Biology subjects

Sieber, S.

Publications and source records attributed to Sieber, S..

15 recordsLinked to original sources

bioRxiv2026

Natural product-derived azaphilones are selective scaffolds for covalent lysine liganding in live cells

Lysine-directed covalent liganding offers a promising strategy to expand the covalently ligandable proteome beyond cysteine. However, broadly applicable and selective lysine-reactive scaffolds remain limited. Here, we designed SCL-alkyne, a simplified azaphilone probe derived from the natural product sclerotiorin (SCL), for the global profiling of lysine reactivity in bacteria. SCL-alkyne displayed high intrinsic selectivity for lysine in vitro and in live Escherichia coli cells. In situ proteome profiling revealed that 84% of engaged sites belong to lysine residues and the protein N-terminus. Reactivity mapping provided an inventory of highly reactive sites showing that only a limited subset of reactive lysines is located in functional regions of proteins. Analysis of proteins enriched via the SCL-alkyne probe unraveled bacterial chemotaxis as a major target pathway which was confirmed by an impaired chemotactic response upon treatment of bacteria with SCL-alkyne. Competitive profiling of three structurally related azaphilone natural products identified 60 protein targets belonging to pathways related to iron-sulfur cluster assembly and antioxidant function. This study not only highlights the diverse modes-of-action of natural product azaphilones, but also establishes its core scaffold as effective lysine-reactive chemotype for identifying ligandable lysines.

biochemistry↗

bioRxiv2026

The glucocorticoid dexamethasone influences motility of the sulfate-reducing bacterium Desulfovibrio desulfuricans by targeting the filament cap protein FliD

Glucocorticoids such as dexamethasone (DXE) are first-line treatments for inflammatory bowel disease (IBD). Beyond their effects on the host immune system, accumulating evidence suggests that glucocorticoids can also influence the gut microbiota. Notably, IBD patients exhibit an increased intestinal colonization by sulfate-reducing Desulfovibrio spp. Here, we show that DXE modulates bacterial motility in the gut commensal Desulfovibrio desulfuricans through a metabolism-independent mechanism. To identify bacterial targets, we developed a DXE-derived chemical probe and performed affinity-based protein profiling, which revealed the flagellar cap protein FliD (Ddes_0530) as a principal binding partner. Structural modeling using AlphaFold3 and Boltz2 predicted DXE binding within a conserved groove of the FliD C-terminal domain. Furthermore, the tip of the flagellum of Desulfovibrio, but not that of Escherichia coli, could be fluorescently labeled with TAMRA-DXE, but not with the structurally related steroid probe TAMRA-norethiosterone, indicating that flagellar labeling is specific to DXE rather than the steroid scaffold itself. As a consequence of this interaction, transmission electron microscopy showed that DXE treatment prevented flagellation in a subpopulation and reduced flagellar length in D. desulfuricans strains ATCC 27774 and CCUG 72978, respectively. Quantitative motility tracking revealed a non-monotonic, dose-dependent modulation of swimming velocity, with peak stimulation at 10 {micro}M DXE, accompanied by straighter trajectories and enhanced net displacement. Together, these findings uncover a previously unrecognized mode of action for DXE which directly perturbs flagellar biogenesis and motility of an important gut microbiome member of IBD patients. SignificanceGlucocorticoids are widely prescribed for inflammatory conditions, yet their direct effects on gut bacteria remain largely unexplored. We demonstrate that dexamethasone, a synthetic glucocorticoid, binds to the flagellar cap protein FliD of the gut commensal Desulfovibrio desulfuricans, affecting flagellar assembly and altering motility behavior. Unlike previously characterized steroid-metabolizing bacteria such as Clostridium steroidoreducens, Desulfovibrio does not degrade dexamethasone, indicating that the observed effects result from direct drug-protein interaction. These findings establish a new paradigm for metabolism-independent drug-microbiome interactions and suggest that glucocorticoid effects on gut bacteria extend beyond enzymatic degradation pathways.

