Biology subjects

Bharat, T.

Publications and source records attributed to Bharat, T..

13 recordsLinked to original sources

bioRxiv2026

Structural basis of biofilm formation mediated by the Pseudomonas aeruginosa fibrillar adhesin CdrA

Many bacteria, including the important human pathogen Pseudomonas aeruginosa, are naturally found in antibiotic-tolerant, multicellular biofilms. Cell-cell interactions within P. aeruginosa biofilms are mediated by a large fibrillar adhesin called CdrA in an extracellular polysaccharide-dependent manner. Here, we report an electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus, which combined with electron cryotomography of focused-ion beam milled specimens, allows us to derive a complete in situ model of the native adhesin. Our structure reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the P. aeruginosa pangenome, with structural similarity to previously reported sugar-binding domains in multiple bacterial species. Inhibitory nanobodies targeting CdrA that reduce biofilm formation bind to epitopes in, or close to, the ADEPT on bacterial cells. Furthermore, structure-guided mutagenesis of residues within the ADEPT abolishes bacterial aggregation, and genomic deletion of the whole ADEPT leads to strong attenuation of biofilm formation. Our data forms a rational basis for future targeted inhibition of pathogenic P. aeruginosa biofilms and elucidates the mechanism of biofilm formation mediated by fibrillar adhesins that are widespread in bacteria.

microbiology↗

bioRxiv2026

Intracellular barriers and receptor masking limit success of a Pseudomonas aeruginosa clinical phage family

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.

microbiology↗

bioRxiv2025

An Asgard archaeon with internal membrane compartments

The emergence of eukaryotes from a merger between an archaeon and a bacterial cell [~]two billion years ago involved a profound change in cellular organisation. While the order in which different features of the eukaryotic cell arose remains a matter of controversy1-3, close archaeal relatives of eukaryotes have recently been identified that possess homologues of eukaryotic trafficking machinery4,5 and a complex cell architecture6,7. The members of this phylum, the Asgard archaea (syn. Prometheoarchaeota) described so far, however, lack internal membrane-bound compartments, and therefore have shed little light on origins of the hallmark eukaryotic endomembrane system. Here we report the cell biological analysis of a member of the Heimdallarchaeia, Candidatus Y. umbracryptum in enriched mixed microbial communities. Possessing a small genome encoding few homologues of eukaryotic membrane remodelling machinery8, Ca. Y. umbracryptum cells in late-stage cultures resemble previously described Asgard archaea with extensive cellular protrusions. Surprisingly, during early stages of culture growth Ca. Y. umbracryptum cells have fewer protrusions but possess numerous intracellular vesicles, most of which have a luminal surface that morphologically resembles the outer coat of the plasma membrane. These data alter our view of eukaryogenesis by identifying a close archaeal relative of eukaryotes with an endomembrane system, whose appearance changes with cell state.

cell biology↗

bioRxiv2025

Sub-cellular chemical mapping in bacteria using correlated cryogenic electron and mass spectrometry imaging

Electron cryomicroscopy (cryo-EM) allows high-resolution spatial visualization of biological specimens, however, it is challenging to chemically identify densities observed in cryo-EM. To overcome this, we combined cryo-EM with chemical imaging using focused ion beam secondary ion mass spectrometry (FIB-SIMS) for integrated spatio-chemical analysis of untagged specimens. We show that our correlative workflow permits sub-cellular localisation of molecules inside bacterial cells and is compatible with cryogenic light microscopy and FIB-milled lamellae of multicellular specimens. To highlight biological insights enabled by the workflow, we studied the uptake of Bisphenol-AF, a widespread chemical pollutant, by environmental bacteria, revealing the storage of these chemicals within cytosolic phase-separated aggregates in pollutant-exposed cells, where they cannot be removed by the bacterial efflux machinery despite its robust upregulation. These mechanistic insights were directly facilitated by the versatile cryo-EM-FIB-SIMS technique, showing that it is an effective avenue to map elemental and molecular signatures in near-native biological samples, which can be extended in the future for multiple applications in cell biology and imaging.

molecular biology↗

bioRxiv2025

The mechanics of a continuous self-assembling surface-layer aids cell division in an archaeon

The surface layer or "S-layer" is a planar lattice of glycosylated proteins that coats a wide range of archaea and bacteria instead of a classical cell wall or capsular polysaccharides, insulating them from the extracellular space and providing the cell membrane with physical support. Although the S-layers role as a mechanical support for the membrane might be expected to hinder cell division, we show that in rapidly dividing Sulfolobus acidocaldarius cells, the S-layer protein SlaA self-assembles into flexible lattice that helps flattens the cytokinetic furrow to accelerate ESCRT-III dependent cell division - a role that is especially important under conditions of mechanical stress. Taken together, these results generated using mutational analysis, live and fixed cellular imaging, along with electron cryomicroscopy, define the rules governing S-layer self-assembly and show how the mechanical properties of flexible lattice coats can enhance membrane functions to both physically support a cell and help to drive ESCRT-III dependent cell division. Significance statementThe bounding membrane of a cell must be protected from environmental insults. In many bacteria and archaea that lack a cell wall, this is achieved by an enveloping S-layer. By virtue of its rigid, planar structure, the S-layer is also expected to act as a barrier to the cell shape changes required for cytokinesis. In this study of the S-layer lattice in the archaeon Sulfolobus acidocaldarius however, we demonstrate a role for this self-assembling mechanical support in re-shaping the cytokinetic furrow as an aid to ESCRT-III-mediated division. In showing how mechanically active and passive structures can work together to give rise to mechanically stable cells that can divide, this reconciles what seems like a trade-off between resilience and flexibility.

