Biology subjects

Ghosal, D.

Publications and source records attributed to Ghosal, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Whole cryo-FIB lamella acquisition at high magnification with rectangular beam montage cryo-electron tomography

The visualisation of large biological samples in cryo-electron tomography requires both few-nanometre resolution and a wide, ideally >10 micrometrre, field of view. For a single image, increasing magnification trades increased resolution for a smaller field of view. Montaging, where high-magnification images are stitched together to increase field of view, addresses the trade-off but is dose-inefficient with round beams that are standard in transmission electron microscopy (TEM), since this illumination shape does not tessellate. Our workflow greatly simplifies montaging schemes for dose-sensitive specimens, allowing large montages of any shape - from individual cells or other features of interest up to whole lamellae - to be collected at high resolution without compromising electron dose efficiency. We demonstrate the workflow by acquiring montage tomograms of whole yeast (Saccharomyces cerevisiae) cells in cryo-FIB lamellae and of human neurons.

biophysics↗

bioRxiv2026

The Spatial Architecture of Human Subgingival Biofilms

Human microbiome studies have transformed understanding of disease-associated microbial communities, yet most approaches collapse polymicrobial biofilms into compositional profiles and lose the spatial relationships through which microorganisms interact in vivo. Periodontitis provides a clinically important model to investigate microbial spatial organisation because the disease is associated with deep, anaerobic periodontal pockets colonized by dense polymicrobial biofilms. Here, using multiplex fluorescence in situ hybridization with spectral and confocal microscopy of subgingival plaque across 174 sites from 36 individuals with periodontitis, we define taxon-resolved spatial architectures of the human subgingival plaque microbiome. We show that subgingival plaque is organized into recurrent multicellular assemblages of microbial consortia rather than unstructured layers of biomass, including assemblages defined as test-tube-brushes, corncobs and additional polymicrobial architectures with distinct taxonomic patterning. Test-tube brushes were enriched in severe periodontal niches, and their detection and burden increased with pocket depth, with distinct patterns observed in smoking-associated disease sites. In a longitudinal treatment subset, sites containing test-tube brushes before therapy lacked detectable test-tube brushes after treatment and instead showed corncobs concomitant with reduced pocket depth and resolution of disease. In addition to canonical oral biofilm structures, we identify previously undescribed polymicrobial assemblages whose occurrence is patterned by pocket depth and anatomical tooth site. These findings show that disease-associated human biofilms are organized into reproducible spatial states and suggest that microbial architecture provides an additional axis for interpreting microbiome-associated disease.

microbiology↗

bioRxiv2026

Viruses, Proviruses and Satellites from Asgard Archaea Enrichments Reveal Complex Microbial Interactions

Asgard archaea are the closest known relatives of eukaryotes and are central to models of eukaryogenesis involving archaeal bacterial symbiosis, yet the contribution of viruses remains unexplored. Here, for the first time, we visualised unique viruses associated with Asgard archaeal cells. Additionally, we identified the first putative Asgard-archaeal virus satellite, which exhibited genomic interactions involving a bacterium, Stromatodesulfovibrio nilemahensis, providing evidence of a virus-mediated interaction between an Asgard archaeon and a bacterium. Additionally, novel proviruses of S. nilemahensis displayed distinct genomic features where predictions of alternate recombination sites suggested the acquisition of horizontally acquired genes associated with biofilm formation. Further, we comprehensively characterise (pro)viruses associated with this co-culture using high resolution cryo-electron tomography, proximity ligation (Hi-C), and metagenomics. Together, these findings expand the known diversity of Asgard archaeal viruses and establish a foundation for investigating the role of viruses in microbial symbiosis relevant to the emergence of eukaryotic life.

microbiology↗

bioRxiv2026

Bacterial nanotubes extend from a tubeosome organelle to establish an intercellular bridge

Bacterial nanotubes are widespread intercellular membranous bridges mediating molecular exchange among neighboring cells, yet their formation and dynamics remain largely unexplored. By combining live-cell imaging with in situ cryo-electron tomography, we describe nanotube biogenesis in the Gram-positive Bacillus subtilis. Nanotubes form elaborate, highly dynamic networks, with extending nanotubes exhibiting multidirectional movement toward nearby cells, repeatedly scanning their surface. Ultimately, nanotubes penetrate through the recipient cell wall to establish an intercellular bridge. Strikingly, nanotubes emerge within milliseconds from lipid-enriched sites representing a previously unrecognized membranous organelle, termed tubeosome, located in an unusual pseudo-periplasmic space beneath the cell wall. We present evidence that tubeosomes comprise proto-nanotube reservoirs that unfold to project nanotubes. Our study uncovers the tubeosome as a novel bacterial organelle and provides the first real-time visualization of intercellular bridge formation. As connecting nanotubes are found across bacteria, archaea, and eukaryotes, this mechanism may reflect a broadly conserved strategy for cellular communication.

microbiology↗

bioRxiv2026

A Tripartite Co-culture System Reveals Defensive Mutualism Between a Nanobdellati Symbiont and Its Host

