The topoisomerase IV ParC subunit colocalizes with the Caulobacter replisome and is required for polar localization of replication origins

Abstract

The process of bacterial DNA replication generates chromosomal topological constraints that are further confounded by simultaneous transcription. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. In addition to topological constraints, the cellular position of the replication origin is strictly controlled during the cell cycle. In Caulobacter crescentus, the origin of DNA replication is located at the cell pole. Upon initiation of DNA replication, one copy of the duplicated origin sequence rapidly appears at the opposite cell pole. To determine whether the maintenance of DNA topology contributes to the dynamic positioning of a specific DNA region within the cell, we examined origin localization in cells that express temperature-sensitive forms of either the ParC or ParE subunit of topoisomerase (Topo) IV. We found that in the absence of active Topo IV, replication initiation can occur but a significant percent of replication origins are either no longer moved to or maintained at the cell poles. During the replication process, the ParC subunit colocalizes with the replisome, whereas the ParE subunit is dispersed throughout the cell. However, an active ParE subunit is required for ParC localization to the replisome as it moves from the cell pole to the division plane during chromosome replication. We propose that the maintenance of DNA topology throughout the cell cycle contributes to the dynamic positioning of the origin sequence within the cell.

Fig. 1.

ParC and ParE are required for positioning the replication origin at the cell pole. (A) Schematic of the C. crescentus cell cycle. Unreplicated chromosomal DNA is labeled purple, newly replicated chromosomal DNA is in green, and the replisome is in blue. The replication origins are shown as red dots. Fluorescence in situ hybridization (FISH) of the origins of replication (1) in parC ts (B) and parE ts (C) strains. Cells were grown in minimal media (M2G) at 28°C to log phase and shifted to 37°C. Samples were taken at 0, 2, 4, 6, and 8 h after the temperature shift, fixed, and hybridized with a Cy3-labeled origin probe. (Upper) Nomarski differential interference contrast (DIC) microscopy images. (Lower) Overlays of the 4′,6-diamidino-2-phenylindole (a DNA-binding dye) and the Cy3 signal. The lower images are a schematic diagram of the intracellular location of the origins for 0 and 2 h after the temperature shift. Origins of replication are pseudocolored red, and DNA stained with 4′,6-diamidino-2-phenylindole is blue. Arrows indicate mislocalized origins. Arrowheads indicate a DNA-partitioning defect. White boxes indicate regions that lack DNA. (White scale bar, 2 μm.) The percentage of cells that show one polar origin and a second origin not at the pole at 0, 2, and 4 h is indicated below the FISH overlays.

Fig. 2.

ParC colocalizes with DnaB component of the replisome. Intracellular localization of ParC-GFP in LS3744(A) and DnaB-YFP in LS3587 (B) during the cell cycle. LS3744 and LS3587 swarmer cells were isolated and allowed to progress synchronously through the cell cycle. Images were collected at the indicated times (in minutes). LS3744 was grown in minimal media and induced with 0.3% xylose for 2 h to induce expression of ParC-GFP before synchronization. (Top) DIC images. (Middle) Either GFP or YFP fluorescence. (Bottom) Schematic diagrams of ParC-GFP fluorescence in red and DnaB-YFP fluorescence in green. (White scale bars, 2 μm.) (C) Measurements of average cell length (solid lines) for LS3744 (blue) and for LS3587 (red). The distance of ParC-GFP foci (blue) and DnaB-YFP foci (red) from the stalked pole at successive time points in a synchronized cell cycle are shown as dashed lines. At least 50 cells were measured for each time point. (D) Colocalization of DnaB helicase and ParC. LS3733 containing parC-cfp at the xyl locus and dnaB-yfp at its endogenous chromosomal locus was grown in minimal media containing 0.3% xylose for 2 h to induce expression of ParC-CFP. Samples of cells from a mixed culture were imaged by fluorescence microscopy. Top row of images show DIC images, second row shows DnaB-YFP foci (pseudocolored red), third row shows ParC-CFP foci (pseudocolored green), and bottom row shows overlays of the YFP and CFP channels. The overlap of red and green signal gives yellow. (White scale bar, 2 μm.)

Fig. 3.

ParC localizes with the replisome whereas ParE is dispersed intracellularly. (A) Time-lapse microscopy of ParC-GFP in strain LS3744. Cells were grown in minimal media with 0.3% xylose for 2 h to induce expression of ParC-GFP. Swarmer cells were isolated and placed on an agarose pad containing xylose and images of the same cells were acquired every 30 min as the cells progressed through the cell cycle. (Left) DIC images. (Center) GFP fluorescence. (Right) Schematic diagrams of the observed fluorescence in red. (White scale bar, 2 μm.) (B) Intracellular localization of GFP-ParE. Cells from a mixed culture of strain LS3745 were grown in 0.3% xylose for 2 h to induce expression of GFP-ParE before being placed on a xylose-containing agarose pad and examined by fluorescence microscopy. (Left) DIC image. (Right) GFP fluorescence. (White scale bar, 2 μm.) (C) Immunoblots of ParC-GFP and GFP-ParE throughout the cell cycle by using antibodies to ParC or ParE (24). Samples were taken from a wild-type (WT) control and from synchronized populations of strains LS3744 and LS3745 at the indicated times (in minutes), after induction with 0.3% xylose for 2 h. Approximately equal amounts of cells were loaded into each lane. Asterisk indicates a cross-reacting band. (D) Schematic of the cell cycle localization patterns of the origin (red), Topo IV ParC (blue) and ParE (purple) subunits, and the replisome components (green) DnaB helicase, and the clamp loaders HolB and HolC (14).

Fig. 4.

Intracellular localization of ParC-GFP requires functional ParE. (A) LS3775 containing parC-gfp in the parE ts strain, was grown at 28°C to log phase, induced with 0.3% xylose for 2 h, and shifted to 37°C. Images were taken at 0, 2, 4, and 6 h after shift to 37°C. (Top) DIC images. (Middle) GFP fluorescence. (Bottom) Schematic diagram of ParC-GFP fluorescence in gray. (White scale bar, 2 μm.)