Transcriptional interference by a complex formed at the centromere-like partition site of plasmid P1

The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for beta-galactosidase, lacZ. There was little interference with lacZ expression when ParA and ParB were supplied in trans. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down. ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription. Mutations in parA and parB that relieved the parAM314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par(PD)) similar to that of parAM314I. Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation. This "permanent" ParA-ParB-parS complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.