Timing of FtsZ assembly in Escherichia coli

Abstract

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

FIG. 1

Cumulative length distribution of E. coli B/rA grown to steady state with a generation time of 130 min. The cells were either fixed (○), fixed and permeabilized (■), or fixed, permeabilized, labeled with MAb F168-12 against FtsZ, and fluorescence stained with a Cy3 secondary antibody (●). Data are for 514, 505, and 723 cells, respectively.

FIG. 2

E. coli B/rA grown to steady state with a generation time of 75 min. (A) Phase-contrast microscopy image of the cells; (B) corresponding Alexa-labeled FtsZ fluorescence. Bar, 2.5 μm. An example of a cell showing an open ring with two distinct dots at midcell is indicated by two opposite arrows; a cell showing a closed FtsZ ring is indicated by one arrow. Two cells (∗) with visible constrictions do not show detectable FtsZ at midcell. The cytosolic nonpolymerized FtsZ is somewhat more apparent in B/rA than in K-12 because of the weaker intensity of the FtsZ ring in the former strain.

FIG. 3

Appearance of the FtsZ ring as a function of cell length in E. coli B/rA grown to steady state at a generation time of 130 min. For 1,422 cells, the number of cells with an open FtsZ ring (○), a closed FtsZ ring (●), or a constriction (▴) in a particular length class was determined. The dotted line shows a sigmoidal fit through the data points for the constricting cells, and the solid lines show Gaussian fits through the FtsZ data points.

FIG. 4

Correlation between the appearance of the FtsZ ring, the termination of DNA replication (i.e., the start of the D period), and the start of a visible constriction. The start of the Z period (●), the start of the D period (or the end of the C period) (○), and the start of the constriction or T period (∗) are plotted as a function of the generation time. The lines are the linear least-squares fits through the data points for the FtsZ ring (a_x = 0.82x − 18.5, with r = 0.993), the D period (a_x = 0.86x − 16.9, with r = 0.982), and the T period (a_x = 0.96x − 13.9, with r = 0.991). The nucleoid separation more or less coincides with the start of constriction (a_x = 0.88x − 8.8, with r = 0.991) (not shown). The lower panel shows a schematic overview of the C, Z, and T periods in an individual cell growing with a generation time of 100 min derived from the linear fits in the upper panel. The values for the D period were obtained from reference . The value for the length of the C period (i.e., time between the initiation and the termination of DNA replication) was obtained from reference .

FIG. 5

E. coli K-12 MC4100 grown to steady state with a generation time of 85 min at 28°C. The upper panels show Alexa-labeled FtsZ fluorescence, the middle panels show the corresponding DAPI fluorescence microscopy images, and the lower panels show the corresponding phase-contrast microscopy images of the cells. Bar, 2.0 μm. (A) Examples of cells showing an open ring with two distinct dots at midcell; (B) cells showing a closed FtsZ ring; (C) cells with visible constrictions and segregated nucleoids without detectable FtsZ at midcell.

FIG. 6

Appearance of the FtsZ ring as a function of cell length in E. coli K-12 grown to steady state at a generation time of 85 min. For 516 cells, the number of cells with an open FtsZ ring (○), a closed FtsZ ring (●), or a constriction (▴) in a particular length class was determined. The dotted line shows a sigmoidal fit through the data points for the constricting cells, and the solid lines show Gaussian fits through the FtsZ data points.

FIG. 7

Schematic representation of the cell cycles of E. coli B/rA and K-12 growing with a generation time of 85 min. The C period (5) shows the duration of the DNA replication cycle. The D period is the time required between the termination of DNA replication and the separation into two daughter cells. The Z period shows the duration of the period in which FtsZ can be detected at midcell. The P period (42) shows the period of septal peptidoglycan synthesis, and the T period is the duration of the constriction.

FIG. 8

E. coli K-12 MC4100 pbpB2158(Ts) grown to steady state with a generation time of 78 min at 28°C. (A) Phase-contrast microscopy image of the cells; (B) the corresponding DAPI-stained nucleoids; (C) the corresponding Alexa-labeled FtsZ fluorescence. Bar, 2.5 μm.