Phase-separated condensates direct selective targeting of xenogeneic silencers

Horizontal gene transfer enables rapid adaptation in prokaryotes but carries the risk of toxic gene expression. Bacterial histone-like proteins, exemplified by H-NS, bind AT-rich xenogenes and silence their expression. Conjugative plasmids autonomously transfer between bacteria, driving the spread of antibiotic resistance, but the transfer genes encode phage receptors and must be silenced. Plasmids frequently carry H-NS homologs, raising a crucial question: why can’t they use the host H-NS, which is abundant and promiscuous? We addressed this paradox by studying Escherichia coli H-NS and its homolog Sfx, encoded on the IncX2 plasmid R6K. Sfx reduces conjugation ~1000 fold, whereas H-NS has no effect, yet both proteins bind DNA similarly in vitro. We show that in E. coli, H-NS and Sfx are selectively targeted to the chromosome and R6K, respectively, even if identical DNA sequences are present. This specificity is underpinned by liquid-liquid phase separation. Our in vitro experiments show that Sfx forms condensates with R6K, whereas H-NS is excluded from these assemblies. This mechanism of selective clustering explains how promiscuous silencers can be precisely delivered to their intended targets and provides a paradigm for understanding other regulatory systems.