Biology subjects

Taschner, M.

Publications and source records attributed to Taschner, M..

13 recordsLinked to original sources

bioRxiv2026

Genome replication and cell division control by the methylation-sensitive GcrA regulator in Agrobacterium tumefaciens

Bacteria with complex genomes including secondary chromosomes and/or megaplasmids face unique challenges when controlling their cell cycle: they not only need to coordinate genome replication with other cell cycle events such as cell growth and cell division, but also to synchronize the replication of different replicons. In Alphaproteobacteria, replication and maintenance of large extrachromosomal replicons are usually dependent on RepABC modules, as seen for the plant pathogen Agrobacterium tumefaciens. In this study, we demonstrate that the conserved GcrA protein is an essential methylation-dependent transcriptional regulator playing at least two critical roles during the A. tumefaciens cell cycle. First, GcrA is required for the on-time and synchronized firing of the three RepABC-dependent origins of replication of A. tumefaciens that are needed for chromosomal and megaplasmid replication. Second, GcrA triggers the expression of several essential cell division genes for the timely assembly of a functional divisome. These findings highlight how this conserved global regulator can control and then synchronize a variety of essential cell cycle events in Alphaproteobacteria with complex genomes, notably coordinating the maintenance of several RepABC-dependent replicons with cell division. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/728657v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@c7f0f7org.highwire.dtl.DTLVardef@9cf8f1org.highwire.dtl.DTLVardef@19baa05org.highwire.dtl.DTLVardef@b73437_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Phage Endolysin Enables Targeted Manipulation of the Small Intestinal Microbiota and Uncovers Niche Overlap Between Oral and Butyrate-Producing Taxa

Oral bacterial overgrowth in the small intestine has been associated with dysbiosis, impaired nutrient absorption, and stunted growth in undernourished children, a condition referred to as small intestinal oral bacterial overgrowth (SIOBO). Here, we explore the use of a phage-derived lysin as a precision antimicrobial to selectively target Streptococcus salivarius within complex microbial communities. Through a newly developed, medium-throughput bioinformatic and wet-lab pipeline we identified, cloned and produced a prophage-encoded lysin from S. salivarius and demonstrated its potent and specific lytic activity against a panel of 49 clinical S. salivarius strains from stunted children, while sparing related species such as S. mitis, S. parasanguinis and S. thermophilus. Application of the lysin to human stool-derived in vitro communities and to mice colonized with S. salivarius led to an approximate 2-3 log reduction in S. salivarius abundance while preserving overall bacterial community composition. However, we observed a negative correlation between S. salivarius and Coprococcus comes in vitro, and Eubacterium xylanophilum, Akkermansia, Lactobacillus and Ruminococcus in vivo. Spent medium assays confirmed niche overlap between 10 clinical strains of S. salivarius and 28 different taxa involved in butyrate production. Together, these results suggest that phage-derived lysins can offer multiple benefits by selectively removing ectopically colonized oral taxa and indirectly promoting beneficial anaerobes.

microbiology↗

bioRxiv2025

Active site assembly by SMG5 as a mechanism for SMG6 endonuclease licensing in nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that eliminates transcripts containing premature termination codons (PTCs). While the recognition phase of NMD is well characterised, the effector step - how targeted mRNAs are nucleolytically degraded - remains incompletely understood. In metazoans, NMD employs an endonucleolytic route mediated by SMG6, a PIN-domain nuclease, alongside SMG5 and SMG7, which act downstream of PTC recognition. SMG5 has recently been proposed to license SMG6 activity, yet the molecular basis of this licensing has remained elusive. Here, we combine AlphaFold structural predictions with biochemical assays to investigate interactions among human SMG5, SMG6, and SMG7. Structural models predict a high-confidence interface between SMG5 and SMG6 PIN domains that forms a composite active site: a conserved SMG5 aspartate (D893) complements the SMG6 acidic triad to reinstate the canonical tetrad required for PIN-domain catalysis. In vitro, SMG6 alone exhibits weak endonucleolytic activity, which is enhanced [~]10-fold by SMG5 PIN. Mutational analyses confirm that conserved residues from both proteins are essential for this composite configuration. Our findings reveal that the SMG5 PIN domain, previously considered catalytically inert, plays a critical role in activating SMG6 by completing its active site. This work provides mechanistic insight into the SMG5-dependent licensing step and uncovers a composite PIN nuclease architecture at the heart of the metazoan NMD effector phase.

