Biology subjects

Liu, L.-N.

Publications and source records attributed to Liu, L.-N..

18 recordsLinked to original sources

bioRxiv2026

Structure and energy transfer of a minimal PSI-LHCI supercomplex with FNR binding from a terrestrial eustigmatophyte

Soil microalgae endure harsh terrestrial stressors, such as intense light. Eustigmatophytes are an independent evolutionary branch within stramenopiles and occupy diverse aquatic and terrestrial environments, but the structural organization of their photosynthetic apparatus remains poorly understood. Here, we determined the cryo-electron microscopy structure of a photosystem I-light-harvesting complex I (PSI-LHCI) supercomplex bound with ferredoxin-NADP+ oxidoreductase (FNR) from the terrestrial eustigmatophyte Vischeria stellata at 2.44 [A] resolution. The supercomplex contains a monomeric PSI core associated with only three LHCI subunits, representing the smallest PSI-LHCI reported among structurally characterized red-lineage PSI complexes composed of violaxanthin-Chl a proteins (VCPs). The three VCPIs with distinct structure features and arrangements form a compact belt along the PsaL-PsaI-PsaM side of PSI. The structure also resolves a 43-residue N-terminal segment of FNR (FNR-N) bound to the PSI stromal surface, which is stabilized by both a eustigmatophyte-conserved insertion in PsaL and the N-terminal region of PsaD. In contrast, the catalytic region of FNR was not resolved, suggesting conformational flexibility. Computational simulations indicate potential excitation-energy-transfer pathways connecting the three VCPI subunits to the PSI core and highlight lineage-specific pigments that maintain energetic connectivity within the exceptionally compact antenna. Our analysis further reveals conservation of FNR tethering despite pronounced diversification of antenna size and organization. These findings uncover a modular evolutionary principle in which PSI acceptor-side organization is retained while the light-harvesting antenna is extensively remodeled, providing a framework for understanding the diversification of photosynthetic energy conversion across ecological transitions.

plant biology↗

bioRxiv2026

The structured hairpin region of the bacterial ESCRT-III protein IM30 orchestrates stress-induced condensate formation

Biomolecular condensates are well characterized in eukaryotes, but their role in bacteria remains largely elusive. In the cyanobacteriumn Synechocystis sp. PCC 6803, the protein IM30, a member of the ESCRT-III superfamily of membrane remodeling proteins, forms stress-induced puncta across diverse environmental challenges, indicating a general adaptive response. Live-cell imaging reveals that IM30 is uniformly distributed throughout the cytoplasm at low concentrations but assembles into puncta upon exceeding a critical saturation threshold, a hallmark of liquid-liquid phase separation. Crucially, stress triggers puncta formation even below this threshold, suggesting stress lowers the phase-separation barrier. Super-resolution microscopy confirms spherical, condensate-like morphologies, while FRAP demonstrates rapid fluorescence recovery, consistent with with a fluid interior and dynamic exchange between puncta and the cytosol. Cellular IM30 levels exceed the in vitro determined critical concentration, placing the protein in a supersaturated state primed for condensation. Domain mapping identifies the structured 1-3 helical hairpin as the minimal phase separation driver; in contrast, the disordered 4-6 segment alone cannot phase-separate. Phase separation occurs within a physiologically relevant pH range (4.5-6.5), matching the localized acidification of the cyanobacterial cytoplasm at damaged thylakoid membranes. This directly links membrane stress, pH changes, and IM30 recruitment. Collectively, these findings establish IM30 puncta as bona fide, stress-responsive biomolecular condensates that function as rapid stress sensors and effectors, providing a mechanistic framework for phase separation and condensate formation by bacterial ESCRT-III proteins during environmental adaptation.

biochemistry↗

bioRxiv2025

Multi-Scale Simulations of Excited-State Energy Transfer Pathways in the C2S2-Type PSII-LHCII Supercomplex of Spinach

Photosystem II-light-harvesting complex II (PSII-LHCII) supercomplex achieves efficient excitation energy transfer (EET), yet how directional transfer emerges from its nearly flat energy landscape has remained unclear. Here, we investigate the C2S2-type spinach PSII-LHCII supercomplex containing 206 pigments by integrating microsecond-scale molecular dynamics, QM/MM site energy calculations, and generalized Forster theory. The computed site energy landscape shows overall downhill gradients from peripheral antennas to the reaction center (RC), with small variations fully explained by dynamic structural fluctuations. Pairwise transfer rates reveal a dense multi-timescale EET network, where fast intra-subunit and interface couplings provide robust channels and slower links maintain global connectivity. Key interface pigments act as energy-funneling hotspots that converge excitations toward the RC. Within the PSII core, both the D1 and D2 branches ultimately transfer energy downhill to ChlD1 and ChlD2, respectively, ensuring thermodynamic feasibility of delivery. Three major transfer pathways were identified, all falling within functional timescales (< 250 ps). Bidirectional dynamics were also observed, with rapid reverse transfers enabling excess excitation to be safely dissipated through peripheral antennas. Together, these results demonstrate that PSII maintains fully downhill energy transfer through the combined effects of structural constraints and dynamic regulation, thereby achieving both efficient energy delivery and photoprotection.

