The sheathed flagellum of Vibrio cholerae is a self-assembling membranous organelle that must coordinate axial assembly, sheath biogenesis, and rapid motor rotation. Here, we determine in-situ near-atomic structures of the sheathed flagellar motor inside intact cells. The motor anchors to the outer membrane through lipidated HL-rings without forming a membrane pore, thereby allowing axial assembly to drive sheath formation. Conserved LP-rings act as slide-rotary bushings that permit high-speed rotation within a dynamic envelope yet can constrict to seal the pore upon stress-induced ejection. We further show that stator activation requires a specific PomB-MotX interaction rather than peptidoglycan engagement. Together, these findings reveal how the distinctive architecture and dynamics of the sheathed flagellum promote V. cholerae motility, environmental survival, and persistent colonization of the human gut.

Single-particle cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling atomic-resolution structure determination of purified macromolecular assemblies. Recent advances in in situ single-particle cryo-EM have extended this capability to near-atomic structural analysis directly within native cellular environments. However, major challenges remain because many cellular targets are low in abundance, structurally heterogeneous, and difficult to detect. In parallel, cryo-electron tomography (cryo-ET) combined with subtomogram averaging enables in situ visualization of macromolecular assemblies while preserving their three-dimensional cellular context, but limited throughput and resolution have constrained high-resolution analysis of rare or heterogeneous complexes. In this review, we discuss recent advances in sample preparation, data acquisition, image processing, and high-resolution refinement that improve the throughput, sensitivity, and resolution of in situ structural biology. We further highlight how integrating in situ single-particle cryo-EM with cryo-ET bridges cellular visualization and near-atomic structure determination, providing a scalable framework for investigating dynamic macromolecular assemblies directly in their native cellular context.

Periplasmic flagella are essential for the distinctive morphology and motility of the Lyme disease spirochete Borrelia burgdorferi , and motility plays a critical role in its pathogenic lifestyle. These flagella are powered by specialized motors that contain a large spirochete-specific multiprotein collar complex, yet the molecular architecture and mechanisms underlying high-torque motility remain poorly understood. Here, we identify the tetratricopeptide repeat (TPR)-containing protein BB0298 as a previously unrecognized flagellar collar component and rename it FlcE. Loss of flcE results in altered morphology and nearly abolishes the spirochete's motility. Using cryo-electron tomography and biochemical analyses, we show that FlcE occupies a unique position within the collar where it surrounds the FliL-stator complex by binding to FliL and the collar protein FlcA. These findings support a model in which FlcE functions as a molecular "latch" that secures stator assemblies to sustain efficient torque generation. Notably, flcE is conserved across most motile members of the Spirochaetales and is located within the division and cell-wall ( dcw ) gene cluster. More broadly, the conservation of TPR-containing structural proteins across diverse bacterial flagellar systems suggests a general architectural principle whereby dedicated scaffolds reinforce stator complexes to maximize motor performance under high mechanical loads. Importance:Spirochetal motility requires the stable engagement of torque-generating stator units, yet the mechanisms that secure these complexes have remained unknown. Here, we identify FlcE as a previously unrecognized collar protein that functions as a molecular latch by restraining the FliL-stator complex. Loss of FlcE destabilizes FliL-stator assemblies and severely impairs motility, demonstrating that peripheral scaffolding is essential for sustaining high torque output in the Lyme disease spirochete Borrelia burgdorferi . The conservation of tetratricopeptide repeat domains in FlcE and other bacterial flagellar proteins further suggests a broader mechanism in which specialized scaffolds anchor and stabilize stator complexes, enabling high torque generation. Because motility is a central virulence determinant in many pathogenic bacteria-including B. burgdorferi during transmission from the tick to the mammalian host and subsequent infection-defining the mechanistic role of FlcE provides important insight into the molecular basis of motility-driven pathogenesis and suggests new strategies for disrupting bacterial infection.

