Biology subjects

Hör, J.

Publications and source records attributed to Hör, J..

6 recordsLinked to original sources

bioRxiv2026

ApeA cleaves genomic RNA to defend against RNA phage infection

To protect themselves against bacteriophage infection, bacteria encode a vast diversity of antiphage defense systems. However, the mechanisms of action of most of these systems have exclusively been studied using phages with DNA genomes as the models, while phages with RNA genomes remain understudied. Here, we investigate how the defense system ApeA confers resistance against RNA phage infection. We show that two ApeA homologs, Ec1ApeA and Ps2ApeA, protect against a variety of single-stranded RNA phages. Focusing on Ec1ApeA, we find that it senses infection through a conserved pocket that likely binds an RNA structure in the phage genome. This activates the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) RNase domain of Ec1ApeA which consequently cleaves the phage genomic RNA to restrict replication. In contrast to many other described defense systems, Ec1ApeA activity directly stops viral replication without inducing cell death, establishing ApeA as a non-abortive defense system that protects against RNA phages. Our results add to the increasingly diverse targets of antiviral HEPN RNases and provide insights into the understudied field of RNA phage defense.

microbiology↗

bioRxiv2023

Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly

Multiple immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defense. Here we studied an anti-phage defense system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fiber, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defense system release a mixture of partially assembled, tailless phage particles, and fully assembled phages in which the central tail fiber is obstructed by the covalently attached ubiquitin-like protein. These phages exhibit severely impaired infectivity, explaining how the defense system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

microbiology↗

bioRxiv2022

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to [~]500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

molecular biology↗

bioRxiv2022

An expanding arsenal of immune systems that protect bacteria from phages

Bacterial anti-phage defense systems are frequently clustered in microbial genomes, forming defense islands. This genomic property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms in bacteria is still unknown. In this study we report the discovery of 21 new defense systems that protect bacteria from phages, based on computational genomic analyses and phage infection experiments. We find multiple systems with protein domains known to be involved in eukaryotic anti-viral immunity, including ISG15-like proteins, dynamin-like proteins, and SEFIR domains, and show that these domains participate in bacterial defense against phages. Additional systems include protein domains predicted to manipulate DNA and RNA molecules, as well as multiple toxin-antitoxin systems shown here to function in anti-phage defense. The systems we discovered are widely distributed in bacterial and archaeal genomes, and in some bacteria form a considerable fraction of the immune arsenal. Our data substantially expand the known inventory of defense systems utilized by bacteria to counteract phage infection.

microbiology↗

bioRxiv2022

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with detection of low abundance transcripts and analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

microbiology↗

bioRxiv2020

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

microbiology↗