Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis

Abstract

Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4-10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

Fig 1. FtsZ domain structure, FtsZ C-terminal tail (CTT) structure and FtsZ C-terminal variable (CTV) mutant constructs.

A. Domain organization of E. coli FtsZ: an unstructured 10 residues at the N-terminal end (squiggly line), a conserved globular core domain containing the nucleotide binding and hydrolysis residues, a flexible variable linker about 50 residues long (squiggly line), and a conserved carboxy terminal peptide (CCTP) which contains both a constant region of ~13 residues (CTC) and a variable region of 4 residues (CTV). B. Structural model of the FtsZ C-terminal residues 367–383 (PDB 1F47) [29]. In a X-ray crystal structure complex with the essential division protein ZipA, the 17 residue FtsZ CCTP binds as an extended β-strand followed by an α-helix. The CTV residue side-chains are identified in the α-helix: K380 (blue), Q381 and A382 (gray) and D383 (red). C. Schematic of the FtsZ CTV mutant constructs used in the study, not drawn to scale.

Fig 2. Sedimentation reactions of purified FtsZ and FtsZ CTV mutant proteins with ZapD.

A. FtsZ and FtsZ CTV mutants (5 ∝M) were incubated alone or combined with purified ZapD at 1:0.2 or 1:1 ratios in a polymerization buffer (50 mM K-MOPS; pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) containing 3 ∝M BSA. Reactions were processed as outlined in the Materials and Methods section in the main text. Equivalent aliquots (5 ∝l) of pellet (bottom panel) and supernatants (top panel) were resolved on a 12.5% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen). A representative gel image of three independent experiments is shown. B. The amounts of FtsZ or FtsZ CTV mutant proteins present in the pellet fractions in reactions with or without ZapD are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments. Of note, FtsZ CTV containing NRNKRG sequences show the highest pelletable amounts of FtsZ under the experimental conditions of this study. C. The amounts of ZapD protein present in the pellet fractions in reactions with FtsZ or FtsZ CTV mutants are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments.

Fig 3. Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.

In vitro reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.

Fig 4. Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in ftsZ84 (Ts) cells.

A. FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the ftsZ84 (Ts) allele. Overnight cultures were normalized to OD₆₀₀, serially diluted, and 3 μl aliquots were spotted on LB and LBNS agar plates with 1 mM IPTG plus appropriate antibiotics, and incubated at 30°C and 42°C as described in the material and methods section. At the permissive condition (30°C LB; left) FtsZ and FtsZ CTV mutants are able to support growth except FtsZ_1-379 and DQAD. At the non-permissive condition (42°C LBNS; right) most FtsZ CTV mutants are able to support growth to WT levels except DQAD. B. FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ_1-379 or DQAD mutants in trans at mid-log phase (OD₆₀₀ = ~0.6) during growth at permissive or restrictive conditions as described in the materials and methods section in the main text. (a) ftsZ84 (Ts) cells grown at 30°C in LB; (b) ftsZ84 (Ts) cells grown at 42°C in LBNS; (c) ftsZ84 (Ts) cells with FtsZ_1-379 expressed in trans grown at 30°C in LB; (d) ftsZ84 (Ts) cells with FtsZ_1-379 expressed in trans grown at 42°C in LBNS; and (e) ftsZ84 (Ts) cells with DQAD expressed in trans grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. C. Overnight cultures of MGZ84 strains bearing FtsZ and FtsZ CTV mutant plasmids were grown in permissive conditions and subcultured into LB at 30°C till OD₆₀₀ = 0.2–0.3 at which point an aliquot was washed, and backdiluted to OD₆₀₀ = 0.05 in LBNS media and transferred to 42°C. After one doubling (~25–30 mins) at 42°C, 1 mM IPTG was added and cells were grown for an additional two doublings (~1 hour). Cells were harvested for whole cell protein preparations and sampled at equivalent optical densities. Protein samples were analyzed by immunoblotting. RpoD was used as a loading and transfer control. ImageStudio software was used to quantify band intensities. Three independent experiments were conducted and a representative blot with relative intensities is shown.

Fig 5. The FtsZ CTV region is required for localization of ZapD-GFP to midcell.

Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion in trans were subcultured in M63 glycerol minimal media in the presence of appropriate antibiotics at the permissive temperature (30°C) till OD₆₀₀ = 0.2–0.3 at which point an aliquot was washed and backdiluted to OD₆₀₀ = 0.05 in the same media and transferred to the restrictive temperature (42°C) for one doubling (~ 1 hour). Expression of FtsZ and ZapD were induced by addition of 1 mM IPTG and grown for an additional one-two doublings (~90 mins) at the same temperature. Fluorescent images were obtained as described in the materials and methods section. Arrows point to midcell ZapD-GFP fusion localization. Bar = 5 μm.