An integrated transcriptomics–functional genomics approach reveals a small RNA that modulatesBacteroides thetaiotaomicronsensitivity to tetracyclines

Abstract

Gene expression plasticity allows bacteria to adapt to diverse environments, tie their metabolism to available nutrients, and cope with stress. This is particularly relevant in a niche as dynamic and hostile as the human intestinal tract, yet transcriptional networks remain largely unknown in gutBacteroidesspp. Here, we map transcriptional units and profile their expression levels inBacteroides thetaiotaomicronover a suite of 15 defined experimental conditions that are relevant in vivo, such as variation of temperature, pH, and oxygen tension, exposure to antibiotic stress, and growth on simple carbohydrates or on host mucin–derived glycans. Thereby, we infer stress and carbon source-specific transcriptional regulons, including conditional expression of capsular polysaccharides and polysaccharide utilization loci, and expand the annotation of small regulatory RNAs (sRNAs) in this organism. Integrating this comprehensive expression atlas with transposon mutant fitness data, we identify conditionally important sRNAs. One example is MasB, whose inactivation led to increased bacterial tolerance of tetracyclines. Using MS2 affinity purification coupled with RNA sequencing, we predict targets of this sRNA and discuss their potential role in the context of the MasB-associated phenotype. Together, this transcriptomic compendium in combination with functional sRNA genomics—publicly available through a new iteration of the ′Theta–Base′ web browser (www.helmholtz-hiri.de/en/datasets/bacteroides-v2)—constitutes a valuable resource for the microbiome and sRNA research communities alike.

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gutBacteroides

“…3a). In accordance with an independent transcriptional activation, published RNA-seq data 12 showed an anticorrelated expression pattern of GibS and the FopS sRNAs (Fig. 3d).…”

“…The bile salt cocktail we used consisted of cholic acid (a primary bile salt) and deoxycholate (a conjugated, secondary bile salt) in equimolar ratio. However, interrogation of previously generated RNA-seq data 12 indicated deoxycholate alone to be insufficient to induce FopS expression (Fig. 3d).…”

“…d, e, Expression profiling of GibS and the FopS sRNAs. Relative expression of the indicated sRNAs over a range of various carbon sources (relative to growth in minimal medium with glucose as the sole carbon source) and stress conditions (relative to an unstressed control sample) based on RNA-seq data from 12 ( d ) and northern blot-based validation ( e ). EEP, early exponential phase; MEP, mid-exponential phase; stat, stationary phase in TYG.…”

“…evolved unique transcription 67 and translation 68 initiation signals. Importantly, although they encode hundreds of noncoding RNAs [11][12][13][14] , these bacteria lack the classical chaperones of pseudomonadotal sRNAs 69 . Despite recent attempts to infer Bacteroidota RNA chaperones from in-silico prediction and in-vitro experimentation 14,15 , up to now the only global RBP that has been functionally characterized in this phylum remained the transcription termination factor Rho 70 .…”

“…Secondary structure was visualized using the R2R software 83 and deposited in the Rfam FopS targets and individual FopS sRNAs upon growth on different nutrient sources and under defined stress conditions based on RNA-seq data from 12 . Plotted in the chart are the Pearson's correlation coefficients (r) for the individual sRNA-mRNA pairs.…”