Nucleoid-independent identification of cell division sites in Escherichia coli

Abstract

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.

FIG. 1

Comparison of ends of ftsA(Ts) dnaA(Ts) (A) and ftsA(Ts) (B) DAPI-stained filaments from strains WC1016 and WC1004 after growth at 42°C for three generations. (C) Cells of strain WC1016 were grown at 42°C for three generations and then shifted to 30°C for 100 min as described in Materials and Methods. The upper panel of each pair is the fluorescence micrograph, and the lower panel is the corresponding Nomarski image. Representative cells are shown. Arrowheads indicate septa. Scale bar, 5 μm.

FIG. 2

Positions of septa and nucleoids at ends of ftsA(Ts) dnaA(Ts) filaments. (A) Diagram showing landmarks. (B, C, and D) Cells that contained septa between the nucleoid and cell pole (illustrated in Fig. 1C) were analyzed for cell length and for septum-to-pole (B), nucleoid-to-pole (C), and septum-to-nucleoid (D) distances. A total of 434 filaments were analyzed.

FIG. 3

Septation in nucleoid-free polar regions of ftsZ dnaB(Ts) filaments. Cells of strain WC1113(λGL100) were grown at 42°C for three generations in the presence of glucose, followed by 30 min in the presence of IPTG, and then fixed with glutaraldehyde and stained with DAPI as described in Materials and Methods. The upper panel of each pair is the fluorescence micrograph, and the lower panel is the corresponding Nomarski image. Representative cells are shown. Arrowheads indicate septa. Scale bar, 5 μm.

FIG. 4

Relationship between septum-to-pole and septum-to-nucleoid distances in ftsZ dnaB(Ts) filaments. Cells of strain WC1113(λGL100) were grown and prepared as described in the legend to Fig. 3, and cells that contained septa between the nucleoid and cell pole (2,857 cells) were analyzed. See Fig. 3A for landmarks.

FIG. 5

Septation over nucleoids. Strains WC1113(λGL100) [ftsZ null dnaB(Ts) sfiA::Tn5 (P_lac-ftsZ)] (A and C) and WC1016 [ftsA(Ts) dnaA(Ts)] (B) were grown and prepared as described in the legends to Fig. 2 and 3. (A and B) Septa that bisected nucleoids. (C) Cells that either lacked nucleoids or that contained nucleoid fragments of different sizes. Scale bar, 5 μm.

FIG. 6

Relationship between septa and nucleoids and poles in ftsZ dnaB(Ts) filaments. Strain WC1113(λGL100) was grown and prepared as described in the legend to Fig. 3. (A) Cells containing septa that were located in the nucleoid-free region adjacent to a pole (type 1) or located over a nucleoid adjacent to a pole (type 2) were analyzed. Frequency represents the number of cells in each class/number of cells in both classes. Cells of all cell lengths were included in the analysis shown in the first column, whereas only cells with a length of <20 μm were included in the analysis shown in the second column. (B) Fifty-seven cells containing a septum located over a nucleoid (type 2 in Fig. 6A) were analyzed as described in the legend to Fig. 2.

FIG. 7

Model for determination of division site position. The diagram illustrates how the position of future division sites might be directed by a signal that is periodically elaborated from the cell poles, leading to establishment of a site at midcell (a), or from both the cell poles and nascent division sites at midcell to establish new sites at the cell quarters (b). In the latter case, the sites at 1/4 and 3/4 cell length are retained at the midpoint of the daughter cells to support septum formation during the next cell cycle (3). See text for further details.