Comprehensive mapping of transcription terminator Rho utilization (Rut) sites across the <i>Bacillus subtilis</i> genome

Recent evidence indicates that the bacterial Rho helicase regulates Bacillus subtilis gene expression in a growth-dependent manner. This regulation, along with extensive in vivo trimming of Rho-dependent transcript 3'-ends, complicates the identification of Rho-dependent transcription terminators using standard transcriptomic approaches. To overcome this challenge, we applied Helicase-SELEX to precisely map Rho utilization (Rut) sites genome-wide. Using B. subtilis Rho (BsRho), we identified 600 putative Rut sites, while the more permissive Escherichia coli Rho (EcRho) revealed 4189 sites, including specimens known to regulate B. subtilis genes. Comparative analysis showed that both enzymes recognize similar pyrimidine-rich sequences, though BsRho favors short unstructured Rut motifs whereas EcRho can act on presumably more structured RNAs without requiring accessory factors. In vivo validation of selected Rut sites confirmed Rho-dependent regulation and extensive PNPase-mediated processing of Rho-terminated transcripts. Collectively, our results reveal a rich and complex Rho-dependent regulatory network in B. subtilis, encompassing the widespread control of antisense transcription and genes/operons of both primary and secondary metabolism. Although nonessential under standard laboratory conditions, Rho thus likely contributes to B. subtilis fitness and survival in more demanding environments. Our comprehensive compendium of Rut sites offers a valuable resource for exploring this adaptive regulatory landscape.