Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human EnteropathogenClostridioides difficile

The emerging human enteropathogenClostridioides difficileis the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view ofC. difficilepromoter structure is still missing. In the genome ofC. difficile630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of theC. difficilegenome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5 '-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5 ' untranslated regions. By following anin silicoiterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors ofC. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components ofC. difficileregulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors ofC. difficile, we defined the SigL regulon by combining transcriptome andin silicoanalyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial forC. difficilegut colonization. Finally, we combined our TSS mapping,in silicoidentification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization inC. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression inC. difficileand will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.