Starts, stops and RNA binding protein interactions of the Bacillus subtilis transcriptome

: (Alternative Format Thesis)

Abstract

Bacteria live in hostile worlds where nutrients are limited and competition is fierce. They constantly adapt to their surroundings and optimise their behaviours in accordance with the environment. When conditions change, species who can rapidly remodel their gene expression landscape to meet emerging demands have an advantage and a better chance of survival. Transcriptional regulation is the starting point of gene expression but is relatively slow and generally has broad effects. On the other hand, post-transcriptional regulation, orchestrated by regulatory RNAs and RNA binding proteins (RBPs), enable rapid, finely tuned, and targeted gene expression changes through effects on transcription progression, RNA stability and translation initiation. High-throughput sequencing techniques have identified hundreds of small non-coding RNAs (sRNAs) and many RBPs with key functions in gene regulatory networks and adaptive responses. Hfq, the most widely studied RBP, has been shown to facilitate hundreds of RNA:RNA interactions with functional roles relating to stress tolerance, motility and virulence across Gram-positive and negative bacteria. However, in Bacillus subtilis, the most widely studied Gram-positive model organism, Hfq have little effect on RNA biology and cellular behaviour. While other RBPs such as CsrA, aconitase, and cold-shock proteins have been explored, none have demonstrated global regulatory functions, leaving post-transcriptional regulation in B. subtilis a largely unresolved question: Are global RNA regulators yet to be identified, or does B. subtilis rely on multiple RBPs with distinct roles across different pathways?

A key limitation in studying post-transcriptional gene regulation has been the lack of high-resolution annotations for non-coding RNAs and limited genome-wide information about sRNA processing sites. To address this, I developed pyRAP, a Python-based pipeline for annotating Rend-seq data, an RNA-seq technique that resolves transcript boundaries and RNA processing sites at single-nucleotide resolution. pyRAP complements existing tools by offering higher sensitivity and scalability for high-throughput analysis. Applied to B. subtilis, Escherichia coli, and Staphylococcus aureus, pyRAP mapped thousands of UTRs, hundreds of sRNAs including transcripts isoforms, several sRNA processing sites, and many novel transcripts. The resulting high-resolution annotation files are freely available on GitHub for use by the microbial research community.
Subsequently, I characterized the functions of four RBPs – SpoVG, Jag, KhpA, and Kre – which have uncharacterized RNA-binding roles in B. subtilis but are known RNA regulators in other Gram-positive bacteria. Using cross-linking and analysis of cDNAs (CRAC) combined with RNA-seq and proteomics I show that all four proteins cross-link RNA in vivo with diverse regulatory outcomes related to protein secretion, carbon catabolite repression, and stationary phase adaptations, including antimicrobial production, biofilm formation and motility development. Preliminary evidence suggests these proteins operate via distinct mechanisms, including modulation of RNA stability, processing, and translation, RNA translocation, and RNA helicase activity.

The combined work of this thesis broadens our understanding of post-transcriptional gene regulation in B. subtilis and should serve as a stepping stone for future studies in Gram-positive bacteria. By presenting four novel RBPs, functioning through different mechanisms in diverse regulatory pathways, we provide supporting evidence, that B. subtilis, unlike other bacterial species, such as E. coli and Salmonella enterica, relies more on localized, pathway-specific RNA regulators rather than global RNA regulators. Furthermore, our novel pipeline for Rend-seq data analysis provides an improved platform for investigations of post-transcriptional gene regulatory events that likely will advance future RNA research.

Date of Award	7 May 2025
Original language	English
Awarding Institution	University of Bath
Supervisor	Emma Denham (Supervisor) & Lauren Cowley (Supervisor)