Contact with the CsrA core is required for allosteric inhibition by FliW in Bacillus subtilis

The RNA-binding protein CsrA is a post-transcriptional regulator that is encoded in genomes throughout the bacterial phylogeny. In the gamma-proteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding.  In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, that non-competitively inhibits CsrA activity but the precise mechanism of antagonism is unclear. Here we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition that fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues that surround the critical regulatory CsrA residue N55 and abolished CsrA interaction. Two loss-of-function alleles however mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA.  One of these two alleles reversed charge at what appeared to be a salt bridge with the CsrA core region, charge reversal of the CsrA partner residue phenocopied the FliW allele, and charge reversal of both residues simultaneously restored antagonism.  We propose a model in which initial interaction between FliW and CsrA is necessary but not sufficient for antagonism which also requires salt bridge formation with, and deformation of, the CsrA core domain to allosterically abolish RNA binding activity.