Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein

Abstract

Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.

FIG. 1

Titration of DEAE-dextran into FtsZ sedimentation assay. (A) SDS-polyacrylamide gel analysis of the supernatant and pelleted FtsZ protein with increasing levels of DEAE (final concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg/ml, respectively, in lanes 1 to 9). The gel lanes are arranged so that the corresponding supernatant and pellet at a specific level of DEAE are vertically aligned. (B) Same as in panel A, but with FtsZ2 mutant protein in place of wild type. (C) Densitometric scans of the gels in the top two panels were used to determine the fraction of FtsZ sedimented and then plotted as a function of the amount of DEAE-dextran added as described in Materials and Methods.

FIG. 2

(A) GTPase of FtsZ2 compared with wild-type FtsZ at 30 and 42°C. (B) Replotted detail (note axis changes) of FtsZ2 at 30 and 42°C, extending the time to 60 min. Each FtsZ protein (Zwt or Z2) was present as 2 μM, and the assay was performed as described in Materials and Methods.

FIG. 3

Electron micrographs of FtsZ polymers induced by DEAE-dextran, each at a magnification of ×59,000. (A to D) Wild-type FtsZ, illustrating tubules (A), protofilaments aligned vertically (B), and minirings (C and D, arrowheads). (E and F) FtsZ2 showing miniring (E) and tubules (F), with inset diagram of proposed tubule structure represented as one light and one dark protofilament. Polymerization reactions were done as described in Materials and Methods with FtsZ proteins present at 5 μM. Scale bar, 100 nm.

FIG. 4

Toxic expression of PrtA-SulA fusions. Strain HMS174(DE3) was transformed with 0.5 μg of the indicated plasmid DNA: pET11-PrtASulAwt (wt), pET11-PrtASulA62H (62H), or pET11-PrtASulA62S (62S). The transformed cells were grown for 2 h at 37°C and then diluted in twofold steps in a 96-well plate. From each dilution 10-μl aliquots were spotted onto each of a series of 10 plates containing Luria-Bertani medium plus ampicillin (50 μg/ml) and 0, 0.0078, 0.0156, 0.0234, 0.0312, 0.0468, or 0.0625 mM IPTG. After incubation overnight at 37°C, colonies were counted for each IPTG concentration. The transformation efficiency was determined relative to that achieved with no IPTG and is plotted as the surviving fraction.

FIG. 5

Effect of PrtA-SulA alleles on cell division. The cloned SulA alleles are as indicated; “none” refers to the vector lacking SulA. All panels show cells after IPTG-induced expression of the plasmid-encoded gene. Strain HMS174(DE3) was transformed with pET11a, pET11-PrtASulAwt, pET11-PrtASulA62H, and pET11-PrtASulA62S plasmids. One colony from each plate was inoculated into minimal medium (33) and 50 μg of ampicillin per ml at 37°C, then grown to an OD₆₀₀ of 0.2, and then induced with 0.5 mM IPTG for 4 h. Cells from 0.5-ml samples of each well were harvested, washed with 100 mM NaCl in 5 mM Tris-Cl (pH 7.4), and resuspended in 0.5 ml of water with 10 μM Syto-17 red fluorescent dye. Aliquots (10 μl) were spread on polylysine-coated glass slides and fixed with Permount as coverslips were mounted. Slides were examined with a Zeiss Axiovert inverted stage microscope with a ×100 oil immersion objective lens and a rhodamine fluorescence filter set. Exposure was for 4 s on ISO 400 film.

FIG. 6

Effects of PrtA-SulA on FtsZ GTPase activity. The indicated PrtA-SulA fusion (5 μM) was each preincubated with 2 μM FtsZ for 5 min before the addition of GTP to the assay. The assays were followed for 20 min, withdrawing and quenching samples for analysis. The GTPase assay was done as described in Materials and Methods.

FIG. 7

Effects of PrtA-SulA fusion proteins on FtsZ polymerization. Wild-type FtsZ (Z) or FtsZ2 (Z2) was present where indicated at a fixed concentration of 2 μM. Increasing amounts of wild-type PrtA-SulA protein (wt) or indicated mutant SulA fusion proteins (62S and 62H) were preincubated with FtsZ or FtsZ2 for 5 min at 37°C before polymerization was initiated by the addition of DEAE-dextran to 100 μg/ml. The sedimentation assay was then done as described in Materials and Methods. The results have been normalized to facilitate comparison of the data for FtsZ2 (95% sedimented in the absence of any SulA) and wild-type FtsZ (86% sedimented in the absence of any SulA).

FIG. 8

Direct comparison of the effects of PrtA-SulA fusion proteins on FtsZ polymerization and on FtsZ GTPase activity. The wild-type PrtA-SulA fusion was titrated into reactions for GTPase or polymerization; in each case reactions were initiated by the addition of FtsZ wild-type protein. The assays were performed as described in Materials and Methods.