Grad-seq identifies KhpB as a global RNA-binding protein in that regulates toxin production.

Much of our current knowledge about cellular RNA-protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA-protein complexes to a major Gram-positive human pathogen, , whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA-protein and protein-protein complexes in and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in , as an uncharacterized, pervasive RBP in . Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in . The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a deletion strain. Moreover, the production of toxin protein is also increased upon deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.