The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.

No abstract

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

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.

Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane’s cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function.

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non–Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo–electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.

SUMMARY The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive. A recent study reported that the increased level of BB0184 (CsrABb; a homolog of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi. In this report, we deciphered the regulatory role of CsrABb on FlaB synthesis and the mechanism involved by analyzing two mutants, csrABb− (a deletion mutant of csrABb) and csrABb+ (a mutant conditionally over-expressing csrABb). We found that FlaB accumulation was significantly inhibited in csrABb+ but was substantially increased in csrABb−. In contrast, the levels of other flagellar proteins remained unchanged. Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrABb in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine-Dalgarno sequence. We found that CsrABb bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrABb specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.

e Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and Leptospira biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique "cap" at the cell end, this report provides structural insights into these fascinating Leptospira species.