The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF

Abstract

The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that are preferentially localized to the replication origin region (ori), close to the long axis of the cell. A replication-dependent increase in the fraction of immobile molecules, together with a proposed catalytic cycle of ~1.8 s, is consistent with the majority of active TopoIV molecules catalyzing decatenation, with a minority maintaining steady-state DNA supercoiling. Finally, we show that the MukB-ParC interaction is crucial for timely decatenation and segregation of newly replicated ori DNA.

Graphical abstract

Figure 1

Tracking PALM of E. coli ParC/E Molecules (A) Example image of a single ParC-PAmCherry molecule at 15 ms exposure (left), super-resolved localizations derived from all frames and for all molecules detected in this cell (middle), and example tracks of individual slow ParC (blue) and immobile ParC (red) molecules (right). Scale bar, 1 μm. (B) Distribution of apparent diffusion coefficients (D^∗) of 73,020 tracked ParC molecules, fitted with a two-species model. Ranges indicate 95% confidence interval. Example cell with individual trajectories colored according to their D^∗ value. (C) Distribution of D^∗ values for 64,551 ParE molecules fitted with a three-species model. Copy numbers of ParC and ParE subunits, normalized for cells 2.5 μm long, were determined by sequentially photoactivating and tracking all available molecules.

Figure 2

MukBEF Clusters Influence TopoIV Diffusion and Organization (A) Left panels: distribution of D^∗ values for ParC molecules fitted with a two-species model with immobile molecules (constrained at D_imm = 0.11 μm²s⁻¹) and slow-moving molecules (constrained at D_slow = 0.35 μm²s⁻¹). Top: ParC molecules in wild-type cells (from Figure 1B). Middle: 18,971 ParC molecules in ΔmukB cells. Bottom: 42,920 ParC molecules after unlabeled ParC-CTD overexpression (3 hr). Ranges give 95% confidence intervals. Right panels: the number of ParC clusters per cell, determined by clustering all localizations using a nearest-neighbor algorithm, in wild-type (2,635 cells) and ΔmukB (387 cells) cells and with ParC-CTD overexpression (214 cells). Error bars indicate SD of three experimental repeats. (B) Example cell with MukB-mYPet foci (top) visualized prior to PALM acquisition and localization of ParC-PAmCherry molecules (middle). Radial distribution of ParC localizations from each MukB focus (717 cells), compared to random distribution (bottom). The radial distribution function shows the probability of finding a ParC localization at distance, r, from a MukB focus. Gray bar shows localization within 200 nm.

Figure 3

MukB-Dependent and Independent ParC Binding Behavior (A) Examples of long ParC trajectories (ten or more localizations) classified according to their D^∗ transitions. Molecules mobile over observation period (blue), immobile (red), and undergoing transition from one state to another (purple). (B) Bar graph of all ParC trajectories for the indicated strains, classified from PALM experiments performed at 15-ms continuous acquisition and time lapse (15-ms exposure + 35-ms delay). (C) Schematic of Markov chain Monte Carlo simulations of molecules inside a typically sized cell volume interconverting between immobile and free diffusion. Cartoon representation of transitions analyzed in simulations. Shown is the time range obtained in simulations that recapitulated the experimental data. (D) Left: example 750-ms exposure frames showing cells with an immobile TopoIV molecule (top) and a mobile molecule (bottom). Right: on-time distributions for immobile ParC with exponential fit (line) and photobleaching-corrected binding time distribution (line with dots). Error bars indicate SD of three experimental repeats.

Figure 4

Catalytically Active TopoIV (A) Left panels: distribution of D^∗ values for ParC molecules in wild-type (387 cells) and ΔmukB (214 cells) cells after ∼10-min treatment with norfloxacin. Control ParC molecules in untreated cells (top, from Figure 1B). Right panels: number of ParC clusters per cell for steady-state populations of cells. Error bars indicate SD of three experimental repeats. Ranges give 95% confidence intervals. (B) Radial distribution of ParC localizations from each MukB focus in cells treated with norfloxacin (726 cells), compared to random distribution. (C) Distribution of D^∗ values for 1,930 ParC molecules in non-replicating cells, as assessed by lack of mYPet-DnaN foci prior to PALM data acquisition. Distributions of D^∗ were fitted with a two-species model with both D values constrained.

Figure 5

The MukB-ParC Interaction Stimulates ori Decatenation (A) Example cells from the time-lapse experiments with wild-type cells transformed with empty expression plasmid (pBAD24). Black arrows show time of ori1 segregation. 0 min time was defined by replisome appearance at ori1. (B) Example cell showing ParC-CTD 3-hr overexpression. (C) Cumulative distribution of times of ori1 locus segregation after replication initiation, marked by appearance of mYPet-DnaN foci at ori1. (D) Snapshot analysis of the number of ori1 foci/cell in steady-state cells. Mean ± SD of three independent experiments (>1,000 cells).