Journal: mBio

Article Title: The SMC Complex MukBEF Recruits Topoisomerase IV to the Origin of Replication Region in Live Escherichia coli

doi: 10.1128/mBio.01001-13

Figure Lengend Snippet: 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).

Article Snippet: Cell outlines were first delineated from a phase image using the MicrobeTracker software run in Matlab (see in the supplemental material).

Techniques: In Vivo, Binding Assay, Fluorescence, Imaging, Microscopy