Biology subjects

Krautwurst, S.

Publications and source records attributed to Krautwurst, S..

15 recordsLinked to original sources

bioRxiv2026

The ArcZ small RNA activates SlyA production to control contact-dependent antibacterial killing and diffusible antifungal activity in Dickeya solani

Bacterial phytopathogens deploy diverse antimicrobial systems, including diffusible secondary metabolites and contact-dependent secretion apparatuses, to compete with coinhabiting microorganisms during host colonization. How the production of these distinct weapons is coordinated remains incompletely understood. Here, we identified a two-tier regulatory cascade by which the Hfq-dependent small RNA (sRNA) ArcZ controls both antibacterial and antifungal antagonism in the phytopathogen Dickeya solani. High-throughput RNA interactome analysis (RIL-seq) shows that ArcZ interacts broadly across the transcriptome and directly base-pairs with the 5' untranslated region of the mRNA encoding the MarR-family transcription factor SlyA, activating its translation. SlyA subsequently binds directly to selected promoter regions within the solanimycin biosynthetic cluster (sol) and the type VI secretion system (T6SS) loci and activates their transcription. The ArcZ-SlyA axis is required for solanimycin mediated antifungal activity against Kluyveromyces lactis and for T6SS-dependent killing of the sympatric pathogen Pectobacterium atrosepticum, demonstrating that the D. solani T6SS mediates interbacterial competition. A compensatory base-pairing mutation between ArcZ and slyA restores both competitive phenotypes, establishing this post-transcriptional interaction as a direct upstream determinant. Together, these results reveal an sRNA controlled regulatory axis that coordinates diffusible antifungal and contact-dependent antibacterial activities.

microbiology↗

bioRxiv2026

A nucleic acid host factor enables optimal phage replication in Escherichia coli

Host acquisition by bacteriophages (phages) often entails modulation, appropriation, or inhibition of components and processes central to bacterial gene expression. Small non-coding RNAs (sRNAs) are major regulators of RNA fate and frequently rely on the conserved RNA chaperone Hfq to engage their cognate targets. Although phages are known to encode specialised proteins and sRNAs to manipulate host gene expression, it has remained unclear whether they also co-opt host-encoded sRNAs for their own regulatory needs. We show that transcriptome-wide Hfq-mediated RNA-RNA interactions are broadly destabilised during T2 phage infection of Escherichia coli. We further demonstrate that the conserved bacterial sRNA ArcZ is co-opted by T2 to promote expression of a conserved phage operon that includes a protein which inhibits a bacterial restriction-modification system. ArcZ achieves this by preventing RNase E-mediated degradation of the transcript originating from the phage operon. Our study provides the first evidence of an evolutionary strategy in which a phage leverages a nucleic acid host factor to fulfil its own gene expression requirements.

microbiology↗

bioRxiv2026

The transcription factor Vca0578 (DsvR) mediated expression of ZapC is required to promote cell division during lytic transglycosylase insufficiency in Vibrio cholerae

The bacterial peptidoglycan (PG) cell wall, a polymer made of amino-acid-bearing glycan strands, maintains cell shape, provides structural integrity, and protects against osmotic lysis. PG maintenance is an active process that requires regulated PG breakdown to make space for insertion of new PG strands. PG breakdown is accomplished by autolysins, i.e. endogenous enzymes with cell wall cleavage activity. The lytic transglycosylases (LTGs), a class of autolysins, for example, cleave glycan strands during PG remodelling. LTGs are broadly conserved and are highly redundant in bacteria, but their physiological role is poorly-defined. In this study, we interrogated physiological consequences of LTG insufficiency in Vibrio cholerae using TnSeq to gain insights about roles of these enzymes. We identify an uncharacterized transcription factor, Vca0578, which alleviates defects associated with the {Delta}6LTG mutant. We demonstrate that Vca0578 positively regulates the expression of zapC, a typically non-essential Z-ring associated protein. In the absence of zapC, cell division was impaired during perturbations of cell envelope homeostasis caused by absence of LTGs, or by exposure to antibiotics inhibiting cell elongation; either condition rendered zapC conditionally essential. This essentiality could be overcome by increasing FtsZ levels. Lastly, we found that ZapC also contributes to both width and length homeostasis during normal growth. This work thus uncovers a novel transcriptional circuit that contributes to effective cell division in{Delta} 6LTG cells, and suggests an essential role for ZapC in cell division under stress conditions that cause perturbation of cell width homeostasis. AUTHOR SUMMARYBacteria must maintain their outer shell (the cell envelope) in the face of changes in the environment. For this, they use elaborate systems that remodel the cell envelope. How some of these systems work is not well understood. In this study, we describe a new gene circuit that is required to keep cells dividing when the cell envelope is compromised. We found that Vca0578, a putative transcription factor, controls expression of the zapC gene. The protein ZapC then helps bacteria grow and divide when the cell envelope is under stress, for example, in the presence of certain antibiotics. Thus, we have discovered a regulatory circuit that promotes bacterial growth and antibiotic resistance under stress.

