Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

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The Toll-Like Receptor 2 pathway Establishes Colonization by a Commensal of the Human Microbiota

Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed1. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes1, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions2. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations3 but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate. Single-cell transcriptomics is revolutionizing the analysis of phenotypic cell-to-cell variation in eukaryotes, but technical hurdles have prevented its robust application to prokaryotes. Here, using an improved poly(A)-independent single-cell RNA-sequencing protocol, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria across all RNA classes and genomic regions. These transcriptomes provide important reference points for single-cell RNA-sequencing of other bacterial species, mixed microbial communities and host-pathogen interactions.

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Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria

The contribution of the large intestine to energy supplies in man [Intestinal absorption, short-chain fatty acids, dietary fiber].

The human body is constantly exposed to microorganisms, which entails manifold interactions between human cells and diverse commensal or pathogenic bacteria. The cellular states of the interacting cells are decisive for the outcome of these encounters such as whether bacterial virulence programmes and host defence or tolerance mechanisms are induced. This Review summarizes how next-generation RNA sequencing (RNA-seq) has become a primary technology to study host–microbe interactions with high resolution, improving our understanding of the physiological consequences and the mechanisms at play. We illustrate how the discriminatory power and sensitivity of RNA-seq helps to dissect increasingly complex cellular interactions in time and space down to the single-cell level. We also outline how future transcriptomics may answer currently open questions in host–microbe interactions and inform treatment schemes for microbial disorders. Interactions between microorganisms and their hosts are highly context dependent and contribute to both normal tissue function and infectious disease pathology. In this Review, Westermann and Vogel describe how advances in RNA sequencing techniques are providing molecular insights into host–microbe interactions, including advances in cross-species and single-cell transcriptomics.

Cross-species RNA-seq for deciphering host–microbe interactions

A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH ( d epletion of a bundant s equences by h ybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.

/pdf/improved-bacterial-rna-seq-by-cas9-based-depletion-of-2kd3s8f17r.pdf

Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads

Bacteria employ noncoding RNAs to maintain cellular physiology, adapt global gene expression to fluctuating environments, sense nutrients, coordinate their interaction with companion microbes and host cells, and protect themselves against bacteriophages. While bacterial RNA research has made fundamental contributions to biomedicine and biotechnology, the bulk of our knowledge of RNA biology stems from the study of a handful of aerobic model species. In comparison, RNA research is lagging in many medically relevant obligate anaerobic species, in particular the numerous commensal bacteria comprising our gut microbiota. This review presents a guide to RNA-based regulatory mechanisms in the phylum Bacteroidetes, focusing on the most abundant bacterial genus in the human gut, Bacteroides spp. This includes recent case reports on riboswitches, an mRNA leader, cis- and trans-encoded small RNAs (sRNAs) in Bacteroides spp., and a survey of CRISPR-Cas systems across Bacteroidetes. Recent work from our laboratory now suggests the existence of hundreds of noncoding RNA candidates in Bacteroides thetaiotaomicron, the emerging model organism for functional microbiota research. Based on these collective observations, we predict mechanistic and functional commonalities and differences between Bacteroides sRNAs and those of other model bacteria, and outline open questions and tools needed to boost Bacteroidetes RNA research.

https://www.degruyter.com/document/doi/10.1515/hsz-2020-0230/pdf

An RNA-centric view on gut Bacteroidetes.

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mmi.14793

Comparative genomics provides structural and functional insights into Bacteroides RNA biology

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 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.

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An integrated transcriptomics–functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines

https://www.nature.com/articles/s41467-023-39787-6.pdf

Gianluca Prezza

Papers

Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads

An RNA-centric view on gut Bacteroidetes.

Comparative genomics provides structural and functional insights into Bacteroides RNA biology

An integrated transcriptomics–functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines

Cytosolic Ptbp2 modulates axon growth in motoneurons through axonal localization and translation of Hnrnpr