The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization

Abstract

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

Figure 1

Effect of MalE-MinC fusions on cell division. (A and B) The MalE-MinC fusion blocks cell division and Z ring formation. A culture of JS964 (Δmin) pZH101 (P_BAD∷malE-minC) was processed for immunofluorescence microscopy before (A) and 1 hr after (B) addition of 0.01% arabinose. The cells were immunostained with antibodies to FtsZ, and a secondary antibody was conjugated to the fluorophore Cy3. The panels to the left are phase contrast micrographs, and those to the right are fluorescence micrographs. (C) A MalE-MinC19 fusion is attenuated for inhibition of cell division. JS964 (Δmin) pZH102 (P_BAD∷malE-minC19) was induced with 0.01% arabinose for 1 hr, and a sample was taken and immunostained for FtsZ.

Figure 2

Monitoring interactions between FtsZ and MalE-MinC fusions by using an optical biosensor. Biotinylated FtsZ was immobilized in a biotin cuvette as described in Materials and Methods. MalE-MinC or MalE-MinC19 was added at the concentrations indicated in a final volume of 60 μl, and the response in arc seconds was measured versus time (Upper). The data obtained from these experiments were analyzed with iasys fastfit software by using single phase association to obtain the equilibrium binding plots and K_d values (Lower).

Figure 3

MalE-MinC does not inhibit FtsZ's GTPase activity. The GTPase activity of FtsZ (5 μM) was measured in polymerization buffer containing increasing amounts of MalE-MinC. The reaction was initiated with the addition of GTP and was incubated at 30°C. At various times, samples were removed, and the amount of P_i hydrolyzed was determined. Shown is FtsZ alone (open circles) and FtsZ with the addition of MalE-MinC at 0.75 μM (filled circles), 3 μM (filled squares), and 12 μM (filled triangles). As a control, the GTPase activity of MalE-MinC (12 μM) was determined in the absence of FtsZ (open squares).

Figure 4

The effect of MalE-MinC on FtsZ polymerization. (A and B) Polymerization reactions (100 μl) were carried out with FtsZ (5 μM) in polymerization buffer and increasing concentrations of MalE-MinC (A) or MalE (B). Polymerization was initiated with the addition of 1 mM GTP, samples were centrifuged, and the pellets were analyzed by SDS/PAGE. A shows the effect of MalE-MinC: lane 1, GDP added; lane 2, GTP added served as controls. The concentrations of MalE-MinC were, by lanes: 3, 50 μg/ml (0.75 μM); 4, 100 μg/ml (1.5 μM); 5, 200 μg/ml (3 μM); 6, 400 μg/ml (6 μM); 7, 800 μg/ml (12 μM); 8, 1200 μg/ml (18 μM); (B) As A except that MalE was added instead of MalE-MinC. The concentrations (in μg/ml) were the same as in A. (C–E) Samples were also taken 10 min after GTP addition and were examined by electron microscopy. C contained FtsZ alone (5 μM); D contained FtsZ (5 μM) with 3 μM (200 μg/ml) of MalE-MinC; E contained FtsZ (5 μM) and MalE at 200 μg/ml; and F contained FtsZ (5 μM) and MalE-MinC19 at 200 μg/ml).

Figure 5

MalE-MinC19 has reduced inhibitory activity in the FtsZ assembly assay. FtsZ was incubated with increasing amounts of MalE-MinC19 in polymerization buffer. Assembly was initiated with 1 mM GTP, and samples were centrifuged and were analyzed by SDS/PAGE. The amount of FtsZ in the pellet was quantitated and plotted along with the results obtained with MalE and MalE-MinC additions (data from Fig. 4). The amount of FtsZ in the pellet in the absence of MalE or the fusions was set at 100%. This represents ≈50% of the FtsZ in the reaction (9, 42).

Figure 6

Comparison of the effects of MinC and SulA on FtsZ. (A) The data obtained previously for SulA's effect on FtsZ (45) are plotted along with the results of MinC obtained in this study. The GTPase activity and amount of polymer obtained in the absence of inhibitor was set at 100%. The fractional values obtained in the presence of the inhibitors are plotted versus the molar ratio of inhibitor to FtsZ. For the purposes of comparison we have assumed the inhibitor is a monomer. The open symbols are GTPase activity, and the filled symbols are for polymerization. (B) This diagram contrasts the different steps affected by the two inhibitors. SulA blocks the GTPase activity and polymerization. In contrast, MinC prevents net assembly of FtsZ without inhibiting the GTPase activity, arguing that it promotes disassembly.