Spatial regulation of cell division by the nucleoid occlusion protein SlmA in Escherichia coli

Spatial regulation of cell division in bacteria occurs at the stage of Z ring formation, a cytoskeletal element that bacterial cells employ for assembly of the cell division machinery. In the model organism Escherichia coli, spatial regulation of Z ring formation is dependent on two partially redundant negative regulatory systems, the Min system, which prevents Z ring formation near the cell poles, and Nucleoid Occlusion (NO), which prevents Z ring formation over the nucleoid. The effector of the Min system, MinC, prevents assembly of the Z ring in its vicinity by antagonizing FtsZ polymerization and membrane attachment, the two essential activities required for FtsZ to assemble into the Z ring. Previous studies have shown that the effector of the NO system, SlmA, is a DNA associated FtsZ inhibitor that is activated by binding to a SlmA binding sequence (SBS). The SlmA binding site on FtsZ has not been identified and how SBS bound SlmA prevents FtsZ assembly into the Z ring in its vicinity is controversial. In this study, we show that SBS bound SlmA acts in a similar manner to MinC, antagonizing FtsZ polymerization and membrane attachment. In the first part of this thesis, two FtsZ mutants were isolated that are resistant to de-localized SBS bound SlmA, which has been shown to block Z ring formation throughout the cell and cause cell death. By characterizing these two FtsZ mutants, we found that SBS activated SlmA antagonizes FtsZ polymerization and the efficacy of SlmA to antagonize FtsZ polymerization depends upon the length of the DNA molecule containing the SBS. The longer the bound SBS DNA molecule (14-30 bp), the more efficiently SlmA disassembles FtsZ polymers; SlmA bound to the shorter SBS DNA molecule is missing several DNA contacts likely explaining the weaker impact on FtsZ polymerization. Even though the isolated ftsZ mutations conferred resistance to the action of SlmA in vivo and in vitro, they did not disrupt FtsZ-SlmA binding. One of the ftsZ mutations increased the bundling of FtsZ polymers in vitro, indicating that it provides resistance to SlmA by increasing FtsZ lateral interactions. The other ftsZ mutation alters a residue in the H7 helix of FtsZ. This helix mediates the conformational change between the two sub-domains of FtsZ during assembly suggesting that SBS bound SlmA antagonizes FtsZ polymerization by reversing this conformational change and that the mutation is resistant to this affect. In the second part of the project, we found that SlmA binds to FtsZ largely through the conserved C-terminal tail of FtsZ, a region critical for FtsZ-ZipA and FtsZ-FtsA interactions and therefore attachment of FtsZ filaments to the membrane. More importantly, we found that SlmA requires the presence of the conserved C-terminal tail of FtsZ to disassemble FtsZ polymers. As the conserved C-terminal tail of FtsZ is not required for FtsZ polymerization, this unexpected finding suggests that SlmA binding to the FtsZ tail allows it to bind to a secondary site in the globular domain of FtsZ to antagonize FtsZ polymerization. This two binding site model is consistent with the observation that SlmA forms a sandwich like complex with FtsZ truncations lacking the conserved C-terminal tail and our finding that ftsZ mutations in the globular domain of FtsZ confer resistance to the action of SlmA. Collectively, our results suggest that SlmA antagonizes Z ring formation in its vicinity in at least two ways: first, SBS bound SlmA competes with ZipA and FtsA for the conserved C-terminal tail of FtsZ preventing membrane attachment of FtsZ filaments; and, second, the binding to the conserved C-terminal tail of FtsZ brings the SBS-SlmA complexes close to FtsZ filaments such that SlmA can actively disassemble FtsZ polymers by reversing the conformational change occurring upon FtsZ assembly. ZipA and FtsA promote Z ring assembly by tethering FtsZ filaments to the membrane through the conserved FtsZ tail. In contrast, MinC and SlmA promote Z ring disassembly by binding the tail because they also have an antagonistic effect on FtsZ polymers. Competition for the FtsZ tail between Z ring promoting factors and Z ring disassembling factors may be an important way to regulate Z ring formation. The remarkable similarity between MinC and SlmA also indicates that antagonizing FtsZ polymerization and FtsZ filaments membrane attachment simultaneously may be a universal mechanism for FtsZ spatial regulators to antagonize Z ring formation in their vicinity

SlmA's mode of action shares features employed by MinCD.

