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Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In walled bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes. 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 superresolution 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 and multilayered composition of the cell envelope of this bacterium, ii) the unusual "sliding doors" septation process and iii) the sequence of events and molecular mechanisms underlying septal closure, including the synthesis of a FtsZ-dependent peptidoglycan layer that rigidifies and straightens the growing septa.

Bacteria use 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 studies showed that Escherichia coli LdcI forms intracellular patches under mild acid stress, and that purified LdcI polymerises into filaments at acidic pH. Here, we investigated the physiological relevance of LdcI filamentation using 3D super-resolution microscopy and an LdcI polymerisation-deficient E. coli mutant strain. We established a semi-automated workflow for intracellular cluster detection and quantitative analysis, and demonstrated predominantly peripheral clustering of LdcI. Disrupting LdcI polymerisation markedly reduced cluster size without significantly affecting localisation, suggesting that clustering is driven by filamentation. Growth and pH measurements revealed that the mutant exhibits reduced fitness and impaired extracellular buffering compared to the wild type, indicating that LdcI polymerisation enhances the E. coli capacity to counteract acid stress without affecting intracellular location of the enzyme. Our findings provide strong evidence that LdcI filamentation regulates acid stress response by spatially optimising enzymatic activity. More broadly, this work supports the functional significance of metabolic enzyme self-assembly in bacterial stress adaptation.

Microtubules (MTs) undergo diverse posttranslational modifications that regulate their structural and functional properties. Among these, polyglutamylation—a dominant and conserved modification targeting unstructured tubulin C-terminal tails—plays a pivotal role in defining the tubulin code. Here, we describe a 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 TTLL11 binding and catalytic domains engage adjacent MT protofilaments. Biochemical and cellular assays identified previously uncharacterized polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of α- and β-tubulin in vitro, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data suggest a cross-talk between polyglutamylation and the detyrosination/tyrosination cycle likely linked to the TTLL11-mediated elongation of the primary α-tubulin chain. These findings unveil an unrecognized layer of complexity within the tubulin code and offer mechanistic insights into the molecular basis of functional specialization of MT cytoskeleton.

We present evidence that supports a 'correct hydrazone tautomer/Dunathan alignment model' for how α-hydrazino analogues of α-amino acids inhibit PLP enzymes. Described is the asymmetric synthesis of l- and d-α-hydrazino acid l-lysine analogues and their inhibition of Hafnia alvei lysine decarboxylase (LdcI) via kinetic analysis, stopped-flow spectrophotometry, and cryo-EM. We describe a similar investigation of the important anti-Parkinsonism drug, carbidopa, with its human DOPA decarboxylase (hDdc) target. Evidence is consistent with these three hydrazino analogues forming the catalytically relevant ketoenamine PLP-hydrazone tautomer in their target active sites, with the α-carboxylate groups, though insulated, aligning with the PLP-π-system in a Dunathan-model-like orientation. High-resolution cryo-EM structures of the H. alvei LdcI holoenzyme (pdb 9E0M-2.1Å) and LdcI-bound l- and d-hydrazones (pdb 9E0O-2.0 Å; pdb 9E0Q-2.3Å) and the first X-ray crystal structure of hDdc-bound carbidopa (pdb 9GNS-1.93Å) support this 'correct tautomer' model. These insights are expected to guide future PLP enzyme inhibitor development.

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 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 cryoET 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, 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 blik's applicability in biological research.

The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer’s disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. 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, releasing the FAD cofactor and converting ACAD9 from a fatty acid β-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-β (Aβ) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aβ toxicity, a hallmark of AD.