A conserved structured non-coding RNA coordinates growth and virulence in Clostridioides difficile. [dRNA-Seq ModT]. 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 (ΔmodT), corresponding to the conserved ModT consensus motif, from C. difficile 630 by homologous recombination and performed comparative transcriptome analysis of Wildtype and ΔmodT 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.

细菌非编码RNA（bacterial non-coding RNAs）可发挥多种细胞功能，例如作为催化剂、多蛋白复合物的结构组分，或在转录及转录后水平调控基因表达。部分RNA在多个细菌门类中展现出异常广泛的保守性，并参与基础且独特的细胞生命过程。因此，鉴定具备深度序列和/或结构保守性的新型RNA家族，有望揭示新的分子与生物学层面的RNA功能。本研究对艰难梭菌（Clostridioides difficile）的‘raiA基序RNA（raiA motif RNA）’进行了系统表征，该RNA在厚壁菌门（Bacillota）与放线菌门（Actinomycetota）的约2500种细菌中均存在保守序列。研究发现，该RNA在指数生长期细菌中的转录本丰度与稳定性可与核糖体RNA（ribosomal RNAs）相媲美。敲除该‘raiA基序RNA’会导致细菌延迟进入稳定期，并改变孢子形成等稳定期相关通路，因此我们将其重命名为ModT（过渡期调节因子，modulator of transition phase）。机制层面，我们证实ModT介导的细胞内环二鸟苷酸（cyclic di-GMP）水平变化与modT突变体显著的孢子形成缺陷密切相关。ModT存在两种同工型（isoforms），二者二级结构基本一致，但体内半衰期以及互补modT缺失相关表型的能力存在差异。值得注意的是，我们发现ModT的表达谱与同工型模式在产气荚膜梭菌（C. perfringens）与索氏梭菌（P. sordellii）中保守，且这些直系同源基因（orthologs）可在艰难梭菌中功能替代ModT。综上，本研究结果表明ModT在细菌生长过渡期的调控中发挥保守的生化功能，为解析其高度保守的三级结构的分子功能提供了关键进展。实验整体设计：为鉴定其细胞功能，我们通过同源重组从艰难梭菌630菌株中敲除modT基因的前215个核苷酸（ΔmodT），该区域对应保守的ModT共有基序；随后对在添加葡萄糖的TY培养基（TY medium）中培养的野生型与ΔmodT菌株开展比较转录组分析。分别在指数生长期（接种后3.5小时）与过渡期（接种后9小时，此时敲除菌株开始表现出生长优势超过野生型）收集样本。