Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes in E. coli  Sota Hiraga, Chiyome Ichinose, Hironori Niki, Mitsuyoshi Yamazoe  Molecular Cell  Volume 1, Issue 3, Pages 381-387 (February 1998) DOI: 10.1016/S1097-2765(00)80038-6

Figure 1 Intracellular Localization of the SeqA Protein in Various Strains Exponentially growing cells in L medium were fixed and immunostained for SeqA. Merged pictures of fluorescent SeqA foci with Cy3 (red fluorescence) and the cell shape obtained by phase-contrast microscopy are shown (except [B] and [C]). (A) The wild-type strain YK1100 was grown exponentially at 37°C. (B and C) Fluorescence microscopy for SeqA (B) and phase-contrast microscopy for cell shape (C) in the dam null mutant KA468. (D and E) Cells of the oriC+ rnhA mutant KHG106 (D) and the oriC-deleted rnhA mutant KHG112 (E) grown at 37°C in minimal glucose medium were incubated further in L medium for 3 hr. Lower picture, DAPI-staining. (F and G) The wild-type strain (F) and the mukB null mutant strain AZ5372 (G) were grown exponentially at 22°C. (H and I) The wild-type strain (H) and the mukB null mutant strain (I) were incubated in the presence of cephalexin (10 μg/ml) for 3 hr at 22°C. Arrows indicate large foci or clusters of fluorescent SeqA. All scale bars, 5 μm. Molecular Cell 1998 1, 381-387DOI: (10.1016/S1097-2765(00)80038-6)

Figure 2 Double Staining of SeqA Protein and the oriC DNA or FtsZ Protein (A and B) Wild-type cells (YK1100) immunostained for SeqA protein with Cy3 and then treated for in situ hybridization with the fluorescein-labeled oriC DNA probe. Left, red fluorescent SeqA foci. Center, green fluorescent foci corresponding to oriC copies. Right, a merged picture of SeqA foci and oriC copies. (C) Wild-type cells were immunostained differentially with Cy2 (green fluorescence) for SeqA protein using rabbit anti-SeqA polyclonal antibody and with Cy5 (near-ultrared fluorescence) for the FtsZ protein using mouse anti-FtsZ monoclonal antibody. Left, SeqA. Center, FtsZ. Right, a merged picture of SeqA and FtsZ. Scale bar, 5 μm. Molecular Cell 1998 1, 381-387DOI: (10.1016/S1097-2765(00)80038-6)

Figure 3 Statistical Analysis of SeqA Foci and oriC DNA Copies in Individual Wild-Type Cells (A and B) Cells (YK1100) grown exponentially in different media. Media from top to bottom are as follows: minimum glucose medium containing L-tryptophan (DT = 55 min), minimum glucose medium containing L-tryptophan and Casamino acids (DT = 35 min), L medium (DT = 30 min), and L medium containing glucose (DT = 20 min). (A) Number of chromosomes (oriC copies) per cell. Amount of chromosomal DNA of individual cells was analyzed with a flow cytometer. (B) Number of SeqA foci in cells. The percentage of the cells with a given number of SeqA foci are plotted in the histogram. (C–E) Cells (YK1100) were grown in M9 medium supplemented with glucose (0.5%) and L-tryptophan (50 μg/ml) at 37°C, fixed, and immunostained for SeqA. (C) The ratio of cells corresponding to each type (the total number of cells, 269). (D) Subcellular positions of SeqA foci in types 1, 2, and 3. The broken line indicates midcell and the solid line indicates the position of a pole. (E) Histogram of positions of SeqA foci in cells of type 1, type 2, and type 3. Molecular Cell 1998 1, 381-387DOI: (10.1016/S1097-2765(00)80038-6)

Figure 4 Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes (A) A scheme of SeqA foci in the cell division cycle for a 55 min doubling time. Blue circles on the chromosomal DNA represent oriC copies. Black circles on the chromosomal DNA represent the replication terminus. Pale blue areas in cells represent nucleoids. Gray areas in cells represent cytosolic areas. Red circles in cells represent fluorescent SeqA foci. A blue bar at midcell represents an FtsZ ring. See the text. (B) Initiation-partition apparatus model. Dark yellow circles, SeqA-associated DNA–protein complexes. Yellow circles, putative specific DNA regions associated with the complexes. Blue circles, oriC DNA regions. Violet triangles, replication forks. See the Discussion in detail. Molecular Cell 1998 1, 381-387DOI: (10.1016/S1097-2765(00)80038-6)