Positions of 112 distinct chromosomal loci in Caulobacter swarmer cells determined by FROS. (A) Positions of the 112 loci tagged either with (lacO)_n or (tetO)_n (see Fig. 5 for detailed information) relative to ori and ter (12 and 6 o'clock, respectively) on the chromosome. (B) The average subcellular position (see Table 1) of the 112 loci in A plotted as a function of their relative location on the chromosome. In both A and B, insertions on the right (12–6 o'clock; •) and left (6–12 o'clock; ▵) arc of the chromosome are depicted.

The positions of four distinct chromosomal loci in Caulobacter swarmer cells. (A) Schematic of the circular Caulobacter chromosome showing the location of the pilA, pleC, and podJ loci relative to that of ori and ter.(B and C) Histograms showing the fraction of cells with ori, pilA, pleC, and podJ at a given position in the cell as determined by FROS (B) or FISH (C). The positions of ori, pilA, pleC, and podJ are shown in green, blue, magenta, and yellow, respectively. (D) Diagram of the average subcellular positions of the four loci. Wavy line, flagellum; straight lines, pili. (E) Average subcellular positions of the pilA, pleC, and podJ loci plotted as a function of their distance from ori on the Caulobacter chromosome as determined by FISH (blue line) and FROS (magenta line).

Rapid and directed movement of (lacO)_n/LacI-CFP-tagged ori during the early stages of the DNA replication cycle. (A) Swarmer cells were isolated and spotted on an agarose pad containing nutrients. Phase contrast and fluorescence images were acquired at 2-min intervals immediately before and during foci duplication and movement to the opposite cell pole. Each image represents an overlay of a phase contrast and a fluorescence image. The number in the upper left corner of each image indicates the time (min) relative to the starting point (0 min) at which the image was taken. Diagrams illustrating the movement of the locus are shown above and below the image frames. (Bar, 1 μm.) (B) Movement of the ori focus within the cell (average of 22 cells) plotted as a function of time. The green line shows the increase in cell length. The blue and magenta lines mark the distance of the “old” and the “new” focus from the stalked cell pole over time.

Rapid, directed, and sequential movement of different (lacO)_n/LacI-CFP-tagged loci during the DNA replication cycle. (A and B) The average movement of the fluorescent foci from at least 20 cells was analyzed as in Fig. 3 for 10 strains, each harboring a (lacO)_n insertion at a distinct chromosomal position. The result of the time lapse experiment shown in Fig. 3B by using the strain that carries (lacO)_n 4 kb from ori are reproduced in A and B (black line) as a reference for the results obtained with the 10 additional strains. (Insets) The relative position of the insertions in the ori-proximal half of the chromosome. The movement of five (lacO)_n/LacI-CFP-tagged loci located 50 (blue), 102 (brown), 166 (turquoise), 442 (pink), and 864 kb (green) to the “right” and five (lacO)_n/LacI-CFP-tagged loci located 43 (blue), 103 (brown), 294 (turquoise), 466 (pink), and 576 kb (green) to the “left” of ori was plotted on the same graph in A and B, respectively. Synchronized swarmer cells were isolated from each strain, spotted on an agarose pad containing nutrients (time point 0 min) and were allowed to proceed through the cell cycle until the movement of the loci began. Images were then acquired at 2-min intervals to record the movement. With increasing distance from ori, the movement of the focus occurred progressively later in the cell cycle. O, ori. (C) The average rates of movement of (lacO)_n/LacI-CFP-tagged chromosomal loci. The strain used in Fig. 3 containing (lacO)_n 4 kb from ori, as well as 10 other strains described were analyzed.