Stochastic Self-Assembly of ParB Proteins Builds the Bacterial DNA Segregation Apparatus

Abstract

Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS. This assembly model and approach to characterizing large-scale, dynamic interactions between macromolecules may be generalizable to many unrelated machineries that self-assemble in superstructures.

DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos

“…We did not consider spontaneous (without ATP hydrolysis) dissociation between ParA dimers and the ParB-rich cargo because it has been shown to be very slow (>10 min) (33). The size of a ParA dimer and ParB-rich cargo were set to 4 and 100 nm, respectively, to match values reported elsewhere (31,44).…”

ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres

“…Spreading is an unusual feature for site-specific DNA binding proteins that is common to HTH ParBs, and it reflects how ParB assembles into higher-order complexes (Rodionov et al, 1999; Murray et al, 2006; Breier and Grossman, 2007; Sanchez et al, 2015). Measured by ChIP approaches, in vivo ParB binding extends beyond parS into the surrounding non-specific DNA, often many kb away from the site.…”

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

“…Our work is fully inspired by studies of one of the most widespread and ancient mechanisms of liquid phase macromolecular segregation and positioning known in nature: bacterial DNA segregation systems. Despite the fundamental importance of these systems in the bacterial world and intensive experimental studies extending over 30-years [3][4][5], no global picture encompasses fully the experimental observations. Partition systems encode only three elements that are necessary and sufficient for active partitioning: two proteins ParA and ParB, and a specific sequence parS encoded on DNA.…”

“…These two species are coupled via a system of reaction-diffusion equations: the rapid species u converts into the slow one with a constant rate k 1 , while the slow species v is hydrolysed in the presence of the ParBS partition complexes located on DNA, with a rate k 2 (typically k 1 ≈ 0.02 s −1 [9] and k 2 ≈ 68.5 s −1 [12]). The ParBS assemblies form 3D-foci complexes [4] and interact with the ParA-slow proteins. The interaction probability is described by the profiles S(r − r i (t)) centered around the ParBS positions r i (t).…”

“…In line with the most recent experimental findings [5], we assume that partition complexes evolve within the nucleoid volume near the axis of the rodshaped bacterial cells, in contrast with the translocation surface mechanism presented in [8][9][10][11] performed on large surfaces coated by ParA, lacking the confinement necessary for equipositioning. Our proposed mechanism integrates explicitly a volumetric interaction [4] with the partition complex (i.e. a length in 1D), placing the system close to the stability threshold for the biological range of parameters.…”