Biology subjects

Repnik, U.

Publications and source records attributed to Repnik, U..

15 recordsLinked to original sources

bioRxiv2026

An SPFH Protein Couples Membrane Stress to Differentiation in Bacillus subtilis

Bacillus subtilis adapts to fluctuating environmental stress, such as membrane perturbation or alkaline conditions, using membrane-associated regulatory complexes. Here, we rename the previously termed pspA-ydjGHI operon to pspA-samGHI (for starvation and motility) to reflect its functional roles in membrane envelope stress signalling. The SamG-SamH membrane proteins recruit SamI, a cytosolic SPFH protein, which stabilizes focal membrane localization and recruitment of PspA, an ESCRT-III homolog. Under normal conditions, this system transiently assembles at the membrane, stabilizing it and allowing proper motility, secretion, and biofilm formation. Loss of SamI ({Delta}samI/{Delta}ydjI) leads to unbalanced SamG-SamH activity leading to a constitutive stress signalling, and global transcriptional changes reminiscent of starvation situations. This, in turn, blocks secretion of the matrix protein BslA, preventing biofilm formation, and reducing motility. Deletion of samH in combination with {Delta}samI restores biofilm formation, while {Delta}pspA mutants form biofilms normally, indicating that PspA is dispensable for the developmental phenotype. Our findings reveal that beside membrane integrity SamGHI coordinates transcriptional homeostasis and multicellular development through formation of a membrane integral stress sensor complex.

microbiology↗

bioRxiv2025

Transcription profiling and functional analysis of spRNAs and their corresponding asRNAs in Methanosarcina mazei

Small proteins (sPs) encoded by small RNAs (sRNAs), the so-called small protein RNAs (spRNAs), and their corresponding antisense RNAs (asRNAs) have emerged as important regulatory elements in microbial stress adaptation, yet remain poorly characterized in archaea. In this study, we identified and characterized five sRNA/asRNA pairs in the methanoarchaeon Methanosarcina mazei. Expression analysis revealed condition-specific regulation of two of those pairs, spRNA23/asRNA93 and spRNA24/asRNA94, under oxidative, temperature, and osmotic stress. Overexpression of the corresponding small proteins, sP23 and sP24, revealed distinct physiological effects: both enhanced growth under high-salt conditions, while sP24 in addition supported cell size maintenance during osmotic stress, as confirmed by transmission electron microscopy. Consistently, overexpression of asRNA94 let to impaired growth under salt stress, suggesting its negative regulatory role towards sP24. The results also imply that the antisense RNA might be crucial for controlling sP24 expression under non-stress conditions. Bioinformatic analysis predicted one transmembrane domain in sP24. Membrane-association of sP24 was experimentally validated in vitro using a cell-free expression system, demonstrating sP24s integration into lipid bilayers. Conservation analysis showed that spRNA24 appears specific for M. mazei strains potentially reflecting niche adaptation, while spRNA23 is widespread within Methanosarcina. These findings demonstrate that archaeal sRNAs can encode functional small proteins, often encoded together with their respective regulatory asRNA, that contribute to environmental stress responses, overall offering new insights into the role of sRNA-derived small proteins in archaea.

microbiology↗

bioRxiv2025

Maternal transmission of a plastid structure enhances offspring fitness

Development in the sea has long been thought to be a nutritional gamble that disproportionately ends in starvation. Here, we unexpectedly show that components of plastids are incorporated into sea urchin eggs and that these, in turn, benefit developmental fitness. We find chromoplast-derived carotenoid crystals and chromoplast-specific metabolites inside of sea urchin eggs. The light-dependent activity of these chromoplast components influence the subsequent abundance of phytohormones that, in turn, regulate the use of energetic lipids that promote development and survival. Offspring that benefit from these chromoplast components are predicted to disperse further, over larger geographic areas, and use a wider range of currents, including those that cross ocean basins. Data presented here challenge the long-held assumption that components of non-metazoan organelles are unable to enter the germline and be passed between generations. We hypothesize that sea urchins manipulate plastids solely for their self-interest by a process that we term machioplasty, with the result of this process being a novel and adaptive form of maternal provisioning. SIGNIFICANCEMaking a fertilizable egg is a complex and carefully regulated process. One long-held assumption is that any component of non-animal organelles are unable to cross the evolutionary valley between non-reproductive and reproductive cells. Here, we unexpectedly show that components of plastids are incorporated into sea urchin eggs and that these, in turn, benefit developmental fitness. Offspring benefit from these plastid components by developing more quickly into larvae and having higher survival due to ability to use phytohormones that influence energetic lipids. This allows offspring to use a wider range of ocean currents, including those that cross entire basins. This challenge the long-held assumption that components of non-metazoan organelles are unable to be passed between generations.

