ABSTRACT
The Gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, little is known about the bacterium’s transcriptome architecture and mechanisms of post-transcriptional control. Here, we have applied a suite of RNA-centric techniques, including transcription start site mapping, transcription termination mapping and Hfq RIP-seq, to generate a single-nucleotide resolution RNA map of C. difficile 630. Our transcriptome annotation provides information about 5’ and 3’ untranslated regions, operon structures and non-coding regulators, including 42 sRNAs. These transcriptome data are accessible via an open-access browser called ‘Clost-Base’. Our results indicate functionality of many conserved riboswitches and predict novel cis-regulatory elements upstream of MDR-type ABC transporters and transcriptional regulators. Recent studies have revealed a role of sRNA-based regulation in several Gram-positive bacteria but their involvement with the RNA-binding protein Hfq remains controversial. Here, sequencing the RNA ligands of Hfq reveals in vivo association of many sRNAs along with hundreds of potential target mRNAs in C. difficile providing evidence for a global role of Hfq in post-transcriptional regulation in a Gram-positive bacterium. Through integration of Hfq-bound transcripts and computational approaches we predict regulated target mRNAs for the novel sRNA AtcS encoding several adhesins and the conserved oligopeptide transporter oppB that influences sporulation initiation in C. difficile. Overall, these findings provide a potential mechanistic explanation for increased biofilm formation and sporulation in an hfq deletion strain and lay the foundation for understanding clostridial ribo regulation with implications for the infection process.
Competing Interest Statement
The authors have declared no competing interest.