Archaeal symbioses remain among the least understood cellular interactions. Ultra-small Nanobdellati (initially called DPANN) archaea rely on larger hosts for survival, yet their effects on host eco-physiology and their interactions with infecting viruses have not yet been examined experimentally. Here, we established the first stable tripartite co-culture comprising a Nanobdellales symbiont (YN4), its archaeal host (YN4HA), and a virus (MTIV4). We analyzed this system using physiological, genomic, transcriptomic, glycoproteomic, and cryo-electron tomography (cryoET) approaches. CryoET analysis revealed that the Nanobdellales archaeon formed cone-like structures to contact the host, as has been observed in other Nanobdellati-host systems. In this system, the host is infected by the virus. The Nanobdellales archaeon mitigated virus-induced growth inhibition of the host without any detectable fitness cost, indicating a defensive mutualism rather than a strictly parasitic relationship. This protective effect may involve Nanobdellati-driven remodeling of host cell surface glycans, suggesting a previously unrecognized glycan-mediated defense strategy. Overall, our tripartite system provides new insight into complex archaeal interactions in extreme environments that cannot be captured through conventional binary co-culture systems.

microbiology↗

bioRxiv2026

A Novel Pilus System in Candidate Phyla Radiation Bacteria

The Candidate Phyla Radiation (CPR) represents a bacterial superphylum estimated to include between 15-50% of all bacterial species, yet CPR bacteria remain challenging to culture and have been primarily identified through metagenomic approaches. Candidatus Mycosynbacter amalyticus is a parasitic CPR bacterium that specifically targets Gordonia amarae, an actinobacterium with a hydrophobic, mycolic acid-rich cell envelope. Previous cryo-electron tomography indicated that Ca. M. amalyticus assembles thin extracellular filaments that are important for host interaction, yet their molecular identity remains unknown. Here, we applied single-particle cryo-electron microscopy to determine high-resolution structures of these filaments (2.8 and 3.6 [A]), enabling the unambiguous identification of two previously uncharacterized pilins from the experimental density maps. These pilins, designated PamA and PamB, assemble into unique helical filaments distinct from all previously characterized filaments in both domain architecture and assembly mechanism. Despite low sequence identity, both PamA and PamB share conserved structural principles, including Ig-like folds and donor-strand exchange-mediated assembly. Phylogenetic analysis indicates that Pam pilins are exclusive to CPR bacteria, with homologues distributed predominantly across the classes Saccharimonadia and Microgenomatia. Analysis of the conserved pam operon identifies putative chaperones (PamC and PamD) and assembly factors structurally homologous to chaperone-usher pili components, suggesting an analogous but distinct assembly pathway. These findings expand the known diversity of bacterial pilus systems and demonstrate the power of structural approaches for characterizing uncharacterized proteins encoded within CPR genomes.

microbiology↗

bioRxiv2026

Structural basis for curvature generation and functional specialization in spirochete flagella

Spirochetes are a distinctive phylum of spiral-shaped bacteria, defined by their curved periplasmic flagella, which drive motility by deforming the cell body and enabling efficient corkscrew-like propulsion through viscous environments. Despite their clinical importance, the molecular mechanisms underlying flagellar assembly, curvature and thus motility, remain poorly understood. Here we used cryo-electron microscopy combined with visual proteomic analysis to determine near-atomic resolution structures of two distinct flagellar filaments natively isolated from T. denticola, a major oral pathogen. Our structures reveal that filament curvature is generated by the asymmetric decoration of a conserved FlaB core by multiple sheath proteins, including FlaA1/2/3 and two previously uncharacterized proteins, termed FlaL1 and FlaL2. We show that the sheath imposes differential axial compaction on the FlaB core: FlaA1 expands the lattice at the outer curvature, FlaA2/3 compress the lattice at the inner curvature, and FlaL proteins stabilize these asymmetric interactions. Incorporation of distinct FlaB homologs contributes to assembled filament identity, with FlaB3 forming thin filaments and FlaB1/2 interacting with the sheath to form thick filaments. Comparative analysis reveals that FlaL proteins are conserved amongst some Treponema species and several other spirochetes, indicating that asymmetric assembly represents a modular solution to the mechanical demands of periplasmic flagella. These findings provide a structural framework for understanding functional specialization in bacterial filaments.

microbiology↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

An Asgard archaeon from a modern analog of ancient microbial mats

It has been proposed that eukaryotic cells evolved via symbiosis between sulfate-reducing bacteria and hydrogen-producing archaea. Here we describe a highly enriched culture of a novel Asgard archaeon, Nerearchaeum marumarumayae, with a bacterium Stromatodesulfovibrio nilemahensis from a stromatolite-associated microbial mat. The N. marumarumayae genome indicates it produces H2, acetate, formate, and sulfite, while S. nilemahensis synthesizes amino acids and vitamins, which can be exchanged in a syntrophic partnership. Electron cryotomography revealed N. marumarumayae cells produce chains of budded envelope vesicles attached to the coccoid cell body by extracellular fibers, and intracellular tube- and cage-like structures. Furthermore, the two species were observed interacting via intercellular nanotubes assembled by the bacterium. These characteristics and interactions may reflect an early step in the symbiotic evolution of eukaryotic cells.