developmental biology↗

bioRxiv2025

Membrane-binding of the bacterial dynamin-like protein DynA is essential for effective phage defence

Bacterial dynamin-like proteins are large GTPases that play crucial roles in membrane dynamics. DynA in B. subtilis, a two-headed bacterial dynamin-like protein, possesses membrane-binding and tethering functions in trans. The formation of large DynA clusters on membranes in response to pore-forming antibiotics and phages demonstrates its potential role in maintaining membrane integrity under various environmental stresses. In this study, we identified the membrane-binding site of B. subtilis DynA within the D1 domain of the protein that includes positively charged lysine residues K360 and K367, as well as hydrophobic phenylalanine residues F363, F364, and F365. For experimental validation, recombinant proteins with mutations in the lysine or phenylalanine residues were produced and used in liposome binding assays. Non-conservative mutations lead to a complete loss of DynAs membrane-binding capability. In vivo data showed that the membrane-binding mutants exhibit significantly increased susceptibility to phage infection compared to wild-type strains, further emphasizing the importance of DynAs membrane interaction in conferring phage resistance. Our findings bridge the gap between the structural characteristics of DynA and its functional implications in maintaining membrane integrity and mediating phage resistance.

microbiology↗

bioRxiv2025

Comparative analysis of novel Pseudobdellovibrionaceae genera and species yields insights into the genomics and evolution of bacterial predation mode

Bacteria of the family Pseudobdellovibrionaceae belong to a group of bacteria that kill and feed on other bacteria. The diversity of predation strategies, habitats, and genome characteristics of these bacteria are largely unexplored, despite their ecological and evolutionary importance in microbial communities. Therefore, we characterized new Pseudobdellovibrionaceae strains isolated from the direct environments of three animal hosts: the zebrafish (Danio rerio), the threespine stickleback fish (Gasterosteus aculeatus), and the nematode Caenorhabditis elegans. We used transmission electron microscopy (TEM) and genomic analyses to characterize the morphology and predation modes of our isolates. While most of our isolates exhibited periplasmic (i.e. endoparasitic) predation, one isolate clearly exhibited epibiotic (i.e. exoparasitic) predation and represents only the third confirmed epibiotic strain within its clade. Of our isolates, six are members of five new species in the genus Bdellovibrio and two strains likely represent new genera within the family Pseudobdellovibrionaceae. From metabarcoding data, we found indications that Pseudobdellovibrionaceae are widespread among our three animal hosts. Genomic analyses showed that epibiotic predators lack genes involved in host independence (i.e. prey-independent feeding) and peptidoglycan modification. However, genes unique to epibiotic predators may underlie this predation mode, particularly those involved in cell wall remodeling and recycling. With robust phylogenomic analyses, we show that our novel isolates cluster with previously described Pseudobdellovibrionaceae isolates according to predation mode. Further, by placing Pseudobdellovibrionaceae predators within a wider evolutionary history including other predatory and non-predatory bacteria, we postulate periplasmic predation as the ancestral mode, with more derived epibiotic predators exhibiting genome streamlining.

genomics↗

bioRxiv2024

Assembly of a functional neuronal circuit in embryos of an ancestral metazoan is influenced by environmental signals including the microbiome

Understanding how neural populations evolve to give rise to behavior is a major goal in neuroscience. However, the complexity of the nervous system in most invertebrates and vertebrates complicates the deciphering of underlying fundamental processes. Here, we explore the self-assembly of neural circuits in Hydra, an organism with a simple nervous system but no centralized information processing, to improve the understanding of nervous system evolution. The N4 neuronal circuit in embryos develops through activity-driven self-assembly, where neurons in distinct regions increase connectivity and synchronization. Gap junctions and vesicle-mediated communication between neuronal and non-neuronal cells drive rapid assembly, with the embryos prospective oral region exhibiting the highest neuronal density. An artificial electrical circuit-based model demonstrates dynamic increases in synchronization over time, along with predictions for selective dynamic adaptions of connections. Environmental factors, like temperature and an absent microbiome, modify neural architecture, suggesting the existence of a certain plasticity in neural development. We propose that these fundamental features originated in the last common bilaterian ancestor, supporting the hypothesis that the basic architecture of the nervous system is universal.