Spirochetes are evolutionarily distinct bacteria defined by their spiral morphology, unique means of motility, and periplasmic flagella (PFs). Because these filaments reside within the periplasm and are mechanically integrated with the cell body, their assembly must be precisely coordinated with cell growth and cytokinesis. However, the mechanism that couples flagellar biogenesis to cell division in spirochetes remains unclear. Using the Lyme disease spirochete Borrelia burgdorferi as a model, we identify FlhG (BB0269), a MinD-like ATPase, as a spatial regulator that links cell division to flagellar patterning. In wild-type cells, 7-11 long helical PFs originate from cell poles and assemble into ribbon-like bundles that wrap around the cell cylinder to drive motility. Deletion of flhG disrupts this ordered architecture, causing marked heterogeneity in flagellar number, defective ribbon assembly, aberrant septation, and severe motility impairment. Mechanistically, FlhG dynamically localizes to the poles and midcell during division, where it directs the positioning of FlhF, a signal recognition particle (SRP) -type GTPase controlling flagellar number and placement, and FliF, the MS-ring protein that nucleates flagellar assembly. Through this spatial regulation, FlhG coordinates flagellar assembly with cytokinetic progression. Together, these findings reveal a spatial regulatory mechanism coupling cell division to flagellation, providing insight into understanding how spirochetes coordinate their distinctive morphogenesis, flagellation and motility. Significance:Spirochetes such as Borrelia burgdorferi , the causative agent of Lyme disease, rely on periplasmic flagella for motility and cell shape, yet how these structures are coordinated with cell division has remained unclear. We identify a MinD-like ATPase, FlhG, as a spatial regulator that couples flagellar assembly to cytokinesis. In contrast to its homologs in other bacteria, FlhG does not regulate flagellar protein levels but instead directs subcellular positioning of key assembly factors. By dynamically redistributing between the cell poles and division site, FlhG synchronizes flagellar patterning with septum formation. These findings uncover a previously unrecognized mechanism linking cell morphogenesis to the cell cycle and reveal how conserved ATPases can be repurposed to organize complex bacterial architectures.

Spirochetes exhibit a distinctive corkscrew-like motility driven by periplasmic flagella that wrap around the cell body in a supercoiled configuration, yet the structural basis of this unique propulsion remains poorly understood. Here we combine cryo-electron microscopy, cryo-electron tomography, and genetic and biochemical analyses to determine the assembly and adaptation principles of the supercoiled flagellar filament in Treponema denticola , a major periodontal pathogen. Near-atomic structures reveal a glycosylated FlaB flagellin core encased by a previously unrecognized asymmetric sheath. The major sheath protein FlaA forms the bulk of the sheath and mechanically couples to the core through defined interfaces required for efficient motility, whereas four minor sheath proteins (FlaA1, FlaA2, FlaAP1, and FlaAP2) assemble along the concave side of the filament to accommodate intrinsic curvature. Disruption of this asymmetric core-sheath organization compromises force transmission and impairs motility, establishing coordinated asymmetric assembly as a fundamental mechanism underlying spirochetal motility.

The transmembrane export apparatus is a conserved core of bacterial type III secretion systems, shared by the flagellum and injectisome. Powered by the proton-motive force (PMF), this machinery translocates protein substrates across the bacterial envelope to support motility and virulence. However, its assembly and operation within native membranes remain poorly understood. Here, using in-situ single-particle cryo-electron microscopy, we resolve near-atomic-resolution structures of the flagellar export apparatus within the periplasmic flagellar motor of the Lyme disease spirochete Borrelia burgdorferi . Structural and genetic analyses suggest that coordinated, stepwise assembly of the flagellar export apparatus and MS-ring drives local deformation of the cytoplasmic membrane through extensive protein-lipid-protein interactions, constructing a funnel-shaped conduit optimal for substrate translocation. In addition, a conserved hydrophilic pathway lies within core export protein FlhA, with key acidic residues essential for export activity and supporting PMF-driven substrate translocation. Together, these findings establish a mechanistic framework for export apparatus assembly and energy transduction, defining core principles of type III secretion.

Programmed lytic cell death, including pyroptosis and necroptosis, involves intracellular enzymes that form membrane-rupturing pores. Tumor-associated ectoenzymes such as alkaline phosphatase (ALP), however, offer the potential to initiate lytic death extrinsically. Here, we design a phospho-biphenyl-capped peptide precursor that is selectively dephosphorylated by ALP on cancer cell surfaces, triggering enzyme-instructed peptide self-assembly (EISA) into in situ peptide filaments. These supramolecular filaments physically breach the plasma membrane, overwhelm ESCRT-dependent membrane repair, and induce catastrophic calcium influx, cytoskeletal collapse, and organelle dysfunction. While cryo-EM uncovers 2.5-2.9 Å resolution details of ordered dimeric packing that underlies their mechanical rigidity and membrane-rupturing capability, cryo-electron tomography (cryo-ET) reveals the filament penetration of the plasma membrane in live cells. By reprogramming ALP from an immune checkpoint ectoenzyme into a pro-death catalyst, this work establishes a molecular mechanism linking enzymatic catalysis to supramolecular order and membrane failure. More broadly, it outlines a supramolecular chemical-biology framework in which enzyme-triggered assemblies function as programmable executors of cell death.

