Biology subjects

Barth-Weber, S.

Publications and source records attributed to Barth-Weber, S..

5 recordsLinked to original sources

bioRxiv2026

Predicted Bacterial uRBSs Reveal Translational Coupling and Ribosome Mediated RBS Occlusion as Gene-Controlling Mechanisms

Upstream open reading frames (uORFs) in the 5' leader of bacterial mRNAs can modulate gene expression, yet genome-wide identification remains limited. We combined bioinformatic prediction of ribosome-binding sites (RBSs) - a Shine-Dalgarno sequence and a start codon - with experimental validation to uncover new uORFs in Sinorhizobium meliloti 2011. From totally 1106 predicted upstream RBSs (uRBSs), we first examined 15 candidates using eGFP reporters and integrating existing RNA-seq and Ribo-seq data. Translation was detected at 13 sites, with fluorescence intensity broadly correlating with predicted initiation rates. Two uRBSs correspond to gene start sites, thereby refining gene annotations. In nine cases, uRBS mutations affected downstream gene expression in reporter fusions. Among others, the data suggests that a Type I secretion system operon, the RNA chaperone gene hfq, and metabolic genes are regulated by uORFs. Four uORFs acted through translational coupling. We also identified uRBSs that were ribosome-occupied yet (nearly) silent in eGFP assays, and closely spaced to the downstream main RBS (mRBS). These uRBSs probably mediate ribosomal occlusion downregulating lacR and SM2011_RS36230. A re-screen of the prediction set revealed 335 close uRBS/mRBS pairs. Three of them were analyzed, supporting the proposed ribosomal occlusion mechanism for SM2011_RS03630 and SM2011_RS22110, while for glnK translational coupling to an uORF was suggested. These results indicate that uORFs are more widespread in bacteria than previously recognized and suggest that direct ribosomal occlusion of the mRBS is a novel mechanism for down-regulating protein synthesis.

molecular 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↗

bioRxiv2019

Trans-acting role of the leader peptide peTrpL in posttranscriptional regulation of multiresistance

Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbour small upstream ORFs (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the soil-dwelling plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in the cell, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. We provide evidence that peTrpL plays a role in the differential posttranscriptional regulation of the smeABR operon encoding the major multidrug efflux pump SmeAB and the TtgR-type transcription repressor SmeR. We show that peTrpL is involved in a tetracycline-dependent smeR mRNA destabilization and forms an antibiotic-dependent ribonucleoprotein (ARNP) complex with smeR and its antisense RNA (asRNA). Induction of asRNA transcription, ARNP formation and smeR downregulation were promoted by several antibiotics and the flavonoid genistein, and the resistance to these antimicrobial compounds was increased by peTrpL. The role of peTrpL in resistance is conserved in other soil Alphaproteobacteria.

molecular biology↗

bioRxiv2019

Posttranscriptional regulation of ribosomal and multiresistance genes by the bacterial leader peptide peTrpL

The ribosome-dependent attenuator located upstream of bacterial tryptophan biosynthesis genes harbors a small ORF trpL containing tryptophan codons. When tryptophan is available, efficient trpL translation causes transcription termination and release of the attenuator RNA rnTrpL. In Sinorhizobium meliloti, rnTrpL is a trans-acting sRNA. Here, we identified an evolutionary conserved function for the trpL-encoded 14-aa leader peptide peTrpL. Upon exposure to tetracycline, the cellular peTrpL levels were increased and rnTrpL was generated independently of tryptophan availability. Both peTrpL and rnTrpL were found to be involved in tetracycline-dependent destabilization of rplUrpmA mRNA encoding ribosomal proteins L21 and L27. We provide evidence for redirection of the sRNA rnTrpL from its antibiotic-independent target trpDC to rplUrpmA by formation of an antibiotic-dependent ribonucleoprotein complex (ARNP). ARNPs comprising peTrpL, rnTrpL, rplUrpmA and antisense RNA were also observed for other translation-inhibiting antibiotics, suggesting that bacteria evolved mechanisms to utilize antibiotics for mRNA destabilization.

microbiology↗