biochemistry↗

bioRxiv2025

Membrane binding of a cyanobacterial ESCRT-III protein crucially involves the helix α1-3 hairpin conserved in all superfamily members

IM30, the inner membrane-associated protein of 30 kDa (also known as Vipp1) is essential for thylakoid membrane biogenesis and/or maintenance in chloroplasts and cyanobacteria. IM30 and its bacterial homolog PspA belong to the ESCRT-III superfamily, proteins previously thought to be restricted to eukaryotes and archaea. Despite low sequence similarity, IM30 shares key structural and functional features with eukaryotic ESCRT-IIIs, including a conserved 1-2 helical hairpin core and the ability to form oligomeric barrel- or rod assemblies that mediate membrane remodeling. Using IM30 variants, we now show that initial membrane recruitment of IM30 is driven by electrostatic interactions between the positively charged 1-3 helical hairpin and negatively charged lipid surfaces, paralleling the role of charged helical regions in some eukaryotic ESCRT-IIIs. This likely initiates lateral assembly of IM30 into higher-order barrel or rod structures on the membrane. Once assembled, 0 helices within these oligomers engage and stabilize internalized membrane tubules, mirroring membrane interaction strategies of eukaryotic ESCRT-IIIs, which use both N-terminal sequences and charged residues on 1/2. Thus, our findings demonstrate a conserved membrane binding and remodeling mechanism across the ESCRT-III superfamily, underscoring an evolutionary link in membrane dynamics between pro- and eukaryotes. SignificanceIM30, a membrane-associated protein found in cyanobacteria and chloroplasts, along with its bacterial homolog PspA, belongs to the ESCRT-III superfamily. Despite low sequence conservation, these proteins share structural and functional features with eukaryotic ESCRT-III proteins. We show that IM30 binds membranes via a conserved structural motif, followed by lateral assembly into higher-order complexes. This supports a mechanism of membrane remodeling that is conserved in prokaryotic and eukaryotic members of the ESCRT-III superfamily.

biochemistry↗

bioRxiv2025

Structural basis for the RC-LH1 supercomplex of Dinoroseobacter shibae for anaerobic anoxygenic photosynthesis

Aerobic anoxygenic phototrophic (AAP) bacteria are essential for oceanic carbon cycling. However, the architecture and structural adaptations of their photosynthetic systems to ensure adequate light harvesting, electron transport, and oxidative resilience in oxygen-rich environments remain poorly understood. In this study, we present the 2.4-[A] cryo-EM structure of the reaction center-light-harvesting 1 (RC-LH1) supercomplex from Dinoroseobacter shibae DFL-12, a marine AAP bacterial symbiont of benthic dinoflagellates. This RC-LH1 supercomplex features a closed LH1 ring comprising 17 {beta}-subunits, each containing two spheroidenones per {beta}-heterodimer--a previously unreported configuration in phototrophic bacteria. The cytochrome subunit of the RC is truncated to three hemes, in contrast to the four-heme configuration found in anaerobic relatives. The structure also reveals elongated bacteriochlorophyll (BChl) spacing, accounting for its blue-shifted absorption maximum that is optimized for low-light benthic environments. Furthermore, we identify a previously unknown protein-LRC (Light-Respiratory Connector), which structurally bridges the RC and LH1 components and exhibits homology to NADH oxidoreductase subunit E, suggesting a functional coupling between photochemical and respiratory electron transport. Collectively, these specific structural features allow AAP bacteria to balance anoxygenic photosynthesis and protection against oxidative damage, providing a mechanistic framework for them to thrive in oxygenated marine environments. Our study provides insights into the structural and functional variability of bacterial photosynthesis in response to oxygenated marine environments.

biochemistry↗

bioRxiv2024

Rubisco packaging and stoichiometric composition of a native β-carboxysome

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within {beta}-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the {beta}-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in {beta}-carboxysomes, which is instrumental for Rubisco encapsulation and {beta}-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire {beta}-carboxysome. This study and recent findings on the {beta}-carboxysome structure provide insights into the assembly principles and structural variation of {beta}-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

biochemistry↗

bioRxiv2024

Engineering Rubisco Condensation in Chloroplasts to Manipulate Plant Photosynthesis