neuroscience↗

bioRxiv2024

An ancient lysozyme in placozoans

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and produced in gland cells of the ventral epithelium. PLys contains a protective and non-conserved cysteine-rich domain N-terminal of the conserved GH23 lysozyme domain. A truncation of this N-terminal domain in the maturation process of PLys leads to a drastic increase in enzymatic activity at the cost of stability. As the lysozyme is most active under acidic conditions, we investigated the pH trajectories during extracellular digestion in situ. Using a pH-senstive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied a structure-based phylogenetics approach to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla, including Porifera, Cnidaria, Placozoa and chordates, acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

evolutionary biology↗

bioRxiv2024

The major nucleoid-associated protein WHIRLY1 promotes chloroplast development in barley

WHIRLY1 is a DNA-binding protein of high abundance in chloroplast nucleoids, which have a complex proteome consisting of proteins involved in gene expression and unexpected proteins indicating links to energy production and biosynthetic activities of chloroplasts. In addition, WHIRLY1 has a second localization in the nucleus making it an excellent candidate for chloroplast-to-nucleus communication. To unravel the role of WHIRLY1 for structure and protein composition of nucleoids and its potential involvement in retrograde signaling during chloroplast development, knockout mutants of HvWHIRLY1 were prepared by site-directed mutagenesis using Cas9 endonuclease. In contrast to mutants of rice and maize, which die after the seedling stage, the barley why1 mutants survive and produce grains. Leaves of the mutants are initially pale and get green with time (xantha-to-green phenotype). However, the chlorophyll content of primary leaves stayed distinctly lower than that of the wild-type leaves, coinciding with a rather heterogeneous plastid population, whereby only 50% developed a rather normal thylakoid membrane system. For comparison, mature foliage leaves had almost normal levels of chlorophyll but a severely reduced photosynthetic capacity. A proteome analysis of chloroplasts isolated from mature foliage leaves revealed that in the absence of WHIRLY1, the abundances of a considerable fraction of proteins were downregulated. The fraction included multiple nucleoid-associated proteins including components of the transcriptional apparatus. Furthermore, ribosomal proteins, subunits of pyruvate dehydrogenase, CLP protease, ATP synthase, Rubisco and chaperons/chaperonins were found to be downregulated. In conclusion, the characterization of the barley why1 mutant plants revealed that WHIRLY1 is not essential for chloroplast development. Rather, it ensures a fast and failure-free progression of chloroplast development by remodeling nucleoids, which serve as assembly platforms for a concerted workflow of the numerous processes required for chloroplast development. Gene expression analyses revealed that the disturbance of chloroplast development is signaled to the nucleus, indicating that WHIRLY1 is not part of the biogenic retrograde signaling of plastids.

plant biology↗

bioRxiv2024

Hepatoblast iterative apicobasal polarization is regulated by extracellular matrix remodeling

Hepatocytes have a unique multiaxial polarity with several apical and basal surfaces. The prevailing model for the emergence of this multipolarity and the coordination of lumen formation between adjacent hepatocytes is based on asymmetric cell division. Here, investigating polarity generation in liver cell progenitors, the hepatoblasts, during liver development in vivo and in vitro, we found that this model cannot explain the observed dynamics of apical lumen formation in the embryonic liver. Instead, we identified a new mechanism of multi-axial polarization: We found that polarization can be initiated in a cell-autonomous manner by re-positioning apical recycling endosomes (AREs) to the cell cortex via fibronectin sensing through Integrin V. Using live cell imaging we showed that this process repeats, leading to multiaxial polarity independently of cell division. We found that establishment of oriented trafficking leads to secretion of the metalloprotease MMP13, allowing neighboring hepatoblasts to synchronize their polarization by sensing extracellular matrix (ECM) distribution and enabling lumen opening. Finally, active remodeling of ECM in proximity of nascent apical surfaces closes a positive feedback loop of polarization, whereas disruption of this loop by either blocking MMP13 or downregulating Integrin V prevents formation of the bile canaliculi network. Integration of this feedback loop into a simple mathematical model reproduces the observed dynamics of bile canaliculi network formation during liver development quantitatively. Our combined findings thus suggest a new mechanism of polarization coupling to self-organization at the tissue scale.

