Biology subjects

Scheuer, R.

Publications and source records attributed to Scheuer, R..

3 recordsLinked to original sources

bioRxiv2026

NGS-RFA/FRA: A High Throughput Experimental and Computational Pipeline for Selective Mutational Scanning in Parallel

Influenza viruses evade vaccine and infection mediated immunity by accumulating mutations in their hemagglutinin (HA) protein. Predicting this evolution might be possible via selective mutational scanning (SMS) - the generation of many specific mutants of interest from currently circulating viruses and characterizing their escape potential and fitness with high accuracy (Mogling 2016). However, this task is challenging, even when focusing on a reduced set of key HA positions (Koel et al. 2013). Here we describe a high-throughput SMS method to address this challenge. Our approach consists of a three-stage pipeline: (1) a parallel optimized virus rescue process that generates balanced target mutant virus libraries (2) an assay to assess replicative fitness and neutralisation of these variants as a mixture, and (3) a bespoke statistical model to quantify statistically significant differences between these observables. We tested the pipeline on libraries of up to 134 variants finding excellent correlation to classical hemagglutination inhibition (HI) and plaque growth assays used to assess antigenic phenotype and replicative fitness respectively, as well as remarkable repeatability overall. Notably, the method reduces the timeline required to carry out such assessments with classical methods from about a year to several weeks. By enabling rapid and efficient characterization of influenza virus variants, this approach has the potential to greatly enhance surveillance efforts, transforming reactive monitoring into proactive forecasting.

evolutionary biology↗

bioRxiv2025

Early posttranscriptional response to tetracycline exposure in a gram-negative soil bacterium reveals unexpected attenuation mechanism of a DUF1127 gene

The Gram-negative, soil-dwelling plant symbiont Sinorhizobium meliloti shares its free-living habitat with antibiotic producers. To learn about early steps of its adaptation to antibiotics, we analyzed transcriptome changes after 10 min exposure to subinhibitory amount of tetracycline (Tc). RNA-seq revealed 297 differentially expressed genes. Besides ten upregulated ribosomal genes, there was no recognizable functional pattern in the observed changes. Instead, upregulated genes were mostly first in operons and often small, while downregulated genes were downstream in operons. Furthermore, we detected mRNA stabilization upon Tc exposure for several up- and down-regulated genes. Thus, mRNA stabilization contributed to increased mRNA levels, but for downstream genes its effect was counteracted by premature transcriptional termination caused by disrupted coupling between transcription and translation. Using reporter constructs, we found that a DUF1127 gene, showing highest mRNA increase, is controlled by transcription attenuation depending on the translation of an upstream ORF (uORF). Our data suggest the following model: The attenuation strongly depends on the accessibility of C-rich codons at the begin of the uORF. The accessibility is guaranteed by translation of the uORF, and is possible in a time window after a ribosome moves downstream and before a next ribosome occupies the ribosomal binding site (RBS). The accessibility is blocked either by impaired translation initiation or, in the absence of ribosome binding, by base-pairing between the RBS and the C-rich codons. We propose that this is used by bacteria to monitor ribosome availability and translation efficiency, and to ensure reciprocal expression of the DUF1127 gene.

molecular biology↗

bioRxiv2022

Small proteome of the nitrogen-fixing plant symbiont Sinorhizobium meliloti

The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated, and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, cataloging the full complement of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with [≤] 70 amino acids was predicted with high confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search databases (iPtgxDBs). Searches against a standard and a 20-fold smaller Ribo-seq data-informed custom iPtgxDB confirmed many annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by applying MS and Ribo-seq as complementary approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

molecular biology↗