Although Rubisco is the most abundant enzyme globally, it is inefficient for carbon fixation because of its low turnover rate and limited ability to distinguish CO2 and O2, especially under high O2 conditions. To address these limitations, phytoplankton, including cyanobacteria and algae, have evolved CO2-concentrating mechanisms (CCM) that involve compartmentalizing Rubisco within specific structures, such as carboxysomes in cyanobacteria or pyrenoids in algae. Engineering plant chloroplasts to establish similar structures for compartmentalizing Rubisco has attracted increasing interest for improving photosynthesis and carbon assimilation in crop plants. Here, we present a method to effectively induce the condensation of endogenous Rubisco within tobacco (Nicotiana tabacum) chloroplasts by genetically fusing superfolder green fluorescent protein (sfGFP) to the tobacco Rubisco large subunit (RbcL). By leveraging the intrinsic oligomerization feature of sfGFP, we successfully created pyrenoid-like Rubisco condensates that display dynamic, liquid-like properties within chloroplasts without affecting Rubisco assembly and catalytic function. The transgenic tobacco plants demonstrated comparable autotrophic growth rates and full life cycles in ambient air relative to the wild-type plants. Our study offers a promising strategy for modulating endogenous Rubisco assembly and spatial organization in plant chloroplasts via phase separation, which provides the foundation for generating synthetic organelle-like structures for carbon fixation, such as carboxysomes and pyrenoids, to optimize photosynthetic efficiency.

plant biology↗

bioRxiv2024

Molecular basis of the biogenesis of a protein organelle for ethanolamine utilization

Many pathogenic bacteria use proteinaceous ethanolamine-utilization microcompartments (Eut BMCs) to facilitate the catabolism of ethanolamine, an abundant nutrient in the mammalian gut. The ability to metabolize ethanolamine gives pathogens a competitive edge over commensal microbiota which can drive virulence in the inflamed gut. Despite their critical functions, the molecular mechanisms underlying the synthesis of Eut BMCs in bacterial cells remain elusive. Here, we report a systematic study for dissecting the molecular basis underlying Eut BMC assembly in Salmonella. We determined the functions of individual building proteins in the structure and function of Eut BMCs and demonstrated that EutQ plays an essential role in both cargo encapsulation and Eut BMC formation through specific association with the shell and cargo enzymes. Furthermore, our data reveal that Eut proteins can self-assemble to form cargo and shell aggregates independently in vivo, and that the biogenesis of Eut BMCs follows a unique "Shell-first" pathway. Cargo enzymes exhibit dynamic liquid-like organization within the Eut BMC. These discoveries provide mechanistic insights into the structure and assembly of the Eut BMC, which serves as a paradigm for membrane-less organelles. It opens up new possibilities for therapeutic interventions for infectious diseases.

cell biology↗

bioRxiv2024

Uncovering the roles of the scaffolding protein CsoS2 in mediating the assembly and shape of the α-carboxysome shell

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, Rubisco, alone with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of -carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the -carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted indepth examinations on the function of CsoS2-M in the assembly of the -carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the -carboxysome shell and plays an important role in shaping the -carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into -carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes.

synthetic biology↗

bioRxiv2023

Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly

Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In -carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineered -carboxysome shells using minimal shell components and determined cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal "molecular thread" stitching through multiple shell protein interfaces. We further uncovered in CsoS2 a remarkable highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-govern carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

microbiology↗

bioRxiv2022

Tales of Two α-Carboxysomes: the Structure and Assembly of Cargo Rubisco

Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. It encapsulates carbonic anhydrase and Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalysing carbon fixation inside a proteinaceous shell. How Rubisco packs into the carboxysomes is unknown. Using cryo-electron tomography and subtomogram averaging, we present 3D organization of Rubisco inside two types of native -carboxysomes from a marine -cyanobacterium Cyanobium sp. PCC 7001 and a chemoautotrophic bacterium Halothiobacillus neapolitanus. We determined the structures of Rubiscos within native Halothiobacillus and Cyanobium carboxysomes at 3.3 [A] and 3.8 [A] resolution respectively and further identified an associated CsoS2 segment. Interestingly, CsoS2 is only associated with a sub-population of Rubiscos that are close to the shell in Halothiobacillus, but with all Rubiscos throughout Cyanobium carboxysome. Moreover, Rubiscos in Cyanobium carboxysomes are organized in three concentric layers whereas Rubiscos in Halothiobacillus carboxysomes are arranged in spiral arrays. Calcium treatment induced a drastic re-organization of Rubiscos, converting these two distinct assemblies into ordered lattice arrays in both -carboxysomes. Our findings provide critical knowledge of the assembly principles of -carboxysomes, which may aid in rational design and repurpose of carboxysome structures for new functions.

