An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important Gram-positive bacterium

The Gram-positive bacterium Clostridium difficile, a leading cause of antibiotic-associated pseudomembranous colitis, has received increasing attention due to a rising incidence of clinical C. difficile infections (CDI). Despite progress understanding bacterial factors that promote CDI-associated morbidity and mortality, many fundamental aspects of C. difficile biology remain to be explored. Compared to other Gram-positive pathogens, little is known about the bacterium’s transcriptome architecture and in particular mechanisms of post-transcriptional control. To close this knowledge gap, we have applied a suite of transcriptome-focused techniques, including transcription start site mapping (dRNA-seq), transcription termination mapping, and Hfq RIP-seq, resulting in a single-nucleotide resolution RNA map of C. difficile strain 630. Differential RNA-seq (dRNA-seq) was performed for C. difficile strain 630 in either different media and different growth stages to identify transcriptional start sites, 5'UTRs and sRNAs. The RNAtag-seq protocol was used to determine 3'UTRs of transcripts. RIP-seq was carried out to identify RNAs bound by Hfq in C. difficile.