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The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome 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 an in silico iterative 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 of C. 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 of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile , we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies. [ABSTRACT FROM AUTHOR]

Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are overexpressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1. dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1. Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread. [ABSTRACT FROM AUTHOR]