Stable inheritance of the Streptomyces linear plasmid SCP1 by dual ParABS partition systems [ChIP-Seq]

Low copy number plasmids must encode maintenance mechanisms, such as partitioning systems, to ensure that the plasmid is sustained through host generations. Plasmid partition systems segregate sister plasmid copies and are subdivided into different types based on the NTPase they encode. The characterisation and distribution of partition system types is well understood in Enterobacteriaceae plasmids. However, how these systems maintain plasmids and are distributed across wider bacterial diversity is poorly understood. We used the Streptomyces coelicolor A3(2) plasmid SCP1, which encodes two type Ia partition systems, as a model to investigate this. Sequence analysis of the SCP1 partition systems revealed that both ParB proteins contain less conserved CTP-binding pockets, suggesting one or both proteins may not behave like canonical ParB proteins. However, using a combination of chromatin immunoprecipitation with deep sequencing (ChIP-seq) we demonstrate that both the SCP1 ParB proteins, ParB1 and ParB2, bound to distinct parS sites on SCP1, and accumulate, or spread, on DNA approximately 20 kb away from their initial parS loading site. Together, our findings further our understanding of Streptomyces plasmid maintenance by providing the first functional characterisation of two type Ia partition systems coexisting on a single plasmid and offer new insights into the diversity and distribution of plasmid partition systems. Overall design: ChIP-seq experiment to determine the DNA-binding sites of the ParB1 and ParB2 proteins of the linear plasmid SCP1 in its host, Streptomyces coelicolor A3(2). ParB1-3xFLAG and ParB2-3xFLAG were constructed by inserting 3x-FLAG tags at the 3' ends of the parB1 and parB2 genes at their native loci on the SCP1 plasmid, allowing for detection of the FLAG-tagged proteins under the production of their native promoters using the ReDirect gene editing method which inserts the tag and an apramycin resistance casette.Non-tagged alleles were included as negative controls. Two biological replicates were carried out per strain. Anti-FLAG agarose beads were used for the ChIP experiment.