-associated proteins (HAPs) (HAP1 and HAP3). The filament is composed of thousands of subunits of flagellin (FliC) and is the most abundant component of this organelle. HAP2 acts as a scaffold protein that helps flagellin subunits to polymerize at the distal end of the filament (3).Th...

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“…Amino acid sequence analysis of B. burgdorferi MotA and MotB indicates that they share 24% and 45% identity, respectively, with those in E. coli (21). Genome sequence analyses as well as cryoelectron tomography (cryo-ET) data suggest that spirochetal periplasmic flagella are similar to E. coli flagella in many aspects (22)(23)(24), although the regulation of motility and chemotaxis genes are distinctive (11,25). While B. burgdorferi motility and chemotaxis genes identified to date are transcribed by the housekeeping 70 factor of RNA polymerase, those genes in E. coli are regulated in a hierarchical manner (11,25,26).…”

mentioning

confidence: 99%

“…While B. burgdorferi motility and chemotaxis genes identified to date are transcribed by the housekeeping 70 factor of RNA polymerase, those genes in E. coli are regulated in a hierarchical manner (11,25,26). A periplasmic flagellum is composed of at least 50 proteins that can be subdivided into three major parts: the basal body-motor-switch complex, the hook, and the filament (24,27).…”

mentioning

confidence: 99%

Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

Sultan

Sekar

Zhao³

et al. 2015

Infect Immun

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c Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ⌬motB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ⌬motB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ⌬motB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. Borrelia burgdorferi, the Lyme disease spirochete, shuttles principally between the Ixodes ticks and a vertebrate host during its natural infection cycle (1-5). In the tick, spirochetes reside primarily in the midgut until the introduction of the host blood meal. During tick feeding on a vertebrate host, spirochetes replicate, and a subset of the motile organisms cross the midgut epithelium into the hemocoel before reaching the salivary glands, where they subsequently are deposited into the host dermis (6-8). Once in the host, B. burgdorferi must migrate through the complex skin tissues, including some hematogenous dissemination, and eventually colonize distant tissues, where they often produce disease symptoms in certain hosts. Subsequently, when a naive tick feeds on an infected reservoir host, spirochetes in the skin migrate into the arthropod to complete the enzootic cycle (3, 4, 9). During the spirochete's migration within and between the hosts, periplasmic flagellar motor rotation and, by extension, motility are thought to be crucial for infection (10, 11). Indeed, nonmotile, reduced-motility, or nonchemotactic mutant spirochetes were found to be attenuated in mouse infection (12)(13)(14). Additionally, our data indicate that B. burgdorferi remains constantly motile in the mouse dermis, even 2 years after needle inoculation (P. Sekar, R. M. Wooten, and M. A. Motaleb, unpublished results). In fact, this Lyme dise...

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Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi

Cited by 105 publications

References 50 publications

Visualization of the type III secretion sorting platform of Shigella flexneri

Visualization of the type III secretion sorting platform of Shigella flexneri

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

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