Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon [ChIP-Seq]
Conjugative plasmids drive bacterial evolution and the spread of antibiotic resistance, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that despite sharing similar DNA motifs and a preference for negative supercoiling, Sfx and H-NS occupy distinct niches: Sfx binds weakly to the chromosome but is enriched on the R6K vir operon while H-NS is excluded. We hypothesize that this selective targeting is mediated by Sfx-vir DNA interactions and phase separation. We show that Sfx binding to vir DNA critically depends on DNA topology but not on the target location. Our results suggest that Sfx forms stable nucleoprotein filaments that are impermeable to competitors and phase separates with R6K DNA to exclude H-NS. Our results suggest how functionally distinct histone-like proteins may achieve target specificity despite broad recognition motifs. Overall design: ChIP-seq assays for comparing the binding sites of H-NS and its homolog Sfx encoded on R6K plasmid in Escherichia coli. The sequences of all plasmids used in this study can be found in the accompanying manuscript. ChIP-seq of RNA polymerase in E. coli carrying the Sfx deletion R6K plasmid to identify potential promoters in R6K. RNA ChIP-seq assay to identify RNAs ChIPed by FLAG-tagged Sfx.