Biology subjects

Wagner, G. E.

Publications and source records attributed to Wagner, G. E..

3 recordsLinked to original sources

bioRxiv2025

Decoding bacterial methylomes in four public health-relevant microbial species: Nanopore sequencing enables reproducible analysis of DNA modifications

Investigating bacterial methylation profiles provides essential complementary information to the native DNA sequence, significantly extending our understanding of how DNA modifications influence virulence, antibiotic resistance, and the ability of bacteria to evade the immune system. Recent advancements in real-time Nanopore sequencing and basecalling algorithms have enabled the direct detection of modified bases from raw signal data, eliminating the need for bisulfite treatment of DNA. However, decoding methylation signals remains challenging due to rapid technological and methodological progress. In this study, we focus on public health-relevant bacterial strains to analyze their methylation profiles and identify methylation motifs. Our dataset includes samples from Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecium, and Klebsiella pneumoniae, sequenced on the Nanopore GridION platform using the latest flow cell chemistry (R10.4.1) and modification basecalling models (Dorado basecalling SUP model v5). We investigated distinct methylation patterns within and between species, focusing on heavily modified genes or genomic regions. Our results reveal distinct species-specific methylation profiles, with each strain exhibiting unique modification patterns. We developed a modular pipeline using Nextflow and the Nanopore Modkit tool to streamline the detection of methylated motifs. We compared the results with outputs from MicrobeMod, a recent toolkit for exploring prokaryotic methylation and base modifications in nanopore sequencing. Our pipeline is publicly available for further use (github.com/rki-mf1/ont-methylation). We identified known methylation motifs already described in the literature and novel de novo motifs, providing deeper insights into the diversity of bacterial DNA modifications. Furthermore, we identified genomic regions that are extensively methylated, which could have implications for bacterial behavior and pathogenicity. We also assess improvements in basecalling accuracy, specifically how methylated bases can influence neighboring basecalls. Recent advances in basecalling models, particularly v5 models as part of Dorado, have reduced these issues, improving the reliability of methylation detection in bacterial genomes. In conclusion, our study highlights the potential of current nanopore sequencing tools for detecting DNA modifications in prokaryotes. By making our pipeline and results publicly available, we facilitate further research into bacterial DNA modifications and their role in microbial pathogenesis.

bioinformatics↗

bioRxiv2023

Nanopore sequencing for accurate bacterial outbreak tracing

Our study investigated the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a three-year-long Klebsiella pneumoniae outbreak with Illumina short read sequencing data as the point of reference. We detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae. Based on this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA is freely available on GitHub under the GNUv3 license: github.com/replikation/MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investing outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either PCR-based sequencing or using our provided bioinformatic workflow. We would advise that read mapping-based quality control of genomes should be provided when publishing results.

molecular biology↗

bioRxiv2023

Vibrio cholerae's ToxRS Bile Sensing System

Cholera represents a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence causing recurring sudden outbreaks is enabled by V. choleraes rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies e.g. bile resistance and virulence regulation. Currently, transcription factor ToxR is described as main environmental sensor for bile acid, whose activity is enhanced by binding to ToxS. Here, the presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unraveling the intricate mechanisms governing ToxRSs environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrios survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

biochemistry↗