molecular biology↗

bioRxiv2024

Tissue-Like Multicellular Development Triggered by Mechanical Compression in Archaea

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once observed in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity become akin to animal tissues and give rise to two cell types--radial peribasal and central apicobasal cells-- with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

cell biology↗

bioRxiv2024

Punctuated and continuous structural diversity of S-layers across the prokaryotic tree of life

Surface layers (S-layers) are two-dimensional (2D) crystalline lattices that frequently coat prokaryotic cells, playing a crucial role in protection, maintaining cellular integrity, and mediating environmental interactions. However, the molecular landscape of these abundant proteins has remained underexplored due to a lack of structural data. By employing AlphaFold2multimer together with planar symmetry constraints in a workflow validated by electron cryomicroscopy structure determination, we have elucidated the lattice structures of over 150 S-layers from diverse archaea and bacteria. Our findings unveil a multifaceted evolutionary landscape for S-layer proteins, highlighting key differences in the evolution of bacterial and archaeal S-layers. Our study allows us to discover underlying patterns in S-layer structure, organisa-tion, and cell anchoring mechanisms across the prokaryotic tree of life, deepening our understanding of the intricately complex microbial cell surfaces, which appear to have evolved proteinaceous S-layers independently on multiple occasions. This work will open avenues for rational manipulation of prokaryotic cellular interactions in multicellular microbiomes, as well as for innovative 2D biomaterial design.

molecular biology↗

bioRxiv2024

Structure of the Pseudomonas aeruginosa PAO1 Type IV pilus

Type IV pili (T4Ps), which are abundant in many bacterial and archaeal species, have been shown to play important roles in both surface sensing and twitching motility, with implications for adhesion, biofilm formation and pathogenicity. While Type IV pilus (T4P) structures from other organisms have been previously solved, a high-resolution structure of the native, fully assembled T4P of Pseudomonas aeruginosa, one of the major human pathogens, is not available. Here, we report a 3.2 [A]-resolution structure of the P. aeruginosa PAO1 T4P determined by electron cryomicroscopy (cryo-EM). PilA subunits constituting the T4P exhibit a classical pilin fold featuring an extended N-terminal -helix linked to a C-terminal globular {beta}-sheet-containing domain, which are packed tightly along the pilus. The N-terminal helices constitute the pilus core where they stabilise the tubular assembly via hydrophobic interactions. The -helical core of the pilus is surrounded by the C-terminal globular domain of PilA that coats the outer surface of the pilus, mediating interactions with the surrounding environment. Comparison of the P. aeruginosa T4P with T4P structures from other organisms, both at the level of the pilin subunits and the fully assembled pili, allows us to enumerate key differences, and detect common architectural principles in this abundant class of prokaryotic filaments. This study provides a structural framework for understanding the molecular and cell biology of these important cellular appendages mediating interaction of prokaryotes to surfaces.

microbiology↗

bioRxiv2022

Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, partly because of its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research into this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjoining hexamer, leading to a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting shared traits in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.

microbiology↗

bioRxiv2022

A multi-domain connector links the outer membrane and cell wall in deep-branching bacteria

Deinococcus radiodurans is a deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report an electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded {beta}-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Using the power of structure prediction and sequence-based bioinformatics, we further show that SlpA-like proteins are widespread in deep-branching Gram-negative bacteria, plausibly constituting an ancestral superfamily of OM-PG connectors, important for organizing the cell envelopes of many bacteria. Finally, combining our atomic structures with tomography of cell envelopes, we report a model for the cell surface of D. radiodurans, with implications on understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria. Furthermore, the widespread occurrence of SlpA-like OM-PG connectors in deep-branching bacteria will help in understanding the evolutionary transition between Gram-negative and Gram-positive bacteria.

microbiology↗

bioRxiv2022

Molecular architecture of the TasA biofilm scaffold in Bacillus subtilis

Many bacteria in nature exist in multicellular communities termed biofilms. Cells within biofilms are embedded in a primarily self-secreted extracellular polymeric matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model biofilm-forming bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. The structure of TasA fibres, however, and how fibres scaffold the biofilm at the molecular level, is not known. Here, we present electron cryomicroscopy structures of TasA fibres, which show that rather than forming amyloid fibrils, TasA monomers assemble into filaments through donor strand complementation, with each subunit donating a {beta}-strand to complete the fold of the next subunit along the filament. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA filaments congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. This study explains the previously observed biochemical properties of TasA and shows, for the first time, how a bacterial extracellular globular protein can assemble from monomers into {beta}-sheet-rich fibres, and how such fibres assemble into bundles in biofilms. We establish a hierarchical, atomic-level assembly mechanism of biofilm scaffolding that provides a structural framework for understanding bacterial biofilm formation.

microbiology↗

bioRxiv2022

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.

biophysics↗

bioRxiv2021

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, where cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused-ion beam milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt pre-existing biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

microbiology↗