microbiology↗

bioRxiv2026

Active Site-Directed Probes for targeting Bacterial Phosphoarginine Phosphatases

Canonical protein phosphorylation patterns are a thoroughly studied post-translational modification (PTM) driving distinct regulatory mechanisms in both prokaryotes, and eukaryotes. In contrast, the identification and investigation of essential components that regulate non-canonical phosphorylation has received considerably less attention, although these PTMs are associated with important functions. One notable example is arginine phosphorylation which modulates processes such as protein degradation, transcriptional regulation and spore germination in bacteria. Herein we introduce the first in class covalent activity-based probes to study phosphoarginine-phosphatases. We identify unsaturated phosphonamidic acids as bespoke electrophilic phosphoarginine (pArg) mimics, which allowed to uncover a series of unprecedented pArg-phosphatases, which in part had been previously annotated as low molecular weight tyrosine-phosphatases across phylogenetically distinct microbial species. This work, which serves as the first example of proteome-wide activity-based profiling of pArg phosphatases will help inform the development of new therapeutic modalities and expand our understanding of bacterial signal transduction.

microbiology↗

bioRxiv2025

Structural Elucidation and Revised Biosynthetic Pathway of the Membrane Vesicle-Associated Antifungal Compound AFC

Bacterial membrane vesicles (MVs) serve as delivery vehicles for hydrophobic and membrane-associated secondary metabolites, enhancing their solubility, stability, and bioactivity. Here, we show that the antifungal compound AFC-BC11 (AFC), produced by Burkholderia cenocepacia K56-2, is selectively packaged into and released via MVs. Using HR-MS/MS, NMR, and stable isotope feeding experiments, we determined the chemical structure of AFC and analyzed its biosynthesis. Our results confirm that the structure largely matches the recent report by Zhong et al., with a key difference: the double bond in the fatty acid moiety is positioned between C11 and C12. We provide compelling evidence that this constitution reflects the direct incorporation of cis-vaccenic acid, the most abundant fatty acid in B. cenocepacia, rather than a tailoring modification. Comparative analysis of afcU, afcF, and afcS mutants suggests a biosynthetic pathway involving {omega}-modification of cis-vaccenic acid, revising previous proposals of citric acid conjugation to myristic acid and opening avenues for acyl chain engineering. Together, these findings establish AFC as an MV-associated antifungal metabolite, provide a refined structural and biosynthetic model, and highlight the role of MVs in the dispersal of hydrophobic bioactive compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/685075v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f4d736org.highwire.dtl.DTLVardef@15d2683org.highwire.dtl.DTLVardef@8b891corg.highwire.dtl.DTLVardef@d6b693_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Signal Antagonists Suppress Pseudomonas syringae Pathogenicity

The bacterial plant pathogen Pseudomonas syringae causes significant damage to economically important crops worldwide. These bacteria coordinate their behavior and virulence through specific signaling compounds, such as the diazeniumdiolate leudiazen. Conventional antibacterial treatments enable the development of resistant strains. A more attractive treatment strategy would involve antagonists that suppress the expression of virulence factors without killing the pathogen, potentially reducing the risk of resistance development. Herein, we present the design and synthesis of analogs of leudiazen, which positively regulates the production of mangotoxin in P. syringae pv. syringae (Pss). Several compounds display inhibitory activity towards mangotoxin production, and a lead compound abolishes necrosis in infected tomato leaves, without significantly affecting bacterial growth. Thus, this study represents a promising advance towards developing effective and sustainable methods for bacterial disease control.

microbiology↗

bioRxiv2024

Functional Biosynthetic Stereodivergence in a Gene Cluster via a Dihydrosydnone N-oxide

Chirality features a critical role in the biochemistry of life and often only one enantiomeric series is observed (homochirality). Only few natural products have been obtained as racemates, e.g. the quorum-sensing signal valdiazen produced by Burkholderia cenocepacia H111. In this study, we investigated its biosynthetic gene cluster and discovered that both the enantiomerically pure (R)-fragin and the racemic valdiazen are obtained from the same pathway. This stereodivergence is based on the unusual heterocycle dihydrosydnone N-oxide intermediate, as evident from gene knockout, stable isotope feeding experiments, and mass spectrometry experiments. Both non-enzymatic racemisation via keto-enol tautomerisation and enzyme-mediated dynamic kinetic resolution were found to be crucial to this stereodivergent pathway. This novel mechanism underpins the production of configurationally and biologically distinct metabolites from a single gene cluster. Our findings highlight the intricate design of an intertwined biosynthesis pathway, providing a deeper understanding of microbial secondary metabolism related to microbial communication. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/591611v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@11b63corg.highwire.dtl.DTLVardef@d339e6org.highwire.dtl.DTLVardef@17e34b1org.highwire.dtl.DTLVardef@b58692_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

