RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

A computational approach for the identification of distant homologs of bacterial riboswitches based on inverse RNA folding.

Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches. We developed a novel approach based on inverse RNA folding that attempts to find sequences that match the shape of the target structure with minimal sequence conservation based on key nucleotides that interact directly with the ligand. Then, to support our matched candidates, we expanded the results into a covariance model representing similar sequences preserving the structure. Our method transforms a structure-based search into a sequence-based search that considers the conservation of secondary structure shape and ligand-binding residues. This method enables us to identify a potential structural candidate in fungi that could be the distant homolog of bacterial purine riboswitches. Further, phylogenomic analysis and evolutionary distribution of this structural candidate indicate that the most likely point of origin of this structural candidate in these organisms is associated with the loss of traditional purine riboswitches. The computational approach could be applicable to other domains and problems in RNA research.