microbiology↗

bioRxiv2025

A 3UTR-derived small RNA modulates the life-cycle of the cholera toxin-encoding filamentous phage, CTXphi

Bacteriophages (phages) are well known to be one of the major driving forces in bacterial evolution. This also applies to virulent microorganisms, such as the major human pathogen Vibrio cholerae, whose pathogenic potential and epidemic proliferation largely depends on the interaction with environmental phages. Specifically, integration of the CTX{phi} phage genome into the first chromosome of V. cholerae also introduced the ctxAB genes, encoding the primary toxin responsible for the severe acute diarrheal disease, cholera. Whereas the mechanisms underlying CTX{phi}-associated horizontal gene transfer and transcriptional control of the ctxAB genes have been intensively studied over the past years, post-transcriptional regulation affecting the CTX{phi} life-cycle has not been documented. Here, we report the discovery and characterization of the CisR small RNA (sRNA) that is produced from the 3UTR (untranslated region) of the prtV gene and inhibits the expression of the CTX{phi}-encoded cep mRNA. CisR-mediated repression of cep involves Hfq-assisted base-pairing of the two transcripts and results in reduced CTX{phi} production under stress conditions. We further demonstrate that transcription of prtV-cisR requires both the master quorum-sensing regulator HapR and CRP, a global regulator of carbon metabolism. Taken together, our work provides evidence that V. cholerae employs sRNA-mediated post-transcriptional gene regulation to coordinate CTX{phi} activation with both cell density and nutrient availability. SIGNIFICANCE STATEMENTThe integration of the CTX{phi} phage genome, which carries the ctxAB toxin genes, is essential for cholera pathogenesis in humans. While transcriptional control of CTX{phi} and ctxAB has been well-studied, post-transcriptional mechanisms remain unexplored. Here, we identify and characterize CisR, a small RNA derived from the 3' untranslated region of prtV, which inhibits the CTX{phi}-encoded cep mRNA through Hfq-dependent base-pairing. CisR-mediated regulation limits phage production under stress conditions and is co-regulated by the quorum-sensing factor HapR and the metabolic regulator CRP. Our findings reveal a new RNA-based mechanism linking CTX{phi} phage activation to cell density and nutrient status of V. cholerae.

microbiology↗

bioRxiv2024

Phage-phage competition and biofilms reduce the efficacy of a combination of two virulent bacteriophages against Pseudomonas aeruginosa

Combined use of virulent bacteriophages (phages) and antibiotics reduces the severity of difficult-to-treat Pseudomonas aeruginosa infections in many patients. In vitro methods that attempt to reproduce more than one physiological state of the pathogens can provide a valuable assessment of antibacterials like phages. Here, by measuring bacterial killing kinetics and individual replication in different growth conditions, including biofilms and a human lung epithelial cell line, we elucidated factors influencing the efficacy of two virulent phages against P. aeruginosa PAO1. A single administration of phages effectively reduced the P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, however, the emergence of phage-resistant variants occurred subsequently. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem. In contrast, surface-attached biofilms exhibited tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the antibacterial effect. Moreover, the phage with the shorter lysis time lysed P. aeruginosa more rapidly and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage of the combination. The sequential addition of phages resulted in their unimpeded replication with no increase in bacteriolytic activity. These findings highlight biofilm developmental stages, phage-phage competition, and phage resistance as factors restricting the in vitro efficacy of a two-phage combination. Our findings provide a framework for selecting and optimizing phage combinations for enhanced efficacy against P. aeruginosa, a metabolically flexible pathogen that undergoes specific adaptation within the infected lung.