A ubiquitous Microcoleus species causes benthic cyanotoxic blooms worldwide

Recently, proliferations of benthic cyanobacteria producing derivatives of anatoxin-a have been reported in rivers all over the world. In three river systems, in New Zealand, the USA, and Canada, a cohesive cluster of Microcoleus strains was responsible for toxin production. Here, we document a similar toxigenic event that occurred at the mouth of the river Areuse in lake Neuchatel (Switzerland) and caused the death of several dogs. Using 16S RNA-based community analysis, we show that riverine benthic communities are dominated by Oscillatoriales and especially by Microcoleus strains. We correlate the detection of one sequence variant with the presence of anatoxin-a derivatives and use metagenomics to assemble a complete circular genome of the strain. The strain is distinct from the ones isolated in New Zealand, the USA, and Canada, but belongs to the same species; it shares significant traits with them, in particular a relatively small genome and incomplete vitamin biosynthetic pathways. Overall, our results suggest that the major anatoxin-a-associated benthic proliferations worldwide can be traced back to a single ubiquitous species, Microcoleus anatoxicus, rather than to a diversity of cyanobacterial lineages. We recommend that this species be monitored internationally in order to help predict and mitigate similar cyanotoxic events.

microbiology↗

bioRxiv2023

Global meta-analysis reveals overall benefits of silvopastoral systems for biodiversity

Domestic livestock grazing accounts for roughly one quarter of the worlds terrestrial surface and is a leading driver of biodiversity loss. Yet, it also provides a critical livelihood for nearly one billion smallholder farmers, creating a paradox that highlights the need for conservation strategies to balance human and ecological needs. Silvopastoral systems (SPS) integrate trees with livestock pastures, offering a promising solution to boost livestock productivity while safeguarding natural areas and biodiversity. However, evidence for the biodiversity benefits provided by SPS is limited to studies focusing on specific geographic regions or taxa. Through a global meta-analysis of 45 studies spanning 15 countries, four biogeographic regions, and seven taxa, we provide the first quantitative synthesis evaluating how SPS affect biodiversity and community stability relative to treeless pastures and natural forests. Overall, we show that SPS harbor higher levels of biodiversity (i.e., richness, abundance, and diversity) and stability than treeless pastures, and perform comparably to nearby forests. However, variations exist across regions and taxa, with the strongest positive responses in tropical dry regions and for low-mobility taxa like invertebrates and plants. Mammals, birds, and soil microorganisms, on the other hand, showed no significant biodiversity differences between treeless pastures and SPS. Thus, integrating SPS and protected areas as complementary components of a multifunctional landscape will be key to halting multi-taxa biodiversity loss and building sustainable livestock systems. Our findings support the conservation potential of SPS, while underscoring the need for strategic implementation to maximize benefits for biodiversity conservation.

ecology↗

bioRxiv2022

Cyclitol secondary metabolism is a central feature of Burkholderia leaf symbionts

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled seventeen new endophyte genomes representing endophytes from thirteen plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

microbiology↗

bioRxiv2022

Intra-membrane client recognition potentiates the chaperone functions of Calnexin

One third of the human proteome are membrane proteins. They are particularly vulnerable to misfolding, often requiring assistance by molecular chaperones. Calnexin (CNX), one of the most abundant ER chaperones, plays an important role in membrane protein biogenesis and engages clients via its sugar-binding lectin domain. Using mass spectrometric analyses, we show that Calnexin (CNX) interacts with a large number of non-glycosylated membrane proteins, suggesting additional binding modes. We find that misfolded membrane proteins are preferentially bound by CNX and that CNX uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by CNX and identify sequence motifs within the CNX TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of CNX. Together, this study reveals a widespread role of CNX client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

cell biology↗

bioRxiv2021

Development of a Cell Surface Display System in Chlamydomonas reinhardtii

Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Towards this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

synthetic biology↗

bioRxiv2020

A Volatile Signal Controls Virulence in the Plant Pathogen Pseudomonas syringae pv. syringae and a Strategy for Infection Control in Organic Farming

