Swarm-seq Reveals Functional Divergence in CsrA Homologs Across Bacteria.

Bacteria employ sophisticated post-transcriptional regulatory mechanisms to adapt to environmental changes. Carbon storage regulator A (CsrA), a highly conserved small RNA-binding protein, serves as a critical post-transcriptional regulator by recognizing GGA motifs in target transcripts and typically repressing translation. Despite extensive research, how this conserved regulator has evolved diverse species-specific regulatory networks remains unclear. Here, using as an experimental chassis, we have developed Swarm-seq - a high-throughput functional genomics platform capable of assessing CsrA homologs across the Bacterial domain for their ability to regulate flagella-dependent swarming motility. Systematically testing over five-hundred codon-optimized homologs for flagellin (Hag) repression revealed striking functional divergence, i.e., broadly two classes of CsrAs. Class I, exemplified by CsrAHp from and RsmN from strongly inhibited swarming, while Class II, exemplified by CsrAEc from and RsmA from failed despite high sequence conservation. This differential activity occurred despite all proteins targeting identical GGA motifs in the transcript's 5'UTR, indicating evolutionary plasticity in RNA-binding specificity beyond motif recognition. We propose that this plasticity enables a conserved global regulator to develop species-specific regulons through subtle structural adaptations, facilitating diverse physiological responses across bacterial taxa. Our findings establish as a powerful platform for characterizing CsrA homologs from genetically intractable bacteria, providing insights into post-transcriptional regulatory network evolution.