microbiology↗

bioRxiv2024

RNA-Protein Interaction Prediction without High-Throughput Data: An Overview and Benchmark of in silico Tools

RNA-protein interactions (RPIs) are crucial for accurately operating various processes in and between organisms across kingdoms of life. Mutual detection of RPI partner molecules depends on distinct sequential, structural, or thermodynamic features, which can be determined via experimental and bioinformatic methods. Still, the underlying molecular mechanisms of many RPIs are poorly understood. It is further hypothesized that many RPIs are not even described yet. Computational RPI prediction is continuously challenged by the lack of data and detailed research of very specific examples. With the discovery of novel RPI complexes in all kingdoms of life, adaptations of existing RPI prediction methods are necessary. Continuously improving computational RPI prediction is key in advancing the understanding of RPIs in detail and supplementing experimental RPI determination. The growing amount of data covering more species and detailed mechanisms support the accuracy of prediction tools, which in turn support specific experimental research on RPIs. Here, we give an overview of RPI prediction tools that do not use high-throughput data as the users input. We review the tools according to their input, usability, and output. We then apply the tools to known RPI examples across different kingdoms of life. Our comparison shows that the investigated prediction tools do not favor a certain species and equip the user with results varying in degree of information, from an overall RPI score to detailed interacting residues. Furthermore, we provide a guide tree to assist users which RPI prediction tool is appropriate for their available input data and desired output. Contactsarah.krautwurst@uni-jena.de

bioinformatics↗

bioRxiv2023

Maximizing the potential of genomic and transcriptomic studies by nanopore sequencing

Nucleic acid sequencing is the process of identifying the sequence of DNA or RNA, with DNA used for genomes and RNA for transcriptomes. Deciphering this information has the potential to greatly advance our understanding of genomic features and cellular functions. In comparison to other available sequencing methods, nanopore sequencing stands out due to its unique advantages of processing long nucleic acid strands in real time, within a small portable device, enabling the rapid analysis of samples in diverse settings. Evolving over the past decade, nanopore sequencing remains in a state of ongoing development and refinement, resulting in persistent challenges in protocols and technology. This article employs an interdisciplinary approach, evaluating experimental and computational methods to address critical gaps in our understanding in order to maximize the information gain from this advancing technology. Here we present both overview and analysis of all aspects of nanopore sequencing by providing statistically supported insights. Thus, we aim to provide fresh perspectives on nanopore sequencing and give comprehensive guidelines for the diverse challenges that frequently impede optimal experimental outcomes.

bioinformatics↗

bioRxiv2023

Targeted decontamination of sequencing data with CLEAN

Many biological and medical questions are answered based on the analysis of sequence data. However, we can find contamination, artificial spike-ins, and overrepresented rRNA sequences in various read collections and assemblies. In particular, spike-ins used as controls, as those known from Illumina or Nanopore data, are often not considered as contaminants and also not appropriately removed during analyses. Additionally, removing human host DNA may be necessary for data protection and ethical considerations to ensure that individuals cannot be identified. We developed CLEAN, a pipeline to remove unwanted sequences from both long- and short-read sequencing techniques. While focusing on Illumina and Nanopore data with their technology-specific control sequences, the pipeline can also be used for host decontamination of metagenomic reads and assemblies, or the removal of rRNA from RNA-Seq data. The results are the purified sequences and sequences identified as contaminated with statistics summarized in a report. The output can be used directly in subsequent analyses, resulting in faster computations and improved results. Although decontamination seems mundane, many contaminants are routinely overlooked, cleaned by steps that are not fully reproducible or difficult to trace. CLEAN facilitates reproducible, platform-independent data analysis in genomics and transcriptomics and is freely available at https://github.com/rki-mf1/clean under a BSD3 license. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/552089v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@133c638org.highwire.dtl.DTLVardef@1ab448org.highwire.dtl.DTLVardef@1320f21org.highwire.dtl.DTLVardef@17f7a9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

bioinformatics↗

bioRxiv2021

poreCov - an easy to use, fast, and robust workflow for SARS-CoV-2 genome reconstruction via nanopore sequencing

In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.

bioinformatics↗

bioRxiv2019

A comprehensive annotation and differential expression analysis of short and long non-coding RNAs in 16 bat genomes

