Roles for replichores and macrodomains in segregation of the Escherichia coli chromosome

Abstract

Recent work has highlighted two main levels of global organization of the Escherichia coli chromosome. Macrodomains are large domains inferred from structural data consisting of loci showing the same intracellular positioning. Replichores, defined by base composition skews, coincide with the replication arms in normal cells. We used chromosome inversions to show that the dif site, which resolves chromosome dimers, only functions when located at the junction of the replichores, whatever their size. This is the first evidence that replichore polarization has a role in chromosome segregation. We also show that disruption of the Ter macrodomain provokes a cell-cycle defect independent from dimer resolution. This confirms the existence of the Ter macrodomain and suggests a role in chromosome dynamics.

Figure 1

Map of the inversions. Top panel: map of the chromosome linearized at oriC. Coordinates are in kb. Positions of migS, dif, the Ori, Right (R), Left (L) and Ter domains, and the replication terminators (the flags) are shown. Middle and bottom panel: inverted fragments. Each line represents a pair of Plr+ and Plr− inversions from dif (zdd346::lacZ::attB and zdd347::lacZ::attB positions; Fig 2) or a single inversion from zdd370::lacZ::attB (only with attP at sp5, sp39, and purA) and in the case of the Inv(29–78) inversion. The respective positions of the attP-Kn insertions are given. The three shortest inversions (sp17, zdd355 and zdd370) in Fig 3 are not shown.

Figure 2

Inversion engineering. The chromosome is represented linearized at oriC with the two replichores polarized from oriC to dif (the black and white square). Arrowheads symbolize polarization. The grey zone is the DAZ, which is the junction of inversely polarized sequences where dif must lie to be active. Inversions occur between lacZ::attB and attP sites. A couple of Plr+ and Plr− inversions are detailed. Only Plr+ inversions keep dif at the junction of the newly formed replichores.

Figure 3

Intra-Ter inversions. The viability of the strain carrying Plr+ and Plr− inversions is plotted as a function of the distance separating attP from dif (top scale). The respective positions of the attP sites are given (circles: Plr−; diamonds: Plr+). The scale on the left is the viability index (measured using the co-culture assay, 70% for a dif-deleted strain, 100% for the wild type). The scale on the right is the inferred frequency of abortive divisions per generation.

Figure 4

Interdomain inversions. Examples of growth defects provoked by interdomain inversions. (A) Generation times were calculated from growth curves in permissive conditions. (B) Serial dilutions of liquid culture grown overnight in permissive conditions were plated in permissive (P, synthetic medium at 30°C) and nonpermissive (NP, here synthetic medium at 42°C) conditions and incubated for 36 h (wt) or 48 h (inverted strains). Note the similar colony size of Inv(dif-aroA) in P and NP conditions despite the difference in temperature (compare with the wt). Note also the appearance of revertant clones (big colonies) in NP.

Figure 5

Interdomain inversions provoke an over-representation of division figures. (A) Normal cells with one or two nucleoids and abnormal cells (filaments, chains and other abnormal cells such as wider cells apparently containing several chromosomes) were counted on micrographs of bacteria grown in permissive conditions (400 cells per count). (B) Examples of micrographs of cells grown in permissive conditions.