The two Escherichia coli chromosome arms locate to separate cell halves - PubMed (original) (raw)
The two Escherichia coli chromosome arms locate to separate cell halves
Xindan Wang et al. Genes Dev. 2006.
Abstract
DNA replication divides the circular Escherichia coli chromosome into equal arms (replichores). Visualization of pairwise combinations of multiple genetic loci reveals that the two replichores occupy separate nucleoid halves, with the replication origin between; positions of loci on each replichore recapitulate the genetic map. Sequential replication-segregation regenerates the structure by sequentially layering newly replicated replichore DNA to specific inner and outer edges of the developing sister nucleoids. Replication fork-dependent locus positions are imprinted, so that in most generations the chromosome orientation in a mother cell is recreated as a arrangement of sister chromosomes in daughter cells.
Figures
Figure 1.
E. coli chromosome organization recapitulates the genetic map. The left panel shows a schematic of chromosome organization in C. crescentus; left and right replichores are organized about a longitudinal axis (Viollier et al. 2004; Gitai et al. 2005). The right panel shows the E. coli chromosome with the markers used, and its cellular organization below. The left and right replichores flank a transverse axis. The left replichore (L) of each chromosome is shown in blue and the right replichore (R) is shown in orange. We imagine that the bulk of DNA in left and right replichores is organized into independent domains. Normal replication termination sites within the ter region (dotted line; alternating blue–orange) are shown in black below the map and the ectopic site is shown in red. The left replichore is positioned left of ori and the right replichore to the right of ori. The ter region spans the two outer nucleoid edges; much of it can be replicated by either fork. The dif site-specific recombination site (Blakely et al. 1991) is located 18 kb counterclockwise of the Ter locus. Representative FISH (left) and DNA-bound fluorescent repressor (FROS) (right) images are shown for L3 (green) and R3 (red).
Figure 2.
Snapshot analysis. (A) Single-locus FISH survey of the whole genome. Focus pairs (14 loci) were binned into groups depending on pattern (see D). The four patterns shown, which account for >65% of all cells, were most informative with regard to asymmetric–symmetric positioning of sister foci. The frequency of each pattern with respect to genetic map was plotted. For each locus, ∼300 two-focus cells were analyzed. Nucleoid pairs were maximized by treating an exponential culture (τ 100 min) for 60 min with cephalexin to block cell division. This does not significantly perturb segregation pattern. (B) Sequential replication–segregation of eight loci, using FROS snapshot analysis of an exponential culture (τ 100 min). The position of a locus on the _X_-axis with respect to genetic map is shown. Cells were binned into four groups in terms of number and position of the foci: one focus off-center; one focus at mid-cell; two foci close to the mid-cell; and two foci well segregated. Time-lapse analysis (Fig. 3; Supplementary Fig. S3) confirms that loci move to the middle third of the cell prior to duplication, where the duplicated foci appear close together before generally segregating away from the mid-cell region. At least 2000 cells were analyzed for each locus. (C, left panel) Representative L3 (red)–R3 (green) FROS snapshots of τ 100-min and τ 26-min cells (inset). (Right panel) Representative R2 (red)–R3 (green) snapshots of τ 100-min cells. White arrows show closely separated sister foci that appear to have duplicated close to mid-cell. Schematics showing frequencies of different patterns in cells with only one focus for each locus (631 of 1112 cells in L3–R3, and 532 of 1243 in R2–R3) are above the micrographs. (D) Pairwise FISH analysis. Cells were divided into five arbitrary regions (I–V; III was one-third of cell length; others were one-sixth of cell length). Six locus pairs, using L3 or R3 as a reference, were examined (Ori–L3, L1–L3, L2–L3; Ori–R3, R1–R3, R2–R3). For each locus pair, ∼200 four-focus cells (two sisters for the target locus and two for the reference, L3 or R3) in which the L3 or R3 sisters have the pattern (V, I) were extracted and then oriented with respect to the reference pattern (V, I). The relative positions of the target sister loci on the oriented nucleoids were plotted. The pairwise L replichore (plus Ori) analysis is shown on the left and the R replichore (plus Ori) analysis is shown on the right. Further snapshot analysis is shown in Supplementary Figure S2.
Figure 3.
Time-lapse analysis. (A) Representative FROS time-lapse progressions (10-min intervals) are shown for L3 (red)–R3 (green). The right panel shows the position of L3 (red) and R3 (green) as a function of time in the left time lapse. The black line positions the division site when it is visible during invagination and the new poles after division. (B,C) The position of sister L3s are tracked by red and orange lines, and R3s are tracked by green and light-green lines. (B) R2 (red)–R3 (green). (C) L3 (green)–dif (red). Schematics of cells at 0, 20, and 90 min are shown. Further analysis is in Supplementary Figure S3. The dif locus array is located 8 kb clockwise of dif and 11 kb from Ter.
Figure 4.
(A) Schematic of E. coli chromosome organization and segregation. Slow-growing E. coli in which a single pair of sister replication forks replicate the whole chromosome. The major <L–R–L–R> pathway (∼85%) and the minor <L–R–R–L> pathways (∼15%) are indicated by thick and thin arrows. The L replichore is in blue and the R replichore is in orange. The two sister nucleoids, expanding through replication, are shown on top and the contracting mother nucleoid is shown on the bottom. The deposition of nascent strands to both edges of the expanding sister nucleoids is indicated by dashed lines. The relative positions of L1, L5, R1, and R5 on the two sister nucleoids are also indicated. The product of the <R–L–L–R> pathway, which occurs at equal probability to <L–R–R–L>, is shown in parentheses. (B) The “split replisome” model for E. coli sister nucleoid segregation. Organization is directed as a consequence of sister replisomes moving apart along the longitudinal axis. Left panels show the two possible situations for generating <L–R–L–R> (frequent), or <R–L–R–L> (rare) sister nucleoids from a <L–R> mother. Right panels show pathways for generating the minority <L–R–R–L> or <R–L–L–R> sister nucleoids. These may be favored when one of the replication forks stalls or collapses (dotted lines around the replisome, shown in pink).
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