title: 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.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Vecchiarelli
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Taylor
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Mizuuchi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport / Q000502:physiology*; D002474:Cell-Free System; D019915:DNA Primase / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C541712:ParA protein, E coli; C489223:SopB protein, E coli; D019915:DNA Primase; C121791:dnaG protein, E coli; C060971:SopA protein, Bacteria
keywords: Bacterial chromosome segregation; Intracellular transport; ParA ATPase; Plasmid partition; Subcellular organization