Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism

Abstract

Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC. In vivo, SopA ATPase forms dynamic patterns on the nucleoid in the presence of the ATPase stimulator, SopB, which binds to the sopC site on the plasmid, demarcating it as the cargo. To understand the relationship between nucleoid patterning and plasmid transport, we established a cell-free system to study plasmid partition reactions in a DNA-carpeted flowcell. We observed depletion zones of the partition ATPase on the DNA carpet surrounding partition complexes. The findings favor a diffusion-ratchet model for plasmid motion whereby partition complexes create an ATPase concentration gradient and then climb up this gradient toward higher concentrations of the ATPase. Here, we report on the dynamic properties of the Sop system on a DNA-carpet substrate, which further support the proposed diffusion-ratchet mechanism.bacterial chromosome segregation | ParA ATPase | plasmid segregation | spatial pattern organization | chromosome dynamics P roper DNA segregation ensures the faithful inheritance of genomic information for all life forms. In bacteria, this fundamental process is poorly understood. Low-copy bacterial genomes, including plasmids and chromosomes, encode active partition (Par) systems to ensure stability. Par systems are minimalistic in that only three dedicated components are required: a partition site on the DNA, a partition site-binding protein, and a nucleoside triphosphatase (NTPase). Par systems have been classified according to the type of NTPase involved: Walker-type (generically called "ParA"), actin-like, or tubulin-like (reviewed in ref. 1). Reconstitution of purified Par components of R1 plasmid in a cell-free system unveiled the mechanism involving an actin-like ATPase, ParM, in which elongating filaments of the ATPase push plasmids to opposite cell poles (2). Tubulin-like GTPases also appear to function as a filament (3). However, all chromosome-based and most plasmid-based systems use ParAs, and mounting evidence shows that ParA-like ATPases also are responsible for transporting large protein machineries (reviewed in ref. 4). However, the underlying mechanism for reactions of this category remains unresolved.The Sop system (stability of plasmid) of F plasmid is one of the first Par systems to be identified (5, 6) and is considered a paradigm for the study of ParA-mediated DNA segregation. The three plasmid-encoded system components are SopA (the ParAtype ATPase), SopB (or ParB in other systems; i.e., the partition site-binding protein), and sopC (or parS in other systems; i.e., the cis-acting partition site on the plasmid). The first task of a partition system is to identify its DNA cargo. SopB accomplishes this task by loading onto sopC and forming a partition c...

A propagating ATPase gradient drives transport of surface-confined cellular cargo

“…The filament-pulling model is reminiscent of eukaryotic mitosis whereby partition complexes are mobilized by helical (8) or linear (9) contractile filaments composed of the ParA ATPase. The diffusionratchet model, however, proposes that ParA dimers, or small oligomers, independently bind the nucleoid and form concentration gradients in the vicinity of the partition complex, which generates the driving force for cargo movement (6,10,11). Here, we extend this model further by showing that a pulling force on the plasmid cargo can be mechanochemically coupled to the ParA concentration gradient on the nucleoid.…”

“…The ParA ATPase of F plasmid, SopA, is stimulated by SopB that assembles into a partition complex on the centromere-like locus, sopC, on the plasmid cargo (3,4). SopA binds DNA nonspecifically in the presence of ATP and colocalizes with the nucleoid in vivo (5,6). The partition complex locally removes SopA, forming a SopA depletion zone on the nucleoid in the vicinity of the plasmid (7), but how this patterning on the nucleoid results in cargo transport is a subject of considerable debate.…”

Surfing on Protein Waves: Proteophoresis as a Mechanism for Bacterial Genome Partitioning

“…In most cases, these models require other assumptions -such as DNA elasticity [13,14] -as simple reaction-diffusion mechanisms are not sufficient to predict proper positioning. Other reaction-diffusion models considered the dynamics of the partition complex on the surface of the nucleoid [8][9][10][11]. Recent experiments, however, demonstrate that partition complexes and ParA translocate through the interior of the nucleoid, not at its surface [5].…”

“…This process entails the oscillation of ParA from pole to pole and the separation of the ParBS partition complex into two complexes with distinct sub-cellular trajectories and long-term localization. Overall, these interactions result in an equidistant, stable positioning of the duplicated DNA molecules along the cell axis.The specific modeling of ParABS systems falls into two categories: either "filament" (pushing/pulling the cargos, similar to eukaryotic spindle apparatus [3]) or reaction-diffusion models [8][9][10][11][12][13][14][15]. Recent superresolution microscopy experiments have been unable to observe filamentous structures of ParA [5,13], disfavoring polymerization-based models [12].…”

“…The pool of ParB proteins is recruited as a cluster of spherical shape centered around the sequence parS, forming the ParBS partition complex [4]. These ParBS cargos interact with ParA bound onto chromosomal DNA (ParA-slow) [6,7], triggering unbinding of ParA by inducing conformational changes through stimulation of adenosine triphosphate (ATP) hydrolysis and/or direct ParB-ParA contact [8], and thereby allowing ParA diffusion in the cytoplasm (ParA-fast) [5]. This process entails the oscillation of ParA from pole to pole and the separation of the ParBS partition complex into two complexes with distinct sub-cellular trajectories and long-term localization.…”