<p>FtsZ (PDB: 1OFU) filaments are tethered to the membrane by interaction of the conserved tail of FtsZ with the membrane anchors ZipA (PDB:1F47) and FtsA (PDB:4A2A). The conserved tail of FtsZ (green helix or cylinder) is connected to the body of FtsZ by an unstructured linker (black dashed line). MinD (PDB:3Q9L) binds to the membrane and recruits MinC (PDB:1HF2), which interacts with the tail of FtsZ through the MinC^C domain and a second interaction mediated by the MinC^N domain. Binding to DNA activates SlmA (PDB: 4GCL) to bind the tail of FtsZ which we propose results in further interaction disrupting the FtsZ filament. The brown spheres indicate residues in SlmA that affect the binding of FtsZ (21) and may be where the tail binds.</p

SlmA Antagonism of FtsZ Assembly Employs a Two-pronged Mechanism like MinCD

<div><p>Assembly of the Z-ring over unsegregated nucleoids is prevented by a process called nucleoid occlusion (NO), which in <i>Escherichia coli</i> is partially mediated by SlmA. SlmA is a Z ring antagonist that is spatially regulated and activated by binding to specific DNA sequences (SlmA binding sites, SBSs) more abundant in the origin proximal region of the chromosome. However, the mechanism by which SBS bound SlmA (activated form) antagonizes Z ring assembly is controversial. Here, we report the isolation and characterization of two FtsZ mutants, FtsZ-K190V and FtsZ-D86N that confer resistance to activated SlmA. In trying to understand the basis of resistance of these mutants, we confirmed that activated SlmA antagonizes FtsZ polymerization and determined these mutants were resistant, even though they still bind SlmA. Investigation of SlmA binding to FtsZ revealed activated SlmA binds to the conserved C-terminal tail of FtsZ and that the ability of activated SlmA to antagonize FtsZ assembly required the presence of the tail. Together, these results lead to a model in which SlmA binding to an SBS is activated to bind the tail of FtsZ resulting in further interaction with FtsZ leading to depolymerization of FtsZ polymers. This model is strikingly similar to the model for the inhibitory mechanism of the spatial inhibitor MinCD.</p></div

Further analysis of FtsZ-SlmA binding using biolayer interferometry.

<p>A) Single amino acid substitutions in the FtsZ tail abolish FtsZ-SlmA binding. The biotinylated SBS17-30mer was immobilized on a streptavidin biosensor and dipped into tubes containing 4 µM SlmA. Two minutes after incubation, the tips with SlmA bound to the biotinylated SBS17-30me were washed for 10 seconds and then moved to tubes containing FtsZ (4 µM) or its mutants and the association monitored. B) ZipA_185–328 competes with SBS bound SlmA for the conserved C-terminal tail of FtsZ. Streptavidin biosensor tips were loaded with SlmA/SBS17-30mer complexes as before. The tips were washed for 10 seconds and then moved to tubes containing 4 µM FtsZ preincubated with various concentrations of ZipA_185–328 and the association monitored. C) The FtsZ C-terminal tail peptide is sufficient for SBS-SlmA binding. Experiments were performed as in (A). Streptavidin biosensor tips were loaded with 50 nM biotinylated SBS17-30mer for 5 minutes followed by a 10 second wash. SBS17-30mer-SlmA complexes were generated by moving the SBS17-30mer coated tips to tubes containing 4 µM SlmA. The SlmA loaded tips were washed for 10 seconds and then moved to tubes containing FtsZ C-terminal tail peptide (Ztail-WT) or mutants to measure the association. A peptide corresponding to the cytoplasmic N-terminus of FtsN was also used as a nonspecific control. D) The FtsZ C terminal tail peptide competes with FtsZ for binding to SlmA-SBS. Streptavidin biosensor tips containing biotinylated SB17-30mer with bound SlmA were moved to tubes containing 4 µM FtsZ preincubated with various concentrations of the FtsZ C-terminal peptide and the association monitored.</p

FtsZ filaments have the opposite kinetic polarity of microtubules

FtsZ is the ancestral homolog of tubulin and assembles into the Z ring that organizes the division machinery to drive cell division in most bacteria. In contrast to tubulin that assembles into 13 stranded microtubules that undergo dynamic instability, FtsZ assembles into single-stranded filaments that treadmill to distribute the peptidoglycan synthetic machinery at the septum. Here, using longitudinal interface mutants of FtsZ, we demonstrate that the kinetic polarity of FtsZ filaments is opposite to that of microtubules. A conformational switch accompanying the assembly of FtsZ generates the kinetic polarity of FtsZ filaments, which explains the toxicity of interface mutants that function as a capper and reveals the mechanism of cooperative assembly. This approach can also be employed to determine the kinetic polarity of other filament-forming proteins

FtsZ mutants are resistant to SlmA antagonism of FtsZ polymerization.

<p>A) Polymerization of FtsZ with or without the addition of SlmA and SBS17-30mer. Reactions were performed in 50 µl volume containing FtsZ or one of the mutants (2 µM) and GTP (1 mM) with or without the addition of SlmA (2 µM) and SBS17-30mer DNA (2 µM) in FtsZ polymerization buffer (50 mