Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ

Abstract

In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coli FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

FIG. 1

Alignment of C-terminal core domains of diverse FtsZ homologs; the bottom two are plant chloroplast homologs. The invariant proline residue, corresponding to P375 of E. coli FtsZ, is highlighted in boldface. The underlined residues denote residues changed in this study to an alanine. Arabidopsis thaliana FtsZ refers to one of at least two chloroplast homologs; the other homolog lacks the homology to the core. Archaeal FtsZs also lack this domain, as do those from other Mycoplasma species.

FIG. 2

FtsZ truncation and point mutant derivatives. ZC* represents six mutants with single-residue changes in the C-terminal core. Slanted hatches represent the N-terminal conserved domain, horizontal hatches represent the C-terminal core, and the checkered pattern indicates the C-terminal core that contains mutations. The numbers indicate the beginning or ending residues relative to those of wild-type FtsZ. ZΔC2, ZΔC3, ZΔC4, and ZΔN all contain C-terminal GFP fusions (see Materials and Methods).

FIG. 3

Western blot analysis of FtsZ and its derivatives expressed in JM105 (wild type) and JFL101 (ftsZ84). (A) JM105 derivatives were grown exponentially at 37°C and then induced with 5 μM IPTG for 180 min. (B) JFL101 derivatives were grown at 42°C for 30 min before being induced with 10 μM IPTG for 90 min. The cells were then collected, and total protein was quantitated by bicinchoninic protein assay (Pierce). An equivalent amount of protein (25 μg) was loaded onto each lane, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Lane 1, cells containing pWM176; lane 2, pMK4 (wild-type ftsZ); lane 3, pWM1202 (ZL372A); lane 4, pWM738 (ZD373A); lane 5, pWM1203 (ZI374A); lane 6, pWM739 (ZP375A); lane 7, pWM1204 (ZF377A); lane 8, pWM1205 (ZL378A); lane 9, pWM737 (ZΔC1); lane 10, pWM930 (ZΔC1.1); lane 11, pWM931 (ZΔC1.2); lane 12, pWM932 (ZΔC1.3); lane 13, pWM1201 (ZΔC2).

FIG. 4

Localization of FtsZ and its derivatives in JFL101 [ftsZ84(Ts)] cells. JFL101 derivatives containing various plasmids were grown at 42°C for 30 min, induced with 10 μM IPTG for 60 min, and then fixed for staining. Simultaneously, FtsZ was stained by anti-FtsZ and visualized by IFM (A to D), nucleoids were stained with DAPI and visualized by fluorescence microscopy (A′ to D′), and cell morphologies were visualized by phase-contrast microscopy (A" to D"). (A to A") JFL101 containing pWM176; (B to B") JFL101 containing pMK4 (wild-type ftsZ); (C to C") JFL101 containing pWM1202 (ZL372A); (D to D") JFL101 containing pWM932 (ZΔC1.3).

FIG. 5

Localization of FtsZ and its derivatives in WM1099 (ftsZ depletion strain) cells. WM1099 derivatives were grown at 42°C for 5 h before induction with 5 μM IPTG for 40 min and fixation. Simultaneously, FtsZ was stained by anti-FtsZ and visualized by IFM (A to D), nucleoids were stained with DAPI and visualized by fluorescence microscopy (A′ to D′), and cell morphologies were visualized by phase-contrast microscopy (A" to D"). (A to A") WM1099 containing pWM176; (B to B") WM1099 with pMK4 (wild-type ftsZ); (C to C") WM1099 with pWM1202 (ZL372A); (D to D") WM1099 with pWM932 (ZΔC1.3).

FIG. 6

Association of FtsA-GFP with FtsZ spirals in JFL101 [ftsZ84(Ts)]. JFL101 cells containing plasmids synthesizing FtsA-GFP and an FtsZ derivative were grown at 42°C for 30 min and then induced with 200 μM IPTG for 60 min. Parallel samples were fixed and stained with anti-GFP antibodies for FtsA-GFP and stained with anti-FtsZ antibodies for FtsZ, followed by IFM. (A to E) FtsA-GFP; (A′ to E′) FtsZ. (A and A′) WM1234 (JFL101 containing pWM633 [ftsA-GFP] and pWM176); (B and B′) WM1235 (pMK4 [wild-type ftsZ]); (C and C′) WM1242 (pWM738 [ZD373A]); (D and D′) WM1244 (pWM739 [ZP375A]); (E and E′) WM1236 (pWM737 [ZΔC1]).

FIG. 7

Binding of ZipA-GFP to FtsZ rings and spirals in the ftsZ depletion strain WM1099. WM1099 containing pWM1206 (zipA-GFP) and various ftsZ derivatives were grown at 42°C for 4 h and then induced by 0.2% l-arabinose and 10 μM IPTG for 60 min. As described in the legend to Fig. 6, parallel samples were taken. One was stained with anti-GFP for ZipA-GFP and the other was stained with anti-FtsZ for FtsZ. The cells were observed by IFM for ZipA-GFP (A to D) and for FtsZ (A′ to D′). (A and A′) WM1221 (WM1099 containing pWM1206 [zipA-GFP] and pWM176); (B and B′) WM1222 (pWM1206 plus pMK4 [wild-type ftsZ]); (C and C′) WM1231 (pWM1206 plus pWM739 [ZP375A]); (D and D′) WM1223 (pWM1206 plus pWM737 [ZΔC1]).