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.
Highlights
Small regulatory RNAs are key mediators of bacterial gene expression, allowing microbes to rapidly adapt to changing environmental conditions and cope with diverse stresses (Holmqvist & Wagner, 2017)
To emphasize that our current knowledge of bacterial RNA biology is strongly biased toward that of Gammaproteobacteria and Bacilli, whose species have been harnessed as model Gram-negative or Gram- positive organisms, respectively, for decades
Due to technical hurdles associated with experimentation with obligate anaerobic species, as of we only have a vague idea of the RNA complement of most gut microbiota species
Summary
Small regulatory RNAs (sRNAs) are key mediators of bacterial gene expression, allowing microbes to rapidly adapt to changing environmental conditions and cope with diverse stresses (Holmqvist & Wagner, 2017). Swimming assays further revealed that the Bacteroides CSP homolog can efficiently cross-complement the motility defect of Salmonella ΔcspC/E (Figure 1g) Taken together, these observations suggest the CSP protein BT_1884 may have a global RNA-related function and should encourage future studies of Bacteroides RBPs. By genome-wide RNA-seq analysis, we previously identified 124 intergenic sRNAs in B. thetaiotaomicron (Ryan et al, 2020a). ⌀ (cm): 8.1 +/- 0.4 2.2 +/- 0.2 4.5 +/- 0.5 7.1 +/- 0.8 5.6 +/- 0.3 the B. thetaiotaomicron genome has some unique qualities with respect to bacterial models of RNA biology, being both AT-rich (42% GC content) and lacking known RBPs such as FinO-like proteins that tend to bind highly structured targets (Bauriedl et al, 2020; Gonzalez et al, 2017; Holmqvist et al, 2018; Pandey et al, 2020; Stein et al, 2020), we asked if this was reflected in the properties of Bacteroides ncRNAs as a whole We determined their average length (Figure 2a). B. vulgatus B. dorei HS1_L_1 B. dorei HS1_L_3 B. fragilis BOB25 B. fragilis NCTC 9343
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