Reconstituting ParA/ParB-mediated transport of DNA cargo

Abstract

Protein gradients play key roles in subcellular spatial organization. In bacteria, ParA adenosine triphosphatases, or ATPases, form dynamic gradients on the nucleoid surface, which imparts positional information for the segregation, transport, and positioning of chromosomes, plasmids, and large protein assemblies. Despite the apparent simplicity of these minimal and self-organizing systems, the mechanism remains unclear. The small size of bacteria along with the number of physical and biochemical processes involved in subcellular organization makes it difficult to study these systems under controlled conditions in vivo. We developed a cell-free reconstitution technique that allows for the visualization of ParA-mediated cargo transport on a DNA carpet, which acts as a biomimetic of the nucleoid surface. Here, we present methods to express, purify, and visualize the dynamic properties of the SopABC system from F plasmid, considered a paradigm for the study of ParA-type systems. We hope similar cell-free studies will be used to address the biochemical and biophysical underpinnings of this ubiquitous transport scheme in bacteria.

MinE recruits, stabilizes, releases, and inhibits MinD interactions with membrane to drive oscillation

“…Flowcell Assembly. Flowcell assembly (Vecchiarelli et al, 2015) and bilayer coating with E. coli polar lipid extract or monounsaturated (18:1) synthetic lipids (Vecchiarelli et al, 2014) has been previously described. The synthetic lipid mixture was composed of 67% 1,2-dioleoyl-sn-glycero-3-phosphocholine (catalog no.…”

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

“…Flow cell assembly, pacification, and carpeting with sonicated salmon sperm DNA was done as previously described in detail (39).…”

“…Protein expression, purification, and labeling were performed as previously described in detail (39).…”

“…A biotinylated and Alexa 647-labeled DNA fragment (3.36 kb) containing a sopC centromere site was PCR amplified and coupled to MyOne Streptavidin C1 Dynal beads (Invitrogen) as previously described in detail (39).…”

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation

“…In the polymer model, ParB binding to ParA is postulated to cause polymer retraction with the concomitant "dragging" of ParB-DNA cargo in the polymer wake (Ringgaard et al 2009;Gerdes et al 2010). The diffusion ratchet model is based largely on in vitro reconstitution experiments with DNA curtains (Vecchiarelli et al 2010(Vecchiarelli et al , 2013a(Vecchiarelli et al ,b, 2014(Vecchiarelli et al , 2015Hwang et al 2013). According to this model, ParB-stimulated ParA-ATP hydrolysis leads to the dissociation of ParA dimers into monomers with the concomitant DNA dissociation by ParA.…”