developmental biology↗

bioRxiv2023

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope

The mycomembrane (MM) is a hydrophobic layer formed by mycolic acids covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. The MM contributes to the impermeability of the cell envelope and thereby also protects bacteria from antibiotics. This makes biosynthesis of the MM an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) interferes with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. Similarly, the new drug candidate, benzothiazinone 043 (BTZ) affects the synthesis of the AG component of the cell wall. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of a sublethal concentration of BTZ. Our work shows that 1 {micro}g ml-1 BTZ efficiently blocks the apical growth machinery and reduces cell proliferation, however the integrity of the MM is largely preserved and the effects of {beta}-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. It functions as a greasy slide for the lateral flow of mycolic acids in the outer leaflet. This layer was not detectable in the septa of BTZ or EMB treated cells, which suggests that the greasy slide is impaired and the confluency of the MM is thereby reduced. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG) layer consistent with the report that EMB also inhibits the glutamate racemase MurI. We conclude that EMB and BTZ have distinct differences in their modes of action. While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

microbiology↗

bioRxiv2023

Four novel Caudoviricetes bacteriophages isolated from Baltic Sea water infect colonizers of Aurelia aurita

The moon jellyfish Aurelia aurita is associated with a highly diverse microbiota changing with provenance, tissue, and life stage. While the crucial relevance of bacteria to host fitness is well known, bacteriophages have often been neglected. Here, we aimed to isolate lytic phages targeting bacteria that are part of the A. aurita-associated microbiota. Four phages (Pseudomonas phage BSwM KMM1, Citrobacter phages BSwM KMM2- BSwM KMM4) were isolated from the Baltic Sea water column and characterized. Phages KMM2/3/4 infected representatives of Citrobacter, Shigella, and Escherichia (Enterobacteriaceae), whereas KMM1 showed a remarkably broad host range, infecting Gram- negative Pseudomonas as well as Gram-positive Staphylococcus. All phages showed short latent periods (around 30 min), large burst sizes (mean of 128 pfu/mL), and high efficiency of plating (EOP > 0.5), demonstrating decent virulence, efficiency, and infectivity. Transmission electron microscopy and viral genome analysis revealed that all phages are novel species and belong to the tailed, linear double- stranded DNA phage families Siphovirus (KMM3) and Myovirus (KMM1/2/4), with genome sizes between 50 - 138 kbp. Those isolates now allow manipulation of the A. aurita-associated microbiota and will provide new insights into phage impact on the multicellular host.

microbiology↗

bioRxiv2022

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G+C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA-DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM XXX = CCUG XXX) as type strain.

microbiology↗

bioRxiv2021

Bulkhead-like apical membrane structures between hepatocytes are required for anisotropic lumen expansion and liver tissue morphogenesis

Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1-3. The mechanisms governing anisotropic lumen expansion remain elusive4-6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.

cell biology↗

bioRxiv2019

Correlative SMLM and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which however cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional sub-domains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-colour SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.\n\nSynopsisSuborganelle compartmentalization cannot be resolved by diffraction limited light microscopy and not interpreted without knowledge of the underlying ultrastructure. This work shows a novel superCLEM workflow that combines multi-colour single-molecule localization-microscopy with electron tomography to map several functional domains on early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nano-domains largely devoid from cargo molecules Transferrin and EGF and opens new possibilities to perform structure-function analysis of organelles at the nanoscale.

cell biology↗

bioRxiv2019

3D spatially-resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression

Early disease diagnosis is key for the effective treatment of diseases. It relies on the identification of biomarkers and morphological inspection of organs and tissues. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D structural changes that are consequence of functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially-resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of new morphometric cellular parameters correlated with disease progression. Moreover, we found profound topological defects in the 3D bile canaliculi (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and zonated cholestasis, consistent with elevated cholestatic biomarkers in patients sera. Our spatially-resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multi-parametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.

pathology↗