Biology subjects

Gutsche, I.

Publications and source records attributed to Gutsche, I..

16 recordsLinked to original sources

bioRxiv2026

Deinococcus radiodurans HU: a versatile architect of nucleoid structure and plasticity

Nucleoid-associated proteins are known to compact and organise bacterial genomes, but how their local DNA binding shapes higher-order chromatin architecture remains unclear. Using in vitro and in situ approaches, we investigated how Deinococcus radiodurans HU (DrHU) orchestrates nucleoid organisation. DrHU engages DNA through two synergistic binding modes: high-affinity {beta}-hairpin-mediated stabilisation of DNA loops and low-affinity, multivalent interaction via its N-terminal tail, driving parallel, uniformly spaced DNA alignment. In vitro, DrHU organises supercoiled plasmid DNA into regular 2D lattices of double-spirals, while in situ, UV-C light induces a nucleoid transition from a loose DNA mesh to a blue liquid crystalline phase characterised by DNA swirls. The striking structural similarity and shared inter-filament spacing of these two arrangements suggests that DrHU plays a pivotal role in genome organisation through its versatile binding properties that enable DNA loop stabilisation, protection and DNA bridging, linking molecular interactions to 3D genome ordering and stress-induced remodelling.

microbiology↗

bioRxiv2026

A single methylation site regulates HRSV nucleocapsid architecture and replication

The nucleoprotein N of the human respiratory syncytial virus (HRSV) encases the viral genome, forming a flexible N-RNA nucleocapsid helix that serves as template for the viral polymerase L. Recent structural analysis revealed a non-canonical helical nucleocapsid arrangement that modulates RNA accessibility, yet its impact on polymerase function remains unknown. Here, we identified symmetric dimethylation of residue R27 of N as a critical modulator of nucleocapsid architecture and viral replication. We also showed that the methylase PRMT5 interacts with N and likely catalyses R27 methylation. Molecular dynamics simulations of RNA-free N dimers indicate that R27 methylation enhances opening and closing of the RNA-binding cavity, whereas a methylation-mimicking R27M substitution favours a closed state. Cryo-electron microscopy reveals a canonical, markedly straight R27M helix with increased rise and pitch. These findings demonstrate that post-translational modifications fine-tune interactions between N protomers, shaping nucleocapsid assembly, structure and dynamics, and thereby controlling HRSV replication.

microbiology↗

bioRxiv2025

Filamentation-driven peripheral clustering of the inducible lysine decarboxylase is crucial for E. coli acid stress response

Bacteria have evolved numerous sophisticated acid stress response strategies to withstand fluctuating environmental pH, with enterobacterial inducible amino acid decarboxylases playing a major role. The lysine decarboxylase LdcI catalyses lysine-to-cadaverine conversion coupled to proton consumption and carbon dioxide release, thereby buffering cytoplasmic and extracellular pH. Our previous study showed that Escherichia coli LdcI forms intracellular patches under mild acid stress, and that at the corresponding acidic pH, purified LdcI polymerises into filaments, suggesting a potential role for polymerisation in its function. Here, we investigate the physiological relevance of LdcI filamentation in E. coli using 3D super-resolution fluorescence microscopy and a structure-based LdcI polymerisation-deficient E. coli mutant strain. We establish a semi-automated workflow for detection of clusters within a bacterial cell as well as quantitative analysis of their volume and localisation. We demonstrate that LdcI predominantly clusters at the cell periphery. We then show that disrupting LdcI polymerisation markedly reduces cluster size without significantly affecting localisation, suggesting that acid-stress induced LdcI clustering in vivo is driven by the enzymes filamentation. Growth and pH measurements reveal that the mutant strain exhibits reduced fitness and impaired extracellular buffering compared to the wild type, indicating that LdcI polymerisation enhances E. coli acid stress response. Our findings provide strong evidence that LdcI filamentation is a key regulatory feature of the E. coli capacity to counteract acid stress, optimising enzymatic activity by spatially organising LdcI within the cytoplasm. More broadly, this work adds to a growing body of evidence supporting the functional significance of metabolic enzyme self-assembly and its implications for bacterial stress adaptation. Author summaryAmino acid decarboxylases confer enterobacteria the ability to survive under acidic conditions. Kirchner et al. show that polymerisation of the acid-stress inducible lysine decarboxylase LdcI within E. coli cells leads to its peripheral intracellular clustering and fine-tunes its enzymatic activity in vivo.

microbiology↗

bioRxiv2025

Expanding the Tubulin Code: TTLL11 Polyglutamylase Drives Elongation of Primary Tubulin Chains

Microtubules (MTs) undergo diverse post-translational modifications that regulate their structural and functional properties. Among these, polyglutamylation - a dominant and conserved modification targeting the unstructured tubulin C-terminal tails - plays a pivotal role in defining the tubulin code. Here, we uncovered a novel mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11s binding and catalytic domains engage adjacent MT protofilaments. Biochemical assays identified previously unknown polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of - and {beta}-tubulin, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data firmly established a crosstalk between TTLL11-mediated polyglutamylation and other tubulin-modifying processes, notably the detyrosination/tyrosination cycle. This discovery unveils an unrecognized layer of complexity within the tubulin code and offers new insights into the molecular basis of functional specialization of cytoskeleton across diverse cellular contexts.

biochemistry↗

bioRxiv2024

Combining live cell fluorescence imaging with in situ cryo electron tomography sheds light on the septation process in Deinococcus radiodurans

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes, but in all cases, division involves a step of septation, corresponding to the growth of a new dividing cell wall, followed by splitting of the two daughter cells. The radiation-resistant Deinococcus radiodurans is a spherical bacterium protected by a thick and unusual cell envelope. It has been reported to divide using a distinctive mode of septation in which two septa originating from opposite sides of the cell progress with a flat leading edge until meeting and fusing at mid-cell. In the present study, we have combined conventional and super-resolution fluorescence microscopy of live bacteria with in situ cryogenic electron tomography of bacterial lamellae to investigate the septation process in D. radiodurans. This work provides important insight into (i) the complex architecture of the cell envelope of this bacterium, (ii) the sliding doors septation process and (iii) the molecular mechanisms underlying septal growth and closure.

microbiology↗

bioRxiv2023

blik: an extensible napari plugin for cryo-ET data visualisation, annotation and analysis

Powerful, workflow-agnostic and interactive visualisation is essential for the ad-hoc, human-in-the-loop workflows typical of cryo-electron tomography (cryo-ET). While several tools exist for visualisation and annotation of cryo-ET data, they are often integrated as part of monolithic processing pipelines, or focused on a specific task and offering limited reusability and extensibility. With each software suite presenting its own pros and cons and often tools tailored to address specific challenges, seamless integration between available pipelines is often a difficult task. As part of the effort to enable such flexibility and move the software ecosystem towards a more collaborative and modular approach, we developed blik, an open-source napari plugin for visualisation and annotation of cryo-ET data (source code: https://github.com/brisvag/blik). blik offers fast, interactive, and user-friendly 3D visualisation thanks to napari, and is built with extensibility and modularity at the core. Data is handled and exposed through well-established scientific Python libraries such as numpy arrays and pandas dataframes. Reusable components (such as data structures, file read/write, and annotation tools) are developed as independent Python libraries to encourage reuse and community contribution. By easily integrating with established image analysis tools - even outside of the cryo-ET world - blik provides a versatile platform for interacting with cryo-ET data. On top of core visualisation features - interactive and simultaneous visualisation of tomograms, particle picks and segmentations - blik provides an interface for interactive tools such as manual particle picking, surface-based and filament-based particle picking and image segmentation, as well as simple filtering tools. Additional self-contained napari plugins developed as part of this work also implement interactive plotting and selection based on particle features, and label interpolation for easier segmentation. Finally, we highlight the differences with existing software and showcase bliks applicability in biological research.

