show Abstracthide AbstractGene 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 gut Bacteroides spp. Here, we map transcriptional units and profile their expression levels in Bacteroides thetaiotaomicron over 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. Overall design: Samples 1-32: RNA-seq of Bacteroides thetaiotaomicron VPI-5482 (wild type) grown in TYG-medium and exposed to different stress conditions (Cold, Heat, Acid, Aerobe, H2O2, Deoxycholate, Bile salts and Gentamicin) or in minimal medium supplemented with different carbon sources (Glucose, Arabinose, Xylose, Maltose, GlcNAc, Mucin and Starvation). Two biological replicates per strain and condition. Samples 33, 34: Genome-wide mapping of transcription start sites via differential RNA-seq (dRNA-seq) of pooled RNA from the above mentioned samples 1-32. Two biological replicates. Samples 35-38: Identification of RNAs (RNA-seq) co-purified with MS2 aptamer tagged MasB sRNA in a masB deletion background and an untagged MasB control strain. Two biological replicates.