The Slow Mobility of the ParA Partitioning Protein Underlies Its Steady-State Patterning in Caulobacter

Abstract

In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus. Transport of the ParB/parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus, the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers (D ∼10(-3)μm(2)/s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

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

“…This dynamic ParA gradient is distinct from the special case of the steady-state gradient of ParA that is maintained without cargo motion throughout the G1 phase of C. crescentus during which the cargo is physically anchored to a cell pole (20-22, 41, 42 from rare spontaneous (without ATP hydrolysis) dissociation of ParA dimers from DNA, requires minutes to develop (47). This time is too long for this mechanism to play a significant role during cargo translocation, as the cargo crosses the entire nucleoid on this time scale.…”

Brownian ratchet mechanisms of ParA-mediated partitioning

“…The slow rate of dissociated ParA resetting its DNA-binding capability maintains a ParA-depleted zone behind the moving cargo (17). Restricted diffusion of ParA dimers on the DNA substrate due to inter-segmental transfer, or “hopping”, also plays a role in the delayed rebinding behind the cargo (2, 18). The asymmetric ParA distribution, either pre-existing or resulting from cargo movement, perpetuates the forward movement of the cargo.…”

“…For the resulting DNA-tethered ParA dimers, which is essential for the “relay”, their lateral diffusion constant is assumed to be ~ 0.01 μm 2 /sec at the time resolution of 1 millisecond (6), or measured to be ~ 0.001 μm 2 /sec (18). If the DNA tether in the DNA-relay model is too weak to impact the intrinsic diffusive mobility of its cargo, i.e.…”

“…, the chromosome loci, then it will not quench the lateral diffusion constant of the ParA dimer, as the ParA dimer is much smaller in size and hence more diffusive than chromosome loci. However, experiments from the same group of researchers show that the intrinsic diffusion of ParA dimers, while not binding to DNA, is ~ 3 μm 2 /sec (18); 300 – 3000 fold faster than that of the DNA-tethered ParA dimers (6, 18). This suggests that the DNA tether is in fact much more stiffer than that assumed in the DNA-relay model, and can effectively quench cargo diffusion, as predicted in the mechanochemical Brownian ratchet model (14, 16).…”

Molecular architecture of the DNA‐binding sites of the P‐loop ATPases MipZ and ParA fromCaulobacter crescentus

“…Various P-loop ATPases were shown to interact with the nucleoid to slow down their diffusion and thus enable the maintenance of subcellular protein gradients (13,18,19,34). Their DNA-binding activity was suggested to be mediated by positively charged amino acids that are exposed on their surface (4,20,35,36).…”