Stable inheritance of the Streptomyces linear plasmid SCP1 by dual ParABS partition systems [SCP1_PCN]

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 searched a large and diverse plasmid database to identify the distribution of partition system types and found that plasmids encoding multiple partition systems are more widespread than previously realised, composing ~19% of all plasmids predicted to encode partition systems. Many of these plasmids were predicted to encode multiple partition systems of the same type, which had never been studied previously. 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), biochemistry and mutagenesis we show that both ParB proteins behave like bone fide ParB proteins: they bound to distinct parS sites on SCP1, both proteins bound and hydrolysed CTP and both proteins required CTP and their cognate parS site to accumulate, or spread, on DNA. Intriguingly, however, only ParB1 was critical for SCP1 stability. 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: To determine the plasmid copy number per chromosome of the two plasmids of Streptomyces coelicolor A3(2), the wild type strain was grown on solid soya flour mannitol (SFM) agar until visably sporeulated. Spores were harvested and the genomic DNA was extracted from these spores and sequenced in order to determine what the plasmid copy number was compared to the chromosome. Spores were chosen over vegetative mycelium as they are metabolically inactive and therefore should represent the true ratio of the plasmid copy number per spore, as during vegetative growth differences in the replication rate of these genetic elements could artifically inflate/deflate the true plasmid copy number ratio to the chromosome.