Functional characterisation of the RaiA noncoding RNA in Clostridioides difficile

In all bacteria there are non-coding RNAs (ncRNAs) that fulfill a variety of cellular functions, e.g., as catalysts of chemical reactions, as structural components in multiprotein complexes or to control gene expression at the transcriptional and post-transcriptional level. Some RNA regulators display exceptionally broad conservation across bacterial phyla, and these fulfill fundamental cellular functions, such as the ribosomal 5S, 16S and 23S RNAs. Using RNA-seq based transcriptome annotation in C. difficile, we recently observed the expression of a ncRNA that belongs to a family of uncharacterized RNA motifs with complex secondary structure and high conservation across two bacterial phyla, which is termed RaiA (1, 2).

The structural complexity and high conservation point towards a sophisticated biochemical function and an important physiological role, which motivated us to perform a detailed characterization in C. difficile.

To determine the physiological relevance, we created raiA deletion mutants that display an overgrowth phenotype that is particularly pronounced upon addition of monosaccharides. In line with the growth phenotype, transcriptome analyses reveal that deletion of raiA alters expression of many amino acid and carbohydrate transport genes and elevates the levels of many tRNAs during stationary phase. We find that a raiA mutant shows strongly suppressed sporulation while producing elevated levels of toxins, directly implicating RaiA in C. difficile virulence. Complementation of the C. difficile raiA mutant with orthologues from different Firmicutes families restored sporulation and toxin production to wild type levels, indicating that the ncRNA function is preserved across species. In summary, we have established RaiA as substantial regulator of physiology in C. difficile.

(1) Lamm-Schmidt et al., (2021) Microlife

(2) Weinberg et al., (2017) NAR