Comparative genomics provides structural and functional insights into Bacteroides RNA biology

Abstract

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.

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

“…MasB (previously BTnc201; renamed here for reasons to follow) is an ∼100 nt-long, narrowly conserved sRNA (Prezza et al, 2022). In B. thetaiotaomicron , the sRNA is encoded in between an operon composed of genes for a Na + /H + antiporter ( BT_3638 ), a thiazole biosynthesis adenylyltransferase ( thiF ), and a lipoprotein-releasing system ATP-binding protein ( BT_3640 ), and a divergently encoded operon comprising a Na + -dependent transporter ( BT_3641 ) and a hypothetical gene ( BT_3642 ) (Fig.…”

“…Shown are representative read coverages across the masB genomic locus and flanking genes. Lower: Experimentally determined secondary structure of MasB (Prezza et al, 2022). The positions of the inserted transposons are indicated by blue arrowheads.…”

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

“…Rifampicin treatment to halt de-novo transcription RNA stability assay was performed as described in 14 . Briefly, single colonies of B. thetaiotaomicron strains AWS-001 (WT), AWS-323 (ΔrbpB), and AWS-218 (rbpB ++ ) were inoculated in liquid TYG medium and grown for ~16 h, subsequently sub-cultured (1:100 dilution) and grown to mid-exponential phase (OD 600 = 2.0).…”

“…The present study reports the identification of an in-vivo phenotype, the RNA interactome, a metabolism-associated function, and the underlying molecular mechanism of the first global RNA-binding protein (RBP) in Bacteroides. Specifically, we found that a B. thetaiotaomicron mutant devoid of the RNA recognition motif 1 (RRM-1)-containing protein RbpB 14,15 failed to efficiently colonize the mammalian intestine in a diet-dependent manner.…”

“…Specifically, we found that a B . thetaiotaomicron mutant devoid of the RNA recognition motif 1 (RRM-1)-containing protein RbpB 14,15 failed to efficiently colonize the mammalian intestine in a diet-dependent manner. Cross-linking immunoprecipitation and sequencing (CLIP-seq) of B .…”