Identification and characterization of ZapC, a stabilizer of the FtsZ ring in Escherichia coli

Abstract

In Escherichia coli, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with E. coli. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division.

FIG. 1.

Localization analyses of a ZapC-eYFP fusion in various strain backgrounds. Phase and fluorescein isothiocyanate (FITC) channels are shown. (a to d) Colocalization of ZapC-eYFP and FtsZ-eCFP in wild-type cells (JD45). (b) ZapC-eYFP localized at midcell. (c) FtsZ-eCFP localized at midcell. (d) Overlay of ZapC-eYFP and FtsZ-eCFP. (e to h) ZapC-eYFP localization in ftsZ84(Ts) background (JD20). A ZapC-eYFP fusion localizing to midcell at 30°C is shown (f); a ZapC-eYFP fusion shows diffuse localization at 42°C (h). (i and j) ZapC-eYFP localization in wild-type cells (JD16) treated with aztreonam to inactivate FtsI. (j) Fluorescent image showing a ZapC-eYFP fusion localizing at periodic intervals along the length of an aztreonam-induced filament. Scale = 5 μm.

FIG. 2.

FtsZ ring morphologies in cells lacking or overexpressing ZapC. FtsZ-GFP (pZG) localization in wild-type (MC4100) or zapC (JD18) cells. Phase, FITC, or Texas Red channels are shown. (a and b) FtsZ-GFP in wild-type cells. (c and d) FtsZ-GFP in zapC cells. (e to j) FtsZ morphologies as detected by immunolabeling with anti-FtsZ antibodies in cells overexpressing ZapC-GFP (JD10). (e and f) FtsZ in wild-type cells. (g and h) FtsZ containing the uninduced ZapC-GFP plasmid. (i and j) Helical FtsZ ring morphologies in cells overexpressing ZapC-GFP. Scale = 5 μm. (k) Immunoblotting analysis of levels of FtsZ expression in cells lacking ZapC (JD18) or overexpressing ZapC-GFP (JD10), performed using anti-FtsZ antibodies. Lanes 1 and 2, wild type (MC4100); lanes 3 and 4, uninduced ZapC-GFP cells; lanes 5 and 6, ZapC-GFP overexpressed; lanes 7 and 8, zapC. Lanes 2, 4, 6, and 8 were loaded with one-half of the sample loaded in lanes 1, 3, 5, and 7.

FIG. 3.

ZapC-FtsZ interaction in yeast as detected in PIP assays. (a) ZapC-eYFP (pAG415 GAL1 ZapC-eYFP) and μNS-FtsZ (pAG416 GAL1 μNS-FtsZ) fusions form discrete fluorescent foci when coexpressed in yeast. (b) A ZapC-eYFP fusion shows diffuse localization in yeast when expressed alone. Scale = 5 μM.

FIG. 4.

In vitro characterization of ZapC-FtsZ interactions. Both FtsZ and ZapC-His were at 5 μM concentrations when added to a total polymerization reaction volume of 100 μl. (A) Relative concentrations of FtsZ in supernatant (S) and pellet (P) from sedimentation assays were visualized by SDS-PAGE analysis. (B) Transmission electron microscopy images of reactions detailed in the sedimentation assays in part A with FtsZ in the presence of GDP (a), characteristic FtsZ protofilaments in the presence of GTP (b), or FtsZ bundles seen in the presence of ZapC-His (c).

FIG. 5.

Complementation of zapC cell length phenotype by a zapC-his fusion (JD59). Histograms of cell length in μm plotted against the percentage of cells for the wild type (MC4100, black; n = 570) or the zapC (JD18, red; n = 469) or zapC-his (green; n = 676) mutant, where n equals the total number of cells measured.