Global identification of RNA-binding proteins and RNA-binding domains in methicillin-resistant Staphylococcus aureus

Using improved and robust biochemical methods, we present the first global analysis of RNA-binding proteins (RBPs) and RNA-binding domains (RBDs) in clinically relevant and multi-drug resistant Gram-positive bacteria. To validate our results in silico, we developed novel bioinformatics tools that compare RBDome data with ligand-binding site predictions generated by five different algorithms on a large number of S. aureus crystal structures. This revealed that the putative RBDs are highly enriched for predicted RNA-binding sites and basic and aliphatic amino acids, demonstrating the robustness of our approach. Surprisingly, we found that HTH-type DNA-binding and NAD and P-loop type Rossmann-fold proteins may also play a prominent role in post-transcriptional regulation in Gram-positive bacteria and we identified a common mode of RNA recognition for these domains. Subsequent in vivo validation studies showed that the HTH-type transcription factor CcpA, a master regulator of carbon metabolism in Gram-positive bacteria, is also a global post-transcriptional regulator and binds its RNA substrates at very specific distances from transcription terminators. Our novel experimental and computations tools are widely applicable, and our work provides an extremely valuable resource for groups studying post-transcriptional regulation and RNA-binding proteins in bacteria. Overall design: Three biological replicates with three experimental replicates each were used to generate the RNA-seq Data. RNA seq Data experiments were performed with the parental USA300 LAC strain, the ccpA gene null mutant and the ccpA deletion mutant complemented with CcpA expressed from the pCN33 plasmid. S. aureus strains were grown in TSB medium until the post-exponential phase. 10 mL of cells at an OD600 of ~3.0 were harvested by rapid centrifugation and flash-frozen. Total RNA was then extracted through acid guanidinium thiocyanate-phenol-chloroform extraction protocol.