The SMC complex MukBEF recruits topoisomerase IV to the origin of replication region in live Escherichia coli

Abstract

The Escherichia coli structural maintenance of chromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interact in vitro through a direct contact between the MukB dimerization hinge and the C-terminal domain of ParC, the catalytic subunit of TopoIV. The interaction stimulates catalysis by TopoIV in vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV to ori, with fluorescent fusions of ParC and ParE both forming cellular foci that colocalize with those formed by MukBEF throughout the cell cycle and in cells unable to initiate DNA replication. Removal of MukBEF leads to loss of fluorescent ParC/ParE foci. In the absence of functional TopoIV, MukBEF forms multiple foci that are distributed uniformly throughout the nucleoid, whereas multiple catenated oris cluster at midcell. Once functional TopoIV is restored, the decatenated oris segregate to positions that are largely coincident with the MukBEF foci, thereby providing support for a mechanism by which MukBEF acts in chromosome segregation by positioning newly replicated and decatenated oris. Additional evidence for such a mechanism comes from the observation that in TopoIV-positive (TopoIV(+)) cells, newly replicated oris segregate rapidly to the positions of MukBEF foci. Taken together, the data implicate MukBEF as a key component of the DNA segregation process by acting in concert with TopoIV to promote decatenation and positioning of newly replicated oris.

Importance: Mechanistic understanding of how newly replicated bacterial chromosomes are segregated prior to cell division is incomplete. In this work, we provide in vivo experimental support for the view that topoisomerase IV (TopoIV), which decatenates newly replicated sister duplexes as a prelude to successful segregation, is directed to the replication origin region of the Escherichia coli chromosome by the SMC (structural maintenance of chromosome) complex, MukBEF. We provide in vivo data that support the demonstration in vitro that the MukB interaction with TopoIV stimulates catalysis by TopoIV. Finally, we show that MukBEF directs the normal positioning of sister origins after their replication and during their segregation. Overall, the data support models in which the coordinate and sequential action of TopoIV and MukBEF plays an important role during bacterial chromosome segregation.

FIG 1

Association of TopoIV subunits ParC and ParE with MukBEF in vivo. (A) Representative examples of colocalization events between MukB-mCherry and ParC/E-mYPet proteins within E. coli cells. The origin of replication region was identified by the binding of a TetR-CFP protein on a tetO array (ori1) located 15 kb CCW of the replication origin, oriC. The fluorescence profiles of the individual cells show the distribution of fluorescence intensities. Exponential-phase cells were grown in M9-glycerol medium at 30°C prior to imaging. Cells without replication are dnaC(Ts) strains grown at the restrictive temperature for 120 min. Bars, 2 µm. (B) In order to localize the fluorescent foci corresponding to MukB and ParC/E proteins or the ori1 DNA locus within cells, microscopy images were first analyzed using the MicrobeTracker Suite (http://microbetracker.org/) to detect and outline bacterial cells. Cumulative distributions present the distances between the centroids of MukB foci and the brightest ParC/E pixels (Materials and Methods). Colocalization (gray shaded rectangle) is defined as when the MukBEF focus centroid is 4 or less pixels (516 nm) from the brightest ParC/E pixel. (C) The percentages of colocalization between MukBEF and ParC/E were plotted in a histogram. Error bars represent the 95% confidence interval. Asterisks indicate that the measured values (meas.) are statistically significantly different from the random calculated values (rand.) (Materials and Methods).

FIG 2

MukB is necessary for TopoIV localization at the origin of replication. (A) Colocalization frequencies (percentages of colocalization determined at a 4-pixel distance) between ori1 versus MukB and ori1 versus ParC were recorded during depletion of the MukE protein. Conditions in the absence or presence of l-arabinose (l-Ara) were plotted. The corresponding random curves were also plotted and show that the measured distances are getting closer to the random positioning during the time course of the experiment. Two representative examples of cells after 180 min of MukE depletion are shown. The values in the 95% confidence interval (95% conf. int.) are shown by purple or green shading. Bars, 2 µm. (B) Cumulative curves of the pairwise distances between ori1 and MukB during the time course of MukE depletion. fract., fraction. (C) Cumulative curves of the pairwise distances between ori1 and ParC during the time course of MukE depletion. In panels B and C, the percentages of colocalization are plotted in the histograms below the graphs (as in Fig. 1C).

FIG 3

ParC depletion leads to a dispersion of MukB foci. ParC protein was fused to a degron tag. Upon addition of l-arabinose, ParC was efficiently degraded in less than 1 h (see Fig. S4 in the supplemental material). Images were taken at different time points (from 0 to 180 min) after the addition of l-arabinose, and the pairwise distance between MukBEF foci and ori1 foci was assessed manually using ImageJ software. Means and standard deviations were calculated. Representative examples of cells at each time point are shown. The nucleoid was highlighted by 4′,6′-diamidino-2-phenylindole (DAPI) staining for the examples at t180 (180 min). Bars, 2 µm.

FIG 4

Regular spacing of ori-independent MukBEF foci after TopoIV impairment and after inhibition of replication initiation. (A) Summary of the time-lapse analysis for 3 representative cells released from a parE(Ts) arrest. Blue × symbols represent origin peaks, and red circles represent MukBEF peaks in the line profiles of intensity along the cells. Time moves down the y axis, and the time points are as follows: 10, 30, 60, 90, 120, 150, 180, and 210 min after release from arrest. For each cell, the analysis is shown up until the point where the cells had clearly divided; septa are marked by a vertical black line on the graph at the last time point. The tables to the right of the three graphs show the number of ori and MukBEF peaks at each time point. The example cells represent 3 cell types observed after parE arrest—cells where the number of Muk foci are equal to (or less than double), double or more than double the number of ori foci—these types are equally represented within the population. (B) Time-lapse following ori1 and MukBEF foci after release from dnaC(Ts) arrest. We measured the total distance moved by ori1 and MukBEF foci along the long axis of the cell during the 5-min interval spanning origin segregation, which is illustrated in the top panel (the blue arrow indicates total distance moved by ori, and the two red arrows combined are the total distance moved by Muk). The distances measured are shown in the box plot below the schematic drawing of the cell; the box indicates the interquartile range with the central line of the box indicating the median value. The whiskers of the plot indicate the minimum and maximum measurements. Colocalization was assessed manually, and it was defined as when the foci centers were less than 2 pixels (258 nm) apart.