Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites

Abstract

A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the beta-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.

Figure 1

Subcellular localization of FtsZ in wild-type, ftsZ84, ftsI23, and cephalexin-treated E. coli. (a–c) Localization of FtsZ (green) in exponentially growing wild-type E. coli. (a) FtsZ immunostaining (green) alone. (b) Doubly exposed micrograph of the same field showing both FtsZ (green) and the propidium iodide-stained nucleoids (red). (c) The propidium iodide-stained nucleoids alone. (d) A series of cells depicting FtsZ assembly states during the E. coli division cycle, starting from a newborn cell lacking an FtsZ ring (top) and ending with a fully constricted but as yet unseparated pair of newborn cells. The left column of cells show both FtsZ protein (green) and the nucleoids (red), while the right column of cells show the nucleoids only (red). (e–g) Localization of FtsZ in an ftsZ84 mutant at the nonpermissive temperature. (e) FtsZ staining (green) alone. (f) A double exposure of the same field of cells showing FtsZ (green) and the nucleoids (red). (g) Propidium iodine-stained nucleoids. (h–j) Localization of FtsZ in the ftsI23 mutant at the nonpermissive temperature. (h) FtsZ alone (green). (i) FtsZ (green) and the nucleoids (red). (j) Nucleoids alone (red). (k–m) Localization of FtsZ in cells treated with cephalexin for two generatations. (k) FtsZ staining (green) alone. (l) FtsZ (green) and the nucleoids (red). (m) Nucleoids alone (red). (n) Diagram of the cells in k–m, showing the inferred outlines of the cells (black), and the positions of the nucleoids (red), and the FtsZ rings (green). (o–q) A different field of cells from the same experiment as in k–m, showing additional patterns of FtsZ localization in filaments after two generations of growth in the presence of cephalexin. (o) FtsZ (green) alone. (p) A double exposure showing both FtsZ (green) and the nucleoids (red). (q) Nucleoids alone (red). (r) A diagram of the cells in o–q, showing the inferred outlines of the cells (black), and the positions of the nucleoids (red) and the FtsZ rings (green). (Bar = 5 μm.)

Figure 2

Cell size distribution of FtsZ rings. Individual wild-type (MG1655) cells (389) from several fields were measured and scored for the presence or absence of an FtsZ ring. The numbers of cells with (solid bars) or without (hatched bars) FtsZ rings is plotted versus cell length. Cell length was used to estimate cell age, which is described as percent progression through the division cycle (35). The fraction of cells in each length class containing an FtsZ ring ranged from 1% for the shortest cells (newborns) to 100% for the longer cells.

Figure 5

Cephalexin does not affect FtsZ or FtsI protein levels. (a) Pulse–chase immunoprecipitation analysis of FtsZ in cultures grown in the absence of β-lactam antibiotics (lanes 1 and 2) or grown for two generations in the presence of cephalexin (lanes 3 and 4). Radiolabeled FtsZ and OmpA, an internal control protein, were immunoprecipitated after a 1-min chase (lanes 1 and 3) and a 15-min chase (lanes 2 and 4). After quantitation of the counts in each band, the normalized value for each FtsZ band was 1.4, 1.3, 1.8, and 1.7 (arbitrary units) for lanes 1–4, respectively, indicating that cephalexin does not affect the rates of synthesis of FtsZ (lanes 1 and 3) or its stability (lanes 2 and 4). (b) Immunoblot analysis of FtsI after three generations of growth in the presence of cephalexin (lane 2) or piperacillin (lane 3) or without drugs (lane 1). The normalized values for the FtsI band were 0.69, 0.84, and 0.78 (arbitrary units) for lanes 1–3, respectively, indicating that cephalexin and piperacillin did not affect FtsI protein levels in cells grown in their presence for two (data not shown) or three (shown) generations.

Figure 3

Decreased occurrence of FtsZ rings in filaments produced by cephalexin treatment. FtsZ was localized in wild-type MC4100 cells (solid squares) or in filaments after growth in the presence of cephalexin for one, two, or three generations of growth at 37°C (open symbols). The length of each filament was measured, the numbers of FtsZ rings in each filament were counted, and the data are presented in two different ways. (a) Number of cells of a given length with one, two, three, or four FtsZ rings per cell. (b) The number of FtsZ rings in each cell or filament was normalized to its length and expressed as microns per FtsZ ring. Four filaments longer than 10 μm (13, 14, 17, and 19 μm) with a single FtsZ ring are not shown. Filaments generally contained from one to four FtsZ rings, although one filament with five rings (13 μm long, 2.6 μm per ring), one filament with six rings (18 μm long, 3 μm per ring), and one filament with seven rings (17 μm long, 2.4 μm per ring) were observed (data not shown). Similar results were obtained with MG1655, except for the difference in cell lengths observed for the two wild-type strains.

Figure 4

Assembly cycle of FtsZ in wild-type and cephalexin-treated cells. (Left) Model of the assembly cycle of FtsZ during one generation of growth. (Right) Effect on FtsZ assembly of inactivating FtsI with cephalexin for two generations. The newborn cell lacks a detectable FtsZ ring, which assembles soon after birth. The FtsZ ring disassembles after constriction but before cell separation. While wild-type newborn cells lack FtsZ rings (see Figs. 1 a–d and 2), even the shortest filaments (which have four nucleoids) in the cephalexin-treated population contain FtsZ rings (see Fig. 1 k–n). This suggests that newborn cells, although they generally lack detectable FtsZ assembly intermediates, are capable of nucleating FtsZ assembly in the presence of cephalexin. Assembly of additional FtsZ rings in these filaments appears to be inhibited. For example, after cephalexin treatment, many filaments contain only a single FtsZ ring, despite having eight well-separated nucleoids, while cells that are nearly constricted produce filaments with eight nucleoids and with FtsZ rings at the midpoints of the nascent daughter cells (see Fig. 1 o–r). These filaments suggest that the FtsI transpeptidase activity may be required prior to the completion of septation for assembly of FtsZ at the future sites of cell division within each daughter cell.