Comprehensive mapping of transcription terminator Rho utilization (Rut) sites across the Bacillus subtilis genome.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Bacterial Rho helicase regulates gene expression in Bacillus subtilis in a growth-dependent manner.
• Standard transcriptomic methods struggle to identify Rho-dependent terminators due to in vivo transcript processing.
• Understanding Rho helicase function is crucial for deciphering bacterial gene regulation.

Purpose of the Study:

• To precisely map Rho utilization (Rut) sites genome-wide in Bacillus subtilis.
• To compare the binding specificities of Bacillus subtilis Rho (BsRho) and Escherichia coli Rho (EcRho).
• To investigate the role of Rho-dependent regulation in Bacillus subtilis fitness and survival.

Main Methods:

• Application of Helicase-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to map Rut sites.
• Genome-wide identification of Rut sites using B. subtilis Rho and E. coli Rho.
• In vivo validation of identified Rut sites and analysis of transcript processing.

Main Results:

• Identified 600 putative Rut sites with BsRho and 4189 sites with EcRho.
• Both Rho enzymes recognize pyrimidine-rich sequences, with BsRho favoring shorter motifs.
• In vivo validation confirmed Rho-dependent regulation and PNPase-mediated processing of transcripts.
• Discovered widespread Rho-dependent control of antisense transcription and metabolic genes.

Conclusions:

• A complex Rho-dependent regulatory network exists in Bacillus subtilis, influencing gene expression and metabolism.
• Rho helicase likely enhances Bacillus subtilis fitness and survival in challenging environments.
• The compendium of Rut sites provides a resource for further research into bacterial adaptive regulation.