microbiology↗

bioRxiv2022

Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome

Carboxysomes are proteaceous bacterial microcompartments (BMCs) that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating carbonic anhydrase and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyses the dehydration of bicarbonate into CO2, the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural characterization of native carboxysomes, including the shell and the internal organization, is currently limited to low-resolution tomography studies. Notably, the degree of heterogeneity of the shell, and the internal arrangement of enzymes, remain poorly understood. Here, we report the structural characterization of a native -carboxysome from a marine cyanobacterium by single-particle cryo-EM. We determine the structure of RuBisCO enzyme at 2.9 [A] resolution. In addition, we obtain low-resolution maps of the icosahedral protein shell and the concentric interior organisation. In combination with artificial intelligence (AI)-driven modelling approaches, we exploited these maps to propose a complete atomic model of an intact carboxysome. This study provides insight into carboxysome structure and protein-protein interactions involved in carboxysome assembly. Advanced knowledge about carboxysome architecture and structural plasticity is critical for not only a better understanding of biological carbon fixation mechanism but also repurposing carboxysomes in synthetic biology for biotechnological applications.

biophysics↗

bioRxiv2021

Structural basis for the assembly and electron transport mechanisms of the dimeric photosynthetic RC-LH1 supercomplex

The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC-LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple photosynthetic bacteria. Some species possess the dimeric RC-LH1 complex with an additional polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC-LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC-LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC-LH1 dimer, interlocking association between the components and mediating RC-LH1 dimerization. Moreover, we identify a new transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations enable a mechanistic understanding of the assembly and electron transport pathways of the RC-LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.

biochemistry↗

bioRxiv2021

Biogenesis of a bacterial metabolosome for propanediol utilization

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both "Shell first" and "Cargo first" assembly pathways, unlike the {beta}-carboxysome structural analog which only involves the "Cargo first" strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

microbiology↗

bioRxiv2021

Probing the biogenesis pathway and dynamics of thylakoid membranes

How thylakoid membranes are generated to form the metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

plant biology↗

bioRxiv2020

Identification of a new orthonairovirus associated with human febrile illness in China

The genus Orthonairovirus of the family Nairoviridae includes the important tick-transmitted pathogens, Crimean-Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV), as well as many other poorly characterized viruses isolated from ticks, birds, and mammals1,2. Here we identified a novel orthonairovirus, designated S[o]ngl[i]ng virus (SGLV), from patients who reported being bitten by a tick in China. The genome of SGLV shared similar structural features with orthonairoviruses, with 46.5-65.7% sequence identify. Phylogenetic analysis showed that SGLV belonged to the Tamdy orthonairovirus and formed a unique clade in the Nairoviridae family. Electron microscopy revealed typical morphological characteristics of orthonairoviruses. The isolated SGLVs from the blood samples of patients could induce cytopathic effects in human hepatoma cells. SGLV infection was confirmed in 42 patients in 2017-2018, with the main clinical manifestations of headache, fever, depression, fatigue and dizziness. Serological assays showed that 69% patients generated virus-specific antibody responses in the acute phase. In contrast, neither SGLV viral RNA nor specific antibodies against SGLV were detected in healthy individuals. SGLV was also detected in Ixodes crenulatus, Haemaphysalis longicornis, Haemaphysalis concinna, and Ixodes persulcatus in northeastern China. Collectively, a newly discovered orthonairovirus was shown to be associated with human febrile illness in northeastern China.

microbiology↗

bioRxiv2020

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intend of engineering BMC-based metabolic modules for new functions in synthetic biology.

biophysics↗

bioRxiv2020

Structural visualization of septum formation in Staphylococcus warneri using atomic force microscopy

Cell division of Staphylococcus adopts a "popping" mechanism that mediates extremely rapid separation of the septum. Elucidating the structure of the septum is crucial for understanding this exceptional bacterial cell division mechanism. Here, the septum structure of Staphylococcus warneri is extensively characterized using high-speed time-lapse confocal microscopy, atomic force microscopy, and electron microscopy. The cells of S. warneri divide in a fast "popping" manner on a millisecond timescale. Our results show that the septum is composed of two separable layers, providing a structural basis for the ultrafast daughter cell separation. The septum is formed progressively toward the center with non-uniform thickness of the septal disk in radial directions. The peptidoglycan on the inner surface of double-layered septa is organized into concentric rings, which are generated along with septum formation. Moreover, this study signifies the importance of new septum formation in initiating new cell cycles. This work unravels the structural basis underlying the "popping" mechanism that drives Staphylococcus cell division and reveals a generic structure of the bacterial cell. IMPORTRANCEThis work shows that the septum of Staphylococcus warneri is composed of two layers and the peptidoglycan on the inner surface of the double-layered septum is organized into concentric rings. Moreover, new cell cycles of Staphylococcus could be initiated before the previous cell cycle is complete. This work advances our knowledge about a basic structure of bacterial cell and provides the double layered structural information of septum for the bacterium that divide with the "popping" mechanism.

biophysics↗