Abstract
In Firmicutes, small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP)2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP)2 requires ATP binding but not hydrolysis to localize onto the cell’s nucleoid as a fluorescent cloud. The interaction of (δ:CFP)2 or δ2 bound to the nucleoid with (ω:YFP)2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP)2, leading to the dynamic release of (δ:GFP)2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP)2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP)2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP)2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP)2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.
Highlights
In eukaryotes, much insight has been gained into how chromosomes are segregated
We report here that in vivo (ω:YFP)2 binding to a plasmid-borne parS site causes discrete clustering of plasmid copies and that (δ:GFP)2 bound to the Bacillus subtilis genome forms dynamic clouds over the nucleoid
When δ2 was replaced by2 plasmids were randomly segregated (Fig 2). These results suggest that the C-terminal fusion does not affect the activity of δ2, and that ATP hydrolysis is essential for plasmid segregation
Summary
Much insight has been gained into how chromosomes are segregated. The ParAB partition system, which is the only type present in bacteria, is the most widespread system among low-copy number plasmids. This system relies on four components: ParA and ParB proteins, cis-acting plasmid-borne parS DNA and the host genome [1,2]. In general the ParA and ParB proteins are subdivided in two PLOS ONE | DOI:10.1371/journal.pone.0131943. In general the ParA and ParB proteins are subdivided in two PLOS ONE | DOI:10.1371/journal.pone.0131943 July 10, 2015
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