Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

Abstract

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.

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

“…1 B and C) due to dissociation of ParA from the DNA after ATP hydrolysis stimulated by the ParB-rich cargo. These dynamics and the time scale of the oscillation period are in good agreement with what has been reported for single-plasmid cargo in E. coli (18,23,24). As in experiments (23), the oscillatory motion of individual cargos was not perfectly periodic (Fig.…”

“…Multiple copies of ParB bind to the cargo and stimulate the ATPase activity of ParA upon interaction. Imaging of fluorescently tagged protein fusions has revealed remarkable correlated localization patterns between ParA and the ParB-rich cargo over the nucleoid region, with the cargo moving in the wake of a "ParA wave" (18)(19)(20)(21)(22)(23). In the case of a single-plasmid cargo, the correlated localization dynamics of ParA and ParB results in oscillatory motion of both the cargo and ParA wave over the nucleoid (18,23,24).…”

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

“…"Smooth" translocation, where the plasmid continuously moves close to the receding edge of a ParA (or SopA) depletion zone on the nucleoid, has been observed in vivo for pB171 plasmid and F plasmid (33). More recently, both plasmids have also been shown to support saltatory movements, whereby the plasmids are for the most part immobile and uniformly distributed at relatively fixed positions on the nucleoid (34). For P1 plasmid, ParA distributes uniformly over the nucleoid and also forms colocalized foci with immobile plasmids (16).…”

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

“…Some of these [11,14] failed to observe a stable equipositioning regime because ParA-slow was not allowed to diffuse (D 2 = 0): thus α diverges, setting the system in the unstable regime. In [14], relative positioning occurs only with multiple cargos as a crowding effect, whereas it is known that positioning can occur even with a single plasmid [22], as predicted by certain modeling studies [12,18]. 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.…”