molecular biology↗

bioRxiv2025

Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex

Synthetic nanobodies--also called sybodies--have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane protein complexes. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization by DNA loop extrusion, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the DNA folding process. These findings underscore the potential of sybodies--and, by extension, designed binders--as versatile tools for probing dynamic protein function in living cells, with potentially broad applications in cell and synthetic biology.

molecular biology↗

bioRxiv2025

The SMC Hinge is a Selective Gate for Obstacle Bypass

DNA loop-extruding SMC complexes play vital roles in genome maintenance and DNA immunity. However, the ability of these ring-shaped DNA motors to navigate large DNA-bound obstacles, such as DNA/RNA polymerases and ribosomes in prokaryotes, has remained unclear. Here, we demonstrate that a bacterial SMC Wadjet complex can efficiently bypass obstacles larger than the SMC coiled coil lumen if they are linked to the extruded DNA by single-stranded DNA (ssDNA). This process is mediated by the selective entrapment of the ssDNA linker within the SMC hinge channel, which acts as an obligate gate--permitting ssDNA passage while excluding double-stranded DNA (dsDNA). By threading the linker through the hinge gate as dsDNA is translocated through the SMC motor, the obstacle can follow a trajectory distinct from its associated dsDNA, remaining outside the SMC ring. This mechanism enables continuous dsDNA entrapment while successfully bypassing obstacles. We show that hinges of diverse SMC complexes including eukaryotic condensin and Smc5/6 also selectively accommodate ssDNA, suggesting that hinge bypass is a conserved feature across SMC proteins. We demonstrate that while the hinge domain is essential for obstacle bypass, it is dispensable for loop extrusion, showing that these functions are independent, provided by separate SMC modules. These findings uncover the long-sought function of the SMC hinge toroid and provide the first mechanistic explanation for how DNA-entrapping SMC complexes can extrude loops on chromosomal DNA densely populated with obstacles. Integrating hinge bypass into the DNA loop extrusion model, we propose that the hinge may also facilitate the passage of RNA and peptide, enabling bypass of transcription-associated ribonucleoprotein complexes and other SMC complexes.

molecular biology↗

bioRxiv2024

4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts

Multi-subunit protein complexes are at the heart of many cellular processes, and studying their biochemical activities and structures in vitro requires their reconstitution by recombinant expression and purification. Obtaining targets at sufficient purity and scale typically requires the screening of several protein variants and expression hosts. Existing cloning strategies allow to produce constructs for co-expression of proteins, but are often time-consuming, labour-intensive, host-specific, or involving error-prone assembly steps. Here we present a unique set of vectors together with a novel assembly strategy designed to overcome these limitations. It allows for the generation of expression constructs for multi-subunit protein complexes for various hosts in a single cloning step. Its modular nature allows the system to be easily extended to target additional expression hosts or to include new tags or regulatory sequences. As a proof of principle, we present the parallel construction of expression vectors for several Structural Maintenance of Chromosomes (SMC) complexes, allowing us to devise strategies for the recombinant production of these targets in bacteria, insect cells, and human cells, respectively. This work will help laboratories working on protein complexes to streamline their workflow, increase their productivity and improve the quality of the purified material.

biochemistry↗

bioRxiv2024

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step

Eukaryotes carry three types of Structural Maintenance of Chromosomes (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion. SMCs modulate DNA supercoiling, but it has remained incompletely understood how this is achieved. Here we present a single-molecule magnetic tweezers assay that directly measures how much twist is induced by an individual SMC in each loop-extrusion step. We demonstrate that all three SMC complexes induce the same large negative twist (i.e., a linking number change {Delta}Lk of -0.6 at each loop-extrusion step) into the extruded loop, independent of step size. Using ATP-hydrolysis mutants and non-hydrolysable ATP analogues, we find that ATP binding is the twist-inducing event during the ATPase cycle, which coincides with the force-generating loop-extrusion step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-loop-extrusion mechanism among these SMC complexes.

