Regulatory RNAs and RNA-binding proteins in Bacteroides thetaiotaomicron

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Bacteria employ noncoding RNAs and RNA-binding proteins to maintain cellular metabolism, adapt global gene expression to changing environmental conditions, sense nutrients, and protect themselves against bacteriophages. Bacterial RNA research has already made fundamental contributions to biomedicine and biotechnology. However, our bulk knowledge on bacterial RNA biology stems from the study of a small number of aerobic model organisms, whereas RNA biology in many medically relevant commensal species, e.g. the numerous anaerobic bacteria constituting our gut microbiota, is a largely unexplored field. Conservational analyses suggest that there is only limited overlap in RNA-mediated processes between phylogenetically distant bacteria, and that many regulatory RNAs are family-, genus-, or even strain-specific. This implies that there is much to be learned from studying RNA biology in the commensals that densely populate our intestine.Bacteroides thetaiotaomicron is a major anaerobic human gut symbiont that contributes to host metabolism and colonization resistance against invading pathogens. Previous studies have attributed successful niche colonization largely to the ability to utilize a wide array of dietary glycans. However, investigations of the underlying regulatory networks are sparse and have so far been restricted to protein-based transcriptional control mechanisms. In stark contrast, little is known about B. thetaiotaomicron post-transcriptional control of gene expression, executed e.g. by means of small regulatory RNAs (sRNAs) and their corresponding protein partners. We have recently re-annotated the B. thetaiotaomicron transcriptome and discovered ~200 novel sRNA candidates; several of which could meanwhile be experimentally validated.I propose to identify those Bacteroides sRNAs that are relevant for colonization of its in-vivo niche and for the interaction with host cells. To this end, B. thetaiotaomicron prototype strain VP 5482 will be used to colonize a human primary 3D model of the large intestine under hypoxic conditions. At defined time points of the co-culture, RNA will be extracted and applied to host-microbe transcriptome profiling. For in-depth mechanistic characterization, sRNA candidates will be selected based on their expression patterns, conservation and phenotype of a corresponding deletion mutant during host colonization. For these prioritized sRNAs, target transcripts will be identified by a combination of computational and experimental screens. In parallel, RNA-binding proteins potentially interacting with the selected sRNAs will be identified by pull-down of aptamer-tagged sRNA variants. The molecular determinants for the sRNAs to interact with their target transcripts and protein binding partners will be uncovered using a variety of biochemical assays. Together, this project will identify in vivo-relevant RNA-mediated control mechanisms used by one of the key members of our gut microbiota to colonize its niche.