CsrA of Bacillus subtilis regulates translation initiation of the gene encoding the flagellin protein (hag) by blocking ribosome

The global regulatory Csr (carbon storage regulator) and the homologous Rsm (repressor of secondary metabolites) systems of Gram-negative bacteria typically consist of an RNA-binding protein (CsrA/RsmA) and at least one sRNA that functions as a CsrA antagonist. CsrA modulates gene expression post-transcriptionally by regulating translation initiation and/or mRNA stability of target transcripts. While Csr has been extensively studied in Gram-negative bacteria, until now Csr has not been characterized in any Gram-positive organism. csrA of Bacillus subtilis is the last gene of a flagellum biosynthetic operon. In addition to the previously identified sigma(D)-dependent promoter that controls expression of the entire operon, a sigma(A)-dependent promoter was identified that temporally controls expression of the last two genes of the operon (fliW-csrA); expression peaks 1 h after cell growth deviates from exponential phase. hag, the gene encoding flagellin, was identified as a CsrA-regulated gene. CsrA was found to repress hag'-'lacZ expression, while overexpression of csrA reduces cell motility. In vitro binding studies identified two CsrA binding sites in the hag leader transcript, one of which overlaps the hag Shine-Dalgarno sequence. Toeprint and cell-free translation studies demonstrate that bound CsrA prevents ribosome binding to the hag transcript, thereby inhibiting translation initiation and Hag synthesis.