microbiology↗

bioRxiv2025

Near-atomic structure of the PorKN rings, disulfide bonded to PorG and bound to Attachment Complexes, provide mechanistic insights into the type IX secretion system

The Type IX Secretion System exports proteins across the outer membrane (OM) of bacteria in the Bacteroidota phylum, however, the mechanistic details remain unknown. Here, we present a [~]3.5[A] cryo-EM structure of the periplasmic rings comprising 32-33 subunits each of PorK and PorN. Additionally, we show the presence of a critical disulfide bond between PorK and the PorG OM protein that is essential for protein secretion and demonstrate that the Attachment Complexes bind to and are localized above the PorKN rings. Overall, each ring resembles a cogwheel with PorN forming cog-like projections on the periplasmic side and the flat surface of PorK orienting towards the OM. Given these results, we propose that the PorLM motor drives the rotation of the PorKN cogwheel together with PorG and associated Attachment Complexes, potentially providing the energy to complete protein secretion and the coordinated cell surface attachment of the secreted cargo.

microbiology↗

bioRxiv2024

Novel cell-to-cell interactions revealed by cryotomography of a DPANN coculture system

DPANN is a widespread and highly diverse group of archaea characterised by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for their survival and proliferation. Despite the recent expansion in this clade, the structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we used electron cryotomography (cryo-ET) to reveal that the Candidatus Micrarchaeota (ARM-1) interacts with its host, Metallosphaera javensis through intercellular proteinaceous nanotubes. These tubes ([~]4.5 nm wide) originate in the host, extend all the way to the DPANN cytoplasm and act like tunnels for intercellular exchange. Combining cryo-ET and sub-tomogram averaging, we revealed the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states, providing mechanistic insights into substrate exchange. Additionally, we performed comparative proteomics and genomic analyses to identify host proteomic changes in response to the DPANN attachment. Our results showed striking alterations in host-proteome during symbiosis and upregulation/downregulation of key cellular pathways. Collectively, these results provided unprecedented insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.

microbiology↗

bioRxiv2024

A large attachment organelle mediates interaction between a novel Nanobdellota archaeon YN1 and its host

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (CryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravelled the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.

microbiology↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2023

Structure and Function of the Dot/Icm T4SS

The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the Agrobacterium tumefaciens type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease by the horizontal transfer of oncogenic DNA that is integrated into the hosts genome. The conjugation is mediated by the conjugative VirB/D4 type 4 secretion system (T4SS). A. tumefaciens T4SS assembles an extracellular filament, the T-pilus, that is involved in the formation of a mating pair between A. tumefaciens and the recipient plant cell by a not fully understood mechanism. Here, we present a 3 [A] cryo-EM structure of the T-pilus, solved by helical reconstruction. Our structure reveals that the T-pilus comprises the major pilin protein VirB2 and phosphatidylglycerol (PG) phospholipid at a 1:1 stoichiometric ratio with 5-start helical symmetry. We further show that PG-headgroups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 destabilized the VirB2 protein and completely abolished pilus formation. While our T-pilus structure shows architectural similarity with previously published conjugative pili structures, positively charged sidechains protrude into the lumen and the lumen is narrower, raising questions whether the T-pilus is a conduit for ssDNA transfer. We also show that the VirB2 subunits in T-pilus filament are not cyclic, as previously thought.

microbiology↗

bioRxiv2022

Structural remodeling of Coxiella burnetii during its biphasic developmental cycle revealedby cryo-electron tomography

Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages to cause a disease known as Q fever. It has a biphasic developmental lifecycle where the extracellular and metabolically inactive small cell variant (SCV) transforms, under host acidic environment, into the vegetative large cell variant (LCV). However, the details about the morphological and structural changes that accompany this biphasic cycle are still lacking. Here, we used cryo-electron tomography to image the different cell variants of C. burnetii grown either under axenic conditions in different pH or purified directly from host cells revealing the major developmental, morphological and structural transitions. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably allow a smooth transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities including a tubular structure possibly involved in host interaction or effector delivery. Also, SCVs and cells in the transition state contained spherical multilayered membrane structures of different sizes and locations suggesting that they are not related to a sporulation process as once assumed.

microbiology↗

bioRxiv2021

A novel widespread bacterial structure related to the flagellar type III secretion system

The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.

microbiology↗

bioRxiv2020

Structure of the bacterial cellulose ribbon and its assembly-guiding cytoskeleton by electron cryotomography

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength and stiffness prevents dehydration and mechanical disruption of the biofilm. Bacteria in the Gluconacetobacter genus secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused ion beam milling of native bacterial biofilms to image cellulose-synthesizing G. hansenii and G. xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several microns in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, that we name the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We find that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line, to form higher-order cellulose structures such as sheets and ribbons.

microbiology↗