biophysics↗

bioRxiv2023

SMC motor proteins extrude DNA asymmetrically and contain a direction switch

Structural Maintenance of Chromosomes (SMC) complexes organize the genome via DNA loop extrusion. While some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, while the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. STAG1 stabilizes NIPBL on cohesin, preventing NIPBL turnover and direction switches. The findings reveal that SMCs, surprisingly, contain a direction switch subunit. HighlightsO_LIAll eukaryotic SMC complexes extrude DNA asymmetrically. C_LIO_LIApparent symmetric loop extrusion is the result of frequent direction switches. C_LIO_LIn human cohesin, loop-extrusion direction changes require exchange of NIPBL. C_LIO_LISTAG1 stabilizes NIPBL on human cohesin. C_LI

molecular biology↗

bioRxiv2023

Structural basis for plasmid restriction by SMC JET nuclease

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of circular plasmids through DNA cleavage; yet the mechanisms for target recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage implying that residual unextruded DNA is cleaved. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as principle for non-self DNA recognition and molecular signature for plasmid cleavage. We elucidate key elements of SMC loop extrusion including motor directionality and the structure of a DNA-holding state.

molecular biology↗

bioRxiv2023

A conserved antigen induces respiratory Th17-mediated serotype-independent protection against pneumococcal superinfection

Several vaccines targeting bacterial pathogens show reduced efficacy in the context of intercurrent viral infection indicating a new vaccinology approach is required to protect against such superinfections. To find antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the highly conserved lafB gene as virulence factor. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid we show is important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against all serotypes in a murine model. In contrast to standard pneumococcal capsule-based conjugate vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, suggesting its merit as a universal pneumococcal vaccine antigen that remains effective following influenza infection. One-Sentence SummaryDiscovery of a universal pneumococcal vaccine protective during superinfection.

microbiology↗

bioRxiv2022

DNA segment capture by Smc5/6 holo-complexes

Three distinct SMC complexes facilitate chromosome folding and segregation in eukaryotes, presumably by DNA translocation and loop extrusion. How SMCs interact with DNA is however not well understood. Among the SMC complexes, Smc5/6 has dedicated roles in DNA repair and in preventing a lethal buildup of aberrant DNA junctions. Here, we describe the reconstitution of ATP-dependent topological DNA loading by Smc5/6 rings. By inserting cysteine residues at selected protein interfaces, we obtained covalently closed compartments upon chemical cross-linking. We show that two SMC subcompartments and the kleisin compartment topologically entrap a plasmid molecule, but not the full SMC compartment. This is explained by a looped DNA segment inserting into the SMC compartment with the kleisin neck gate locking the loop in place when passing between the two DNA flanks and closing. This DNA segment capture strictly requires the Nse5/6 loader, which opens the neck gate prior to DNA passage. Similar segment capture events without gate opening may provide the power stroke for DNA translocation/loop extrusion in subsequent ATP hydrolysis cycles. Our biochemical experiments thus offer a unifying principle for SMC ATPase function in loading and translocation/extrusion, which is likely relevant to other members of the family of SMC proteins too.

biochemistry↗

bioRxiv2022

Biochemically validated structural model of the 15-subunit IFT-B complex

Cilia are ubiquitous eukaryotic organelles important to cellular motility, signalling and sensory reception. Cilium formation requires intraflagellar transport for trafficking of structural and signalling components. The large MDa IFT-B complex constitutes the backbone of polymeric IFT trains that carry ciliary cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B sub-complexes leaving >50% of the IFT-B complex structurally uncharacterized. We have used recent advances in protein structure prediction as implemented in Alphafold to assemble a structural model for the 15-subunit IFT-B complex. The model was validated using crosslinking/MS data on reconstituted IFT-B complexes, X-ray scattering in solution and diffraction from crystals as well as site-directed mutagenesis and protein binding assays. The IFT-B structural model reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The >400[A] long IFT-B complex can roughly be divided into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our structural modelling and crosslinking/MS results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of two different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

biochemistry↗

bioRxiv2021

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

biochemistry↗