Localization of fluorescent foci of various chromosomal DNA segments in cells with two foci in exponentially growing cultures. Cells with two fluorescent foci in the same culture as in Fig. 3 were statistically analyzed. (A) Positions of both foci in cells with two fluorescent foci are plotted against cell length. The nearest oriC focus from a cell pole is shown as a blue circle; the other oriC focus from the same pole is shown as a red circle. (B) Histogram showing the distribution frequency of both foci in cells with two foci.

The map of E. coli chromosome with 23 DNA segments used as FISH probes. Positions of DNA probes derived from the Kohara phage library (Kohara et al. 1987) are indicated on the E. coli chromosome with map units in min (outside) and phage clone number (inside) according to EcoMap10 (Rudd 1998). Three DNA probes including oriC, dif, and the mukEFB operon are also shown. Open circles on the chromosome indicate the points of the inversion endpoints in strains KIN273and LN3214 (Cornet et al. 1996).

Localization of fluorescent foci of various chromosomal DNA segments in cells having a single focus in an exponentially growing culture. (A) Cells were grown in M9-glucose containing proline (doubling time: 80 min) to mid-log phase. Cells with a single fluorescent focus were analyzed statistically. The position of the fluorescent focus in cells with a single focus is plotted vs. cell length. The broken lines indicate the middle of the cell and the solid lines indicate the position of a pole. (B) Histogram showing the distribution frequency of the foci in cells with a single focus.

Localization of the oriC and dif segments in the inversion mutant. Statistical analysis of cells with two fluorescent foci of the oriC (A,B) or dif segment (C,D). Wild-type cells (A,C) and the inversion mutant, KIN273 (B,D) were cultivated at 30°C in M9-glucose medium. (A,B)The positions of foci from mid-cell are plotted vs. cell length. In cells with two foci, the nearest oriC focus from a cell pole is shown as a blue circle; the other oriC focus is shown as a red circle. (C,D) Histogram showing the distribution frequency of both foci in cells with two foci. The broken line indicates mid-cell; the solid line indicates the position of a pole.

Localization of the oriC and dif segments on segregating chromosomal DNA in the large inversion cells. Cells were grown to mid-log phase at 30°C in M9-glucose minimal medium. (A) Visualization of segregating chromosomal DNA in large inversion cells. Fixed cells were stained with DAPI to detect chromosomal DNA. (a) Wild-type CB0129 cells, (b–e) KIN273 cells. Cells are shown in Nomarski DIC images combined with fluorescent DAPI images (a, b, e), (c) DIC images, (d) fluorescent DAPI images. Arrowheads indicate anucleate cells (b) or the Guillotine effect (c–e). (B, C) All images were combined of the fluorescent and the phase-contrast micrograph. (B) Localization of the oriC and dif segments in KIN273 is simultaneously shown in images of the fluorescent micrograph for the Cy3-labeled oriC probe and the fluorescein-labeled dif probe. (C) Localization of the oriC and dif segments in KIN273 is simultaneously shown in images of the fluorescent micrograph for the Cy3-labeled oriC probe, the fluorescein-labeled dif probe, and fluorescent DAPI micrograph. Scale bars, 1 μm.

Localization of two segments in the same cells. (A) Cells were grown as described in the legend to Fig. 3 and simultaneously hybridized with the fluorescein-labeled DNA probes (green) and the Cy3-labeled DNA probes (red). (a–c) Cy3–oriC and fluorescein–dif; (d–g) Cy3-20- and fluorescein–70-min segments; (h) Cy3–oriC and fluorescein–20-min segment; (i) Cy3–dif and fluorescein–20-min segment. All photos combine the phase-contrast micrograph with the fluorescent micrograph for FISH in the same cell. We measured distance between the center of the Cy3 focus and the nearest pole and the distance between that of the fluorescein focus from the same pole. (B–E) Cells were analyzed statistically. The histogram shows the distribution frequency of both foci in cells with two foci. (B) Localization of oriC (black) and dif (red); (C) localization of the 20 (black) and 70-min segments (red); (D) localization of oriC (black) and the 20-segment (red); (E) localization of dif (black) and the 20-min segment (red). Scale bar, 1 μm.

Localization of the oriC DNA segment in cells under various growth conditions. (A) Statistical analysis of subcellular localization of a single fluorescent focus in cells. Cells were cultivated at 37°C in M9-glucose medium containing casamino acids with a doubling time of 52 min (a), M9-glucose containing proline with a doubling time of 80 min (b), and M9-sodium acetate containing proline with a doubling time of 280 min (c). The oriC region was detected by hybridization with the Cy-3 labeled oriC DNA probe. The histogram shows the distribution frequency of the focus in cells with a single focus (a–c). Fraction of cells with a single oriC focus against cells with two foci was as follows: 1.1 (52 min), 2.0 (80 min), and 2.3 (280 min). (B) Statistical analysis of cells with two fluorescent foci of the oriC segment. Cells were cultivated at 37°C in M9-glucose medium containing casamino acids with a doubling time of 52 min (a), in M9-glucose and proline (doubling time: 80 min) to mid-log phase (b), and early stationary phase (c). The positions of foci from mid-cell are plotted vs. cell length. In cells with two foci, the nearest oriC focus from a cell pole is shown as a blue circle; the other oriC focus is shown as a red circle. The yellow line indicates the regression line calculated by the Lowess method (tension, 50). The broken line indicates mid-cell; the solid line indicates the position of a pole.

Domains, organization, and dynamics of the bacterial chromosome. (A) Organization of the Ori and Ter domains. The DNA segments that showed similar localization patterns to that of the oriC segment are shown in green. The DNA segments that showed similar localization patterns to that of the dif segment are shown in blue. (B) A model of bacterial chromosome structure in vivo. Long circular bacterial chromosomal DNA may be folded by an unknown mechanism to form a compact ring structure, in which the DNA segments are arranged according to their chromosome map positions. The Ori (green, O) and Ter domains (blue, T) are localized near or at the cell poles in newborn cells (top). In the B period, both the domains migrate and are localized at mid-cell (bottom). Factors (gray circles) may bind specifically to cis-acting sites of the Ori domain; other factors (gray triangles) may bind to another type of cis-acting site of the Ter domain. The two groups of factors may participate in cell cycle-dependent positioning of the Ori and Ter domains. (C) Fluorescent microscopy of DAPI-stained cells. Wild-type (a,b) and mukB-disrupted mutant (c,d) that were grown at 22°C were stained with DAPI according to Hiraga et al. (1989). (a,c,d) Images of fluorescence-phase contrast-combined microscopy. (b) Image of fluorescence microscopy in the same field as a. Scale bars, 1 μm.