Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development in <i>Pseudomonas putida</i>

Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer. ICE transfer is initiated in a small subpopulation of cells that leads to transfer competence. Here we study the role of the ParB-like protein BisD for ICEclc transfer competence formation in Pseudomonas putida. Genetic dissection and single cell microscopy showed that mCherry-BisD fusion proteins form fluorescent foci on parS-like DNA sequences on ICEclc. Chromatin immunoprecipitation followed by DNA sequencing showed that BisD accumulates at two bisS sites, and further enriches around 11 ICEclc transfer competence promoters. BisD enrichments suggest its sliding over ca. 50 kb of ICE-DNA in an asymmetric manner, following the direction of inwards ICE gene transcription. BisD has a unique domain absent from ParB, which is essential for ICE promoter activation in conjunction with the ICEclc encoded factor BisC, and for ICE transfer. Similar to ParB, purified BisD preferentially binds and hydrolyses cytidine triphosphate (CTP). Mutation of a conserved Arg- at position 95 to an Ala-residue abolished nucleotide binding by BisD, and also inhibited ICE promoter activation and transfer. BisD action might thus serve two purposes, orchestrating ICE gene activation and ICE-DNA clamping.