Pseudomonas syringae is an important pathogen of many agriculturally valuable crops. Among the various pathovars described P. syringae pv. syringae (Pss) has a particularly wide host range, infecting primarily woody and herbaceous host plants. The ability of Pss to cause bacterial apical necrosis of mango trees is dependent on the production of the antimetabolite toxin mangotoxin. The production of this toxin was shown to be regulated by a self-produced signaling molecule. In this study, we determined the structure of the Pss signal molecule belonging to the recently described family of diazeniumdiolate communication molecules. Employing a targeted mass spectrometry-based approach, we provide experimental evidence that the major signal produced by Pss is the volatile compound leudiazen, which controls mangotoxin production and virulence in a detached tomato leaflet infection model. Experimental results demonstrate that KMnO4 solution inactivates leudiazen and that treatment of infected leaves with KMnO4 abolishes necrosis. This strategy represents the first example of chemically degrading a signaling molecule to interfere with bacterial communication. The application of KMnO4 solution, which is regulatorily approved in organic farming, may constitute an environmentally friendly strategy to control Pss infections.

microbiology↗

bioRxiv2019

Biodegradable harmonophores for targeted high-resolution in vivo tumor imaging

Optical imaging probes have played a major role in detecting and monitoring of a variety of diseases1. In particular, nonlinear optical imaging probes, such as second harmonic generating (SHG) nanoprobes, hold great promise as clinical contrast agents, as they can be imaged with little background signal and unmatched long-term photostability2. As their chemical composition often includes transition metals, the use of inorganic SHG nanoprobes can raise long-term health concerns. Ideally, contrast agents for biomedical applications should be degraded in vivo without any long-term toxicological consequences to the organism. Here, we developed biodegradable harmonophores (bioharmonophores) that consist of polymer-encapsulated, self-assembling peptides that generate a strong SHG signal. When functionalized with tumor cell surface markers, these reporters can target single cancer cells with high detection sensitivity in zebrafish embryos in vivo. Thus, bioharmonophores will enable an innovative approach to cancer treatment using targeted high-resolution optical imaging for diagnostics and therapy.

bioengineering↗

bioRxiv2019

Cryo-EM structure of the ClpXP protein degradation machinery

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXPs complex mode of action and provides a structural framework for future pharmacological applications.

molecular biology↗

bioRxiv2018

Bone marrow-on-a-chip: Emulating the human bone marrow

Multipotent hematopoietic stem and progenitor cells HSPC reside in specialized stem cell niches within the bone marrow, that provide a suitable microenvironment for lifelong maintenance of the stem cells. Meaningful in vitro models recapitulating the in vivo stem cell niche biology can be employed for both basic research as well as for applied sciences and represent a powerful tool to reduce animal tests in preclinical studies. Recently we published the generation of an in vitro bone marrow niche model, capable of long-term cultivation of HSC based on an organ-on-a-chip platform. This study provides a detailed analysis of the 3D culture system including matrix environment analysis by SEM, transcriptome analysis and system intrinsic differentiation induction. Furthermore, the bone marrow on a chip model can serve to multiply and harvest HSPC, since repeated cell removal not compromised the functionality of the culture system. The prolongation of the culture time to 8 weeks demonstrate the capacity to apply the model in repeated drug testing experiments. The quality of the presented system is emphasized by the differentiation capacity of long-term cultivated HSPC in vitro and in vivo. Transplanted human HSPC migrated actively into the bone marrow of irradiated mice and contributed to the long-term reconstitution of the hematopoietic system after four and eight weeks of in vitro cultivation.\n\nThe introduced system offers a multitude of possible applications to address a broad spectrum of questions regarding HSPC, the corresponding bone marrow niche biology, and pathological aberrations.

bioengineering↗