bioinformatics↗

bioRxiv2023

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid {beta}-oxidation (FAO) to CI assembly. We identify an adjacent -helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylated in vitro and ex cellulo and provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimers disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-{beta} (A{beta}) oligomers. These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by A{beta} toxicity, a hallmark of Alzheimers disease.

molecular biology↗

bioRxiv2023

Structural landscape of the Respiratory Syncytial Virus nucleocapsids

Human Respiratory Syncytial Virus (RSV) is a prevalent cause of severe respiratory infections in children and the elderly. The viral genome, enwrapped by the nucleoprotein N into a helical nucleocapsid (NC), is a template for the viral RNA synthesis and a scaffold for the virion assembly. Although the structures of NC filaments representative of the other major families of the Mononegavirales order have been solved, a detailed understanding of the RSV NCs is missing. This cryo-electron microscopy (cryo-EM) analysis highlights the polymorphism of the RSV nucleocapsid-like assemblies. We reveal in particular the non-canonical arrangement of the RSV NC helix, composed of 16 N per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from recurring longitudinal interactions by the C-terminal arm of the RSV N, whose truncation abrogates the inter-turn contacts. We report the cryo-EM structures of the full-length helical NC filaments, double-headed NCs, ring-capped NCs and double-decameric N-RNA rings, as well as those of the alternative assemblies formed by a C-terminally truncated N mutant. In addition, we demonstrate the functional importance of the interface involved in the formation of the double-headed and the ring-capped interactions. We put all these findings in the context of the RSV RNA synthesis machinery and delineate the structural basis for its further investigation.

microbiology↗

bioRxiv2022

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to antibiotics under anaerobiosis and has a major impact on cell membrane regulation.

microbiology↗

bioRxiv2021

Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction. IMPORTANCELike respiratory syncytial virus (RSV), human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for these two pneumoviruses. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, in particular a pocket on the surface of the N protein that could be used as a template for the design of compounds interfering with N-P complexes and viral replication.

microbiology↗

bioRxiv2021

Alternative architecture of the E. coli chemosensory array

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

biochemistry↗

bioRxiv2021

Tools enabling flexible approaches to high-resolution subtomogram averaging

Cryo-electron tomography and subtomogram averaging are increasingly used for macromolecular structure determination in situ. Here we introduce a set of computational tools and resources designed to enable flexible approaches to subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

biophysics↗

bioRxiv2020

Supramolecular assembly of the E. coli LdcI upon acid stress

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statement Bacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Complete structure of the core signalling unit of the E. coli chemosensory array in an optimised minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2 Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novel E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 [A] resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

cell biology↗

bioRxiv2019

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.

biochemistry↗

bioRxiv2018

Identification of a novel subfamily of bacterial AAT-fold basic amino acid decarboxylases and functional characterization of its first representative: Pseudomonas aeruginosa LdcA

Polyamines are small amino-acid derived polycations capable of binding negatively charged macromolecules. Bacterial polyamines are structurally and functionally diverse, and are mainly produced biosynthetically by PLP-dependent amino acid decarboxylases referred to as LAOdcs (Lysine-Arginine-Ornithine decarboxylases). In a phylogenetically limited group of bacteria, LAOdcs are also induced in response to acid stress. Here, we performed an exhaustive phylogenetic analysis of the AAT-fold LAOdcs which showcased the ancestral nature of their short forms in Cyanobacteria and Firmicutes, and emergence of distinct subfamilies of long LAOdcs in Proteobacteria. We identified a novel subfamily of lysine decarboxylases, LdcA, ancestral in Betaproteobacteria and Pseudomortadaceae {Gammaproteobacteria). We analyzed the expression of LdcA from Pseudomonas aeruginosa, and uncovered its role, intimately linked to cadaverine production, in promoting growth and reducing persistence of this multidrug resistant human pathogen during carbenicillin treatment. Finally, we documented a certain redundancy in the function of the three main polyamines - cadaverine, putrescine and spermidine - in P. aeruginosa by demonstrating the link between their intracellular level, as well as the capacity of putrescine and spermidine to complement the growth phenotype of the IdcA mutant.

microbiology↗