Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.

The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes.

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite's asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Evolution of Archaellum Rotation Involved Invention of a Stator Complex by Duplicating and Modifying a Core Component.

Novelty in biology can arise from opportunistic repurposing of nascent characteristics of existing features. Understanding how this process happens at the molecular scale, however, suffers from a lack of case studies. The evolutionary emergence of rotary motors is a particularly clear example of evolution of a new function. The simplest of rotary motors is the archaellum, a molecular motor that spins a helical propeller for archaeal motility analogous to the bacterial flagellum. Curiously, emergence of archaellar rotation may have pivoted on the simple duplication and repurposing of a pre-existing component to produce a stator complex that anchors to the cell superstructure to enable productive rotation of the rotor component. This putative stator complex is composed of ArlF and ArlG, gene duplications of the filament component ArlB, providing an opportunity to study how gene duplication and neofunctionalization contributed to the radical innovation of rotary function. Toward understanding how this happened, we used electron cryomicroscopy to determine the structure of isolated ArlG filaments, the major component of the stator complex. Using a hybrid modeling approach incorporating structure prediction and validation, we show that ArlG filaments are open helices distinct to the closed helical filaments of ArlB. Curiously, further analysis reveals that ArlG retains a subset of the inter-protomer interactions of homologous ArlB, resulting in a superficially different assembly that nevertheless reflects the common ancestry of the two structures. This relatively simple mechanism to change quaternary structure was likely associated with the evolutionary neofunctionalization of the archaellar stator complex, and we speculate that the relative deformable elasticity of an open helix may facilitate elastic energy storage during the transmission of the discrete bursts of energy released by ATP hydrolysis to continuous archaellar rotation, allowing the inherent properties of a duplicated ArlB to be co-opted to fulfill a new role. Furthermore, agreement of diverse experimental evidence in our work supports recent claims to the power of new structure prediction techniques.

Corrigendum: The "Jack-of-all-Trades" Flagellum From Salmonella and E. coli Was Horizontally Acquired From an Ancestral ß-Proteobacterium.

[This corrects the article DOI: 10.3389/fmicb.2021.643180.].

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump.

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis.

The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor. This loss may be prevented by the formation of a cytoplasmic vesicle around the complex. These observations suggest a relatively loose association of the switch complex with the rest of the flagellar machinery. IMPORTANCE We show in eight different bacterial species (belonging to different phyla) that the flagellar motor loses its cytoplasmic switch complex upon cell lysis, while the rest of the flagellum remains attached to the cell body. This suggests an evolutionary conserved weak interaction between the switch complex and the rest of the flagellum which is important to understand how the motor evolved. In addition, this information is crucial for mimicking such nanomachines in the laboratory.

The "Jack-of-all-Trades" Flagellum From Salmonella and E. coli Was Horizontally Acquired From an Ancestral ß-Proteobacterium.

The γ-proteobacteria are a group of diverse bacteria including pathogenic Escherichia, Salmonella, Vibrio, and Pseudomonas species. The majority swim in liquids with polar, sodium-driven flagella and swarm on surfaces with lateral, non-chemotactic flagella. Notable exceptions are the enteric Enterobacteriaceae such as Salmonella and E. coli. Many of the well-studied Enterobacteriaceae are gut bacteria that both swim and swarm with the same proton-driven peritrichous flagella. How different flagella evolved in closely related lineages, however, has remained unclear. Here, we describe our phylogenetic finding that Enterobacteriaceae flagella are not native polar or lateral γ-proteobacterial flagella but were horizontally acquired from an ancestral ß-proteobacterium. Using electron cryo-tomography and subtomogram averaging, we confirmed that Enterobacteriaceae flagellar motors resemble contemporary ß-proteobacterial motors and are distinct to the polar and lateral motors of other γ-proteobacteria. Structural comparisons support a model in which γ-proteobacterial motors have specialized, suggesting that acquisition of a ß-proteobacterial flagellum may have been beneficial as a general-purpose motor suitable for adjusting to diverse conditions. This acquisition may have played a role in the development of the enteric lifestyle.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

How Did the Archaellum Get Its Rotation?

How new functions evolve fascinates many evolutionary biologists. Particularly captivating is the evolution of rotation in molecular machines, as it evokes familiar machines that we have made ourselves. The archaellum, an archaeal analog of the bacterial flagellum, is one of the simplest rotary motors. It features a long helical propeller attached to a cell envelope-embedded rotary motor. Satisfyingly, the archaellum is one of many members of the large type IV filament superfamily, which includes pili, secretion systems, and adhesins, relationships that promise clues as to how the rotating archaellum evolved from a non-rotary ancestor. Nevertheless, determining exactly how the archaellum got its rotation remains frustratingly elusive. Here we review what is known about how the archaellum got its rotation, what clues exist, and what more is needed to address this question.

CryoEM of bacterial secretion systems: A primer for microbiologists.

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic ß-barrel Outer-Membrane Proteins (OMPs) - are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the ß-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) - an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC - contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression.

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.

Adenita: interactive 3D modelling and visualization of DNA nanostructures.

DNA nanotechnology is a rapidly advancing field, which increasingly attracts interest in many different disciplines, such as medicine, biotechnology, physics and biocomputing. The increasing complexity of novel applications requires significant computational support for the design, modelling and analysis of DNA nanostructures. However, current in silico design tools have not been developed in view of these new applications and their requirements. Here, we present Adenita, a novel software tool for the modelling of DNA nanostructures in a user-friendly environment. A data model supporting different DNA nanostructure concepts (multilayer DNA origami, wireframe DNA origami, DNA tiles etc.) has been developed allowing the creation of new and the import of existing DNA nanostructures. In addition, the nanostructures can be modified and analysed on-the-fly using an intuitive toolset. The possibility to combine and re-use existing nanostructures as building blocks for the creation of new superstructures, the integration of alternative molecules (e.g. proteins, aptamers) during the design process, and the export option for oxDNA simulations are outstanding features of Adenita, which spearheads a new generation of DNA nanostructure modelling software. We showcase Adenita by re-using a large nanorod to create a new nanostructure through user interactions that employ different editors to modify the original nanorod.

Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression.

Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection.

Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella.

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.

Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS.

Technical developments are unifying molecular and cellular biology. A recent electron cryotomography study by von Kügelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.