Although bats are increasingly becoming the focus of scientific studies due to their unique properties, these exceptional animals are still among the least studied mammals. Assembly quality and completeness of bat genomes vary a lot and especially non-coding RNA (ncRNA) annotations are incomplete or simply missing. Accordingly, standard bioinformatics pipelines for gene expression analysis often ignore ncRNAs such as microRNAs or long antisense RNAs. The main cause of this problem is the use of incomplete genome annotations. We present a complete screening for ncRNAs within 16 bat genomes. NcRNAs affect a remarkable variety of vital biological functions, including gene expression regulation, RNA processing, RNA interference and, as recently described, regulatory processes in viral infections. Within all investigated bat assemblies we annotated 667 ncRNA families including 162 snoRNAs and 193 miRNAs as well as rRNAs, tRNAs, several snRNAs and IncRNAs, and other structural ncRNA elements. We validated our ncRNA candidates by six RNA-Seq data sets and show significant expression patterns that have never been described before in a bat species on such a large scale. Our annotations will be usable as a resource (Electronic Supplement) for deeper studying of bat evolution, ncRNAs repertoire, gene expression and regulation, ecology, and important host-virus interactions. Supplementary informationis available at rna.uni-jena.de/supplements/bats, the Open Science Framework (doi.org/10.17605/OSF.IO/4CMDN), and GitHub (github.com/rnajena/bats_ncrna).

bioinformatics↗

bioRxiv2019

Characterization of Amino Acid Recognition in Aminoacyl-tRNA Synthetases

Storage and directed transfer of information is the key requirement for the development of life. Yet any information stored on our genes is useless without its correct interpretation. The genetic code defines the rule set to decode this information. Aminoacyl-tRNA synthetases are at the heart of this process. For the first time, we extensively characterize how these enzymes distinguish all natural amino acids based on the computational analysis of crystallographic structure data. The results of this meta-analysis show that the correct read-out of genetic information is a delicate interplay between the composition of the binding site, non-covalent interactions, error correction mechanisms, and steric effects.

bioinformatics↗

bioRxiv2019

Distributed representations of protein domains and genomes and their compositionality

Learning algorithms have at their disposal an ever-growing number of metagenomes for biomining and the study of microbial functions. We propose a novel representation of function called nanotext that scales to very large data sets while capturing precise functional relationships.\n\nThese relationships are learned from a corpus of 32 thousand genome assemblies with 145 million protein domains. We treat the protein domains in a genome like words in a document, assuming that protein domains in a similar context have similar \"meaning\". This meaning can be distributed by the Word2Vec embedding algorithm over a vector of numbers. These vectors not only encode function but can be used to predict even complex genomic features and phenotypes.\n\nWe apply nanotext to data from the Tara ocean expedition to predict plausible culture media and growth temperatures for microorganisms from their metagenome assembled genomes (MAGs) alone. nanotext is freely released under a BSD licence (https://github.com/phiweger/nanotext).

genomics↗

bioRxiv2018

Nanopore direct RNA sequencing reveals modification in full-length coronavirus genomes

Sequence analyses of RNA virus genomes remain challenging due to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called quasispecies. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus.\n\nUsing DRS, we were able to map the longest ([~]26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by demonstrating the feasibility of intra-sample haplotype separation.\n\nEven though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

genomics↗

bioRxiv2017

Backbone brackets and arginine tweezers delineate class I and class II aminoacyl tRNA synthetases

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes.\n\nAuthor summaryAminoacyl tRNA synthetases (aaRS) are primordial enzymes essential for interpretation and transfer of genetic information. Understanding the origin of the peculiarities observed with aaRS can explain what constituted the earliest life forms and how the genetic code was established. The increasing amount of experimentally determined three-dimensional structures of aaRS opens up new avenues for high-throughput analyses of molecular mechanisms. In this study, we present an exhaustive structural analysis of ATP binding motifs. We unveil an oppositional implementation of enzyme substrate binding in each aaRS Class. While Class I binds via interactions mediated by backbone hydrogen bonds, Class II uses a pair of arginine residues to establish salt bridges to its ATP ligand. We show how nature realized the binding of the same ligand species with completely different mechanisms. In addition, we demonstrate that sequence or even structure analysis for conserved residues may miss important functional aspects which can only be revealed by ligand interaction studies. Additionally, the placement of those key residues in the structure supports a popular hypothesis, which states that prototypic aaRS were once coded on complementary strands of the same gene.

bioinformatics↗