ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes

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.

The interaction of ω2with the RNA polymerase β’ subunit functions as an activation to repression switch

“…Similar results were observed when the P δ -lacZ fusion was replaced by the P ω -lacZ fusion (data not shown). Furthermore, plasmid-borne ω2 and δ genes which were transcribed from P ω ( parS 2) and P δ ( parS 1) (Figure 1A ), were necessary and sufficient for stabilizing of an otherwise unstable plasmid in B. subtilis cells ( 38 , our unpublished results). It is likely that: (i) the dimer is the functional unit of ω2; (ii) ω 2 2 interacts with δ 2 just like ω 2 ; and (iii) the different C-terminal domains of ω and ω2 are not involved in gene repression.…”

Brownian ratchet mechanisms of ParA-mediated partitioning

“…The DNA partition system in C. crescentus might be an atypical case, as it has been shown to function with much fewer ParA molecules inside the cell compared to typical plasmid DNA partition systems (6, 20–22). Furthermore, a uni-polar ParA concentration gradient in C. crescentus is present at the onset of segregation (6).…”

Subcellular Organization: A Critical Feature of Bacterial Cell Replication

“…Equidistant distribution of component copies along the long axis of the cell will also ensure equal inheritance between daughter cells (Figure 3Aiii). Such spatial patterning has been reported for low-copy-number plasmids (Adachi et al, 2006; Ebersbach et al, 2006; Lioy et al, 2015; Sengupta et al, 2010), storage granules in C. crescentus, Pseudomonas aeruginosa and Pseudomonas putida (Galan et al, 2011; Henry and Crosson, 2013; Racki et al, 2017), cytoplasmic chemotaxis protein clusters in Rhodobacter sphaeroides (Thompson et al, 2006), and carboxysomes (protein organelles for carbon fixation) in Synechococcus elongatus (Savage et al, 2010).…”