The high neuroactive potential of metabolites produced by gut microbes has gained traction over the last few years, with metagenomic-based studies suggesting an important role of microbiota-derived γ-aminobutyric acid (GABA) in modulating mental health. Emerging evidence has revealed the presence of the glutamate decarboxylase (GAD)-encoding gene, a key enzyme to produce GABA, in the prominent human intestinal genus Bacteroides. Here, we investigated GABA production by Bacteroides in culture and metabolic assays combined with comparative genomics and phylogenetics. A total of 961 Bacteroides genomes were analyzed in silico and 17 metabolically and genetically diverse human intestinal isolates representing 11 species were screened in vitro. Using the model organism Bacteroides thetaiotaomicron DSM 2079, we determined GABA production kinetics, its impact on milieu pH, and we assessed its role in mitigating acid-induced cellular damage. We showed that the GAD-system consists of at least four highly conserved genes encoding a GAD, a glutaminase, a glutamate/GABA antiporter, and a potassium channel. We demonstrated a high prevalence of the GAD-system among Bacteroides with 90% of all Bacteroides genomes (96% in human gut isolates only) harboring all genes of the GAD-system and 16 intestinal Bacteroides strains producing GABA in vitro (ranging from 0.09 to 60.84 mM). We identified glutamate and glutamine as precursors of GABA production, showed that the production is regulated by pH, and that the GAD-system acts as a protective mechanism against acid stress in Bacteroides, mitigating cell death and preserving metabolic activity. Our data also indicate that the GAD-system might represent the only amino acid-dependent acid tolerance system in Bacteroides. Altogether, our results suggest an important contribution of Bacteroides in the regulation of the GABAergic system in the human gut.

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GABA Production by Human Intestinal Bacteroides spp.: Prevalence, Regulation, and Role in Acid Stress Tolerance.

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

Cross-kingdom communication via non-coding RNAs is a recent discovery. Exogenous microRNAs (exog-miRNAs) mainly enter the host via the diet. Generally considered unstable in the gastrointestinal tract, some exogenous RNAs may resist these conditions, especially if transported in extracellular vesicles. They could then reach the intestines and more probably exert a regulatory effect. We give an overview of recent discoveries concerning dietary miRNAs, possible ways of enhancing their resistance to food processing and gut conditions, their transport in extracellular vesicles (animal- and plant-origin) and possible biological effects on recipient cells after ingestion. We critically focus on what we believe are the most relevant data for future pharmacological development of dietary miRNAs as therapeutic agents. Finally, we discuss the miRNA-mediated cross-kingdom regulation between diet, host and the gut microbiota. We conclude that, despite many obstacles and challenges, extracellular miRNAs are serious candidates to be targeted pharmacologically for development of new therapeutic agents.

Eating microRNAs: pharmacological opportunities for cross-kingdom regulation and implications in host gene and gut microbiota modulation.

Insects are the most diverse group of animals and colonize almost all environments on our planet. This diversity is reflected in the structure and function of the microbial communities inhabiting the insect digestive system. As in mammals, the gut microbiota of insects can have important symbiotic functions, complementing host nutrition, facilitating dietary breakdown or providing protection against pathogens. There is an increasing number of insect models that are experimentally tractable, facilitating mechanistic studies of gut microbiota-host interactions. In this Review, we will summarize recent findings that have advanced our understanding of the molecular mechanisms underlying the symbiosis between insects and their gut microbiota. We will open the article with a general introduction to the insect gut microbiota and then turn towards the discussion of particular mechanisms and molecular processes governing the colonization of the insect gut environment as well as the diverse beneficial roles mediated by the gut microbiota. The Review highlights that, although the gut microbiota of insects is an active field of research with implications for fundamental and applied science, we are still in an early stage of understanding molecular mechanisms. However, the expanding capability to culture microbiomes and to manipulate microbe-host interactions in insects promises new molecular insights from diverse symbioses.

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Mechanisms underlying gut microbiota-host interactions in insects.

Discovering riboswitches: the past and the future

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called ‘Theta-Base’ (
 www.helmholtz-hiri.de/en/datasets/bacteroides
 ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes. Bacteroides thetaiotaomicron is a human gut microbe and an emergent model organism. Here, Ryan et al. generate single-nucleotide resolution RNA-seq data for this bacterium and map transcription start sites and noncoding RNAs, one of which modulates expression of metabolic enzymes.

/pdf/a-high-resolution-transcriptome-map-identifies-small-rna-kmosf9jpz6.pdf

A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron

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

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species, yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the previously uncharacterized sRNA BatR to increase susceptibility to bile salts, likely through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for discovering hidden functions of bacterial sRNAs.

A CRISPR-based genetic screen in Bacteroides thetaiotaomicron reveals a small RNA modulator of bile susceptibility

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in post- transcriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We develop a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 minute, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with CLIP-seq revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3’ untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for both proteins in the control of respiration, and for ProQ in the oxidative stress response. Our findings provide new insights into post-transcriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression. Significance Statement Together with transcription and translation, RNA decay is one of the major processes governing protein production. Here, we have developed a new statistical approach that corrects for confounding effects when estimating RNA decay rates from RNA-seq in bacteria. Our more accurate decay rate estimates indicate that bacterial transcripts have half-lives about three times shorter than previously thought. This approach allowed us to measure the effects of RNA- binding proteins (RBPs) on decay rates, identifying large cohorts of transcripts with changes in stability following RBP deletion and conditions where post-transcriptional regulation affects survival. Our method should lead to a reevaluation of RNA stability estimates across diverse bacteria and new insights into the role of RBPs in shaping the transcriptome.

https://www.biorxiv.org/content/biorxiv/early/2023/06/15/2023.06.15.545072.1.full.pdf

Sarah Reichardt

Papers

A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron

Comparative genomics provides structural and functional insights into Bacteroides RNA biology

A CRISPR-based genetic screen in Bacteroides thetaiotaomicron reveals a small RNA modulator of bile susceptibility

Improved RNA stability estimation through Bayesian modeling reveals most bacterial transcripts have sub-minute half-lives