The LysR-type transcription factor LsrB regulates beta-lactam resistance in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta-lactam antibiotics by utilizing the inducible beta-lactamase AmpC. Our picture on the condition-dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild-type levels by lsrB complementation. By transcriptome profiling via RNA-Seq and qRT-PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR-type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR-mediated regulation of ampC. Our results substantially expand the existing model of inducible beta-lactam resistance in A. tumefaciens by establishing LsrB as higher-level transcriptional regulator.

Inverse folding based pre-training for the reliable identification of intrinsic transcription terminators.

It is well-established that neural networks can predict or identify structural motifs of non-coding RNAs (ncRNAs). Yet, the neural network based identification of RNA structural motifs is limited by the availability of training data that are often insufficient for learning features of specific ncRNA families or structural motifs. Aiming to reliably identify intrinsic transcription terminators in bacteria, we introduce a novel pre-training approach that uses inverse folding to generate training data for predicting or identifying a specific family or structural motif of ncRNA. We assess the ability of neural networks to identify secondary structure by systematic in silico mutagenesis experiments. In a study to identify intrinsic transcription terminators as functionally well-understood RNA structural motifs, our inverse folding based pre-training approach significantly boosts the performance of neural network topologies, which outperform previous approaches to identify intrinsic transcription terminators. Inverse-folding based pre-training provides a simple, yet highly effective way to integrate the well-established thermodynamic energy model into deep neural networks for identifying ncRNA families or motifs. The pre-training technique is broadly applicable to a range of network topologies as well as different types of ncRNA families and motifs.

A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens.

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.

Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions.

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.