A conserved structured non-coding RNA coordinates growth and virulence in Clostridioides difficile. [dRNA-Seq ModT]

Bacterial non-coding RNAs fulfill a variety of cellular functions, for example as catalysts, as structural components in multiprotein complexes or as regulators of gene expression at the transcriptional and post-transcriptional level. Some RNAs display exceptionally broad conservation across bacterial phyla and are involved in fundamental and unique cellular functions. Hence, the characterization of new RNA families with deep sequence and/or structure conservation has the potential to reveal new molecular and biological RNA functions. Here, we characterize the Clostridioides difficile 'raiA motif' RNA, which is conserved in approx. 2,500 bacterial species from the phyla Bacillota and Actinomycetota. We show that its transcript abundance and stability in exponentially growing bacteria rivals that of ribosomal RNAs. Deletion of the 'raiA motif' RNA is associated with delayed transition into stationary phase, and changes in stationary phase pathways such as spore formation, hence we renamed it ModT (modulator of transition phase). Mechanistically, we show that ModT-mediated changes in cellular cyclic di-GMP levels are linked to the pronounced sporulation defect in the modT mutant. ModT is produced in two isoforms that are largely identical in their secondary structure, but differ in their in vivo half-lives as well as their capacity to complement phenotypes associated with modT deletion. Importantly, we show that expression profiles and isoform patterns of ModT are conserved in C. perfringens and P. sordellii, and that these orthologs can functionally replace ModT in C. difficile. In summary, our findings indicate that ModT fulfills a conserved biochemical function in regulating growth transitions in bacteria, and provide a crucial step towards delineating the molecular function of its highly conserved tertiary structure. Overall design: To identify cellular functions, we deleted the first 215 nt of modT (?modT), corresponding to the conserved ModT consensus motif, from C. difficile 630 by homologous recombination and performed comparative transcriptome analysis of Wildtype and ?modT strains grown in TY medium supplemented with glucose. We collected samples in the exponential phase (3.5 hours post inoculation) and during the transition phase (9 hours post inoculation), when the knockout strain begins to outgrow the wild type.