The bacterial flagellum is a complex nanomachine essential for motility, colonization, and invasion in diverse species. Helicobacter pylori has evolved elaborate sheathed flagella that enable migration through the highly viscous gastric mucus layer to reach its colonization niche on the gastric epithelium, yet the molecular basis for these unique adaptations has remained elusive. Here, we use in situ single-particle cryo-electron microscopy to determine near-atomic structures of the flagellar filament within the membranous sheath of H. pylori. The major flagellin FlaA constitutes the bulk of the filament, whereas the minor flagellin FlaB contributes critically to the hook-proximal region. Both FlaA and FlaB form a conserved core surrounded by variable surface-exposed domains. Our structures further reveal that pseudaminic acid glycans decorate these domains, where they mediate inter- and intra-subunit contacts that stabilize the filament and confer a negatively charged surface. Together, these findings support a model in which the filament rotates independently of the membranous sheath to drive H. pylori motility and provide a molecular framework for understanding how the sheathed flagellum enables colonization and persistence within the gastric niche.

Semi-artificial photosynthesis, integrating biocatalysts with photosensitive materials to enable self-photosensitization in non-photosynthetic microorganisms, is a rapidly evolving interdisciplinary field for solar-driven energy and chemical production using air, water, and sunlight. However, the efficiency of such constructed biocatalysts is often impeded by the limited biocompatibility, prevalent biotoxicity, and narrow spectral response associated with photosensitive materials. Quantum dots (QDs), zero-dimensional crystals, exhibit favorable photoexcitation properties and enhanced biocompatibility, providing essential reducing equivalents for microbial metabolisms. This review examines recent advances in semi-artificial photosynthesis, focusing on the self-assembly of microorganisms in conjunction with QDs. It highlights the biocompatible, directional design of QDs and explores the underlying mechanisms of electron and energy transfer within the microbe-QDs complexes. By leveraging the synergies of solar absorption and biocatalytic activity, this review discusses the future trajectory and potential improvements in semi-artificial photosynthesis, offering a paradigm-shifting approach to sustainable solar energy utilization. The solar-powered QDs-biocatalyst biohybrids for semi-artificial photosynthesis are projected to emerge as a transformative technology in advanced energy production.

A wide range of globally-important environmental redox phenomena and biotechnology applications for bioremediation, bioenergy and bioelectronics, require long-range (>10 µm) extracellular electron transfer (EET) via bacterial surface-displayed “nanowires”. Since 2005, Type IV pili (T4P) and recently cytochrome filaments are proposed to function as nanowires, but direct evidence for either claim is lacking due to lack of methods to directly visualize EET. By developing correlated imaging of metabolic electrons and proteins in individual filaments used for EET, here we show that Geobacter sulfurreducens performs EET to soil-abundant minerals and electrodes via polymeric cytochromes nanowires rather than T4P. In contrast to the long-held belief that EET suppression in T4P mutant strains is due to the loss of T4P conductivity, we find that T4P mutations lower filament stability and impair secretion of cytochrome nanowires. Mutagenesis, in vivo interaction studies, and cryo-electron tomography reveal that G. sulfurreducens combines key components of the T4P system (T4PS) and the Type II secretion system (T2SS) for nanowire secretion and EET, in contrast to monomeric cytochromes translocated solely by T2SS. Our study resolves a long-standing controversy regarding nanowire identity and function, establishing a new class of hybrid T4PS-T2SS machinery that evolved for the secretion of cytochrome nanowires.

Motility promotes the complex life cycle and infectious capabilities of Vibrio cholerae and is driven by rotation of a single polar flagellum. The flagellar filament comprises four flagellin proteins (FlaA-D) and is covered by a membranous sheath continuous with the outer membrane. Here we combine in situ cryo-electron microscopy single-particle analysis, fluorescence microscopy and molecular genetics to determine 2.92-3.43 Å structures of the sheathed flagellar filament from intact bacteria. Our data reveal the spatial arrangement of FlaA-D, showing that FlaA localizes at the cell pole and functions as a template for filament assembly involving multiple flagellins. Unlike unsheathed flagellar filaments, the sheathed filament from V. cholerae possesses a highly conserved core but a smooth, hydrophilic surface adjacent to the membranous sheath. A tiny conformational change at the single flagellin level results in a supercoiled filament and curved membranous sheath, supporting a model wherein the filament rotates separately from the sheath, enabling the distinct motility of V. cholerae.

Bacteriophages must recognize host receptors and penetrate the host cell envelope to initiate infection. How the classic phage λ initiates infection is not yet understood. Here, we combine cryo–electron microscopy and tomography to visualize infection initiation by Ur-λ, the original λ isolate that uses side fibers to adsorb rapidly to Escherichia coli . We determine the structure of Ur-λ, resolving the full-length central and side fibers, thus providing a structural basis for host recognition. We show that Ur-λ contains six copies of its tape measure protein. We capture intermediates of the tail tip complex during infection initiation, revealing how extensive conformational changes enable adsorption, and visualize the trans-envelope channel required for genome ejection.

Comprehensive in situ structures of macromolecules can transform our understanding of biology and advance human health. Here, we map protein synthesis inside human cells in detail by combining automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo electron microscopy (cryo-EM). With this in situ cryo-EM approach, we resolved a 2.2 Å consensus structure of the human 80S ribosome and unveiled 23 functional states, nearly all better than 3 Å resolution. Compared to in vitro studies, we observed variations in ribosome structures, distinct environments of ion and polyamine binding, and associated proteins such as EDF1 and NACβ that are typically not enriched with purified ribosomes. We also detected additional peptide-related density features on the ribosome and visualized ribosome-ribosome interactions in helical polysomes. Finally, high-resolution structures from cells treated with homoharringtonine and cycloheximide revealed a distinct translational landscape and a spermidine that interacts with cycloheximide at the E site, one of the numerous polyamines that also bind native ribosomes. These results underscore the value of high-resolution in situ studies in the native environment.

YcgR is a c-di-GMP effector that inhibits chemotaxis and swimming speed in Escherichia coli and Salmonella. Genetic, biochemical, and structural studies suggest that YcgR interacts with both the bidirectional flagellar rotor and the stator to bias rotation toward counterclockwise (CCW) and reduce motor speed, but the underlying mechanism remains unresolved. Recent cryo-electron microscopy structures revealing conformational changes in the rotor-stator complex during directional switching suggested to us a mechanism by which YcgR acts. We call this the Texas 2-step model, after the country dance in which partners move smoothly in a CCW arc with quick steps followed by slow ones. In this model, YcgR first binds a MotA subunit when the rotor adopts the CCW conformation, in which stators are largely displaced from the C-ring. In the next step, the rotating MotA pentamer delivers YcgR to the rotor protein FliG, thereby slowing motor speed. We provide evidence for the first step of this model, offering testable predictions for future work.IMPORTANCEThe mechanism of YcgR action has been investigated by multiple laboratories using diverse approaches, yet no consensus has emerged. Some studies implicate the rotor, others the stator. A key complication is the involvement of four interacting proteins-MotA, FliG, FliM, and YcgR-with multiple contact sites in several of them. Recent rotor-stator cryo-electron microscopy structures revealing conformational changes during directional switching suggested a mechanism that we set out to test. Our experiments show that rotor conformation is crucial for YcgR function.

The Dot/Icm machine of Legionella pneumophila is among the most versatile type IV secretion systems (T4SSs), capable of translocating more than 330 distinct effector proteins across the bacterial envelope into host cells. Assembly and function of the system require at least 27 Dot and Icm proteins, yet its architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy in situ single-particle cryoelectron microscopy to determine near-atomic structures of the Dot/Icm machine and its intimate association with three distinct outer membrane porins in intact bacteria. Notably, two essential yet enigmatic components, DotA and IcmX, form a pentameric protochannel in an inactive state at the central axis of the Dot/Icm machine. Upon Dot/Icm activation with host lysate, this protochannel undergoes extensive rearrangements to generate an extended transenvelope conduit, as visualized by cryoelectron tomography (cryo-ET) and subtomogram averaging. Furthermore, a combination of cryo-ET and cryo-FIB milling of macrophages infected with L. pneumophila reveals tethering of the Dot/Icm machine to the host membrane, suggesting direct translocation of effector proteins from the bacterial cytoplasm into the host. Together, our studies identify the DotA-IcmX complex as a gatekeeper for effector translocation and provide a molecular framework for understanding the assembly and activation of the elaborate Dot/Icm T4SS.

Spirochetes are a widely existing group of bacteria with a distinct morphology. Some spirochetes are important human pathogens that utilize periplasmic flagella to achieve motility and host infection. The motors that drive the rotation of periplasmic flagella have a unique spirochete-specific feature, termed the collar, crucial for the flat-wave morphology and motility of the Lyme disease spirochete Borrelia burgdorferi. Here, we deploy cryo-electron tomography and subtomogram averaging to determine high-resolution in-situ structures of the B. burgdorferi flagellar motor. Comparative analysis and molecular modeling of in-situ flagellar motor structures from B. burgdorferi mutants lacking each of the known collar proteins (FlcA, FlcB, FlcC, FlbB, and Bb0236/FlcD) uncover a complex protein network at the base of the collar. Importantly, our data suggest that FlbB not only forms a novel periplasmic ring around the rotor but also acts as a scaffold supporting collar assembly and subsequent recruitment of stator complexes. The complex protein network based on the FlbB ring effectively bridges the rotor and 16 torque-generating stator complexes in each flagellar motor, thus contributing to the specialized motility and lifestyle of spirochetes in complex environments.

The flagellin-specific chaperone FliS has been studied in externally flagellated bacteria; however, its role in spirochetes, a group of bacteria that possess unique internalized flagella (termed endo- or periplasmic flagella), remains unexplored. Here, we investigate the function of FliS in the Lyme disease spirochete Borrelia burgdorferi . Using loss-of-function studies, combined with biochemical assays and cryo-electron tomography, we demonstrate that FliS deletion selectively reduces FlaB expression, the major flagellin protein, resulting in non-motile mutants with defective flagellar filaments. Mechanistically, we show that FlaB interacts with both FliS and FliW, the latter being an allosteric repressor of the RNA-binding protein CsrA, which inhibits FlaB translation. These four components form a regulatory circuit that fine-tunes FlaB levels and flagellar assembly via a partner-switching mechanism. Deletion of fliS disrupts FlaB secretion, leading to its cytoplasmic accumulation, sequestration of FliW, and subsequent release of CsrA to suppress FlaB synthesis. Accumulation of cytoplasmic FlaB also triggers its degradation to prevent toxicity. Our findings reveal a post-transcriptional regulatory mechanism governing flagellar assembly in B. burgdorferi , an evolutionary outlier that lacks the canonical transcriptional cascade controlling flagellar biosynthesis in most bacteria.

Selecting a suitable cyanobacterial strain and developing easy-to-afford purification processes are two crucial aspects impacting the optimal production yield and appropriate purity of C-phycocyanin (C-PC). Cyanobium sp. MMK01, a highly efficient C-PC-producing bacterium, was identified among four cyanobacterial isolates using morphological characteristics and 16S rRNA gene sequencing. The purification process of C-PC began with ammonium sulfate precipitation, leading to a purity index (PI) of 4.04. Subsequent purification through ion exchange chromatography ultimately resulted in an ultra-highly purified form of C-PC with a significant PI of 5.82. SDS-PAGE analysis of purified C-PC showed the presence of two distinct bands, α (13 kDa) and β (15 kDa). Significantly effective at scavenging free radicals, C-PC also inhibits the viability of human lung cancer cells (Calu-6). Antibacterial, anti-inflammatory, antioxidant, and cancer-preventive compounds were detected in the MMK01 cells’ methanolic extract following GC–MS analysis. The promising results indicate that Cyanobium sp. MMK01 has a great deal of potential for producing C-PC that is on par with strains found in the market, and the tried-and-true two-step purification process proved to work well to achieve an ultra-highly purified form of C-PC.

A photobioreactor is the key to sustainable microalgal industrial processes. Improving the mass transfer in a microalgal photobioreactor is a key factor in modifying a photobioreactor. So-called mass transfer is the diffusion rate between gas–liquid phases, which can be quantified as the mass transfer coefficient or the mass transfer rate. It is also evaluated together with mixing time between liquids. As we know, the microalgal CO2 fixation process is a multiphase interaction process, which involves the microalgal solution, CO2 bubbles, and microalgal cells. Improving the mass transfer coefficient between CO2 bubbles and the microalgal solution as well as mixing time between microalgal solution allows for higher CO2 fixation rate by microalgae and blooming of microalgal biomass. Mass transfer coefficient and mixing time can be estimated from many different theoretical equations, correlations, and analogies that are functions of material properties, intensive properties and flow regime (laminar or turbulent flow). Selection of the most applicable model is dependent on the materials and the system, or environment, being studied. As a result, understanding the definition, measurement method, application field of mass transfer coefficient, and mixing time in photobioreactors is necessary. Here, we will introduce the basic principle of the mass transfer coefficient and the literature that used the mass transfer coefficient as the modification parameter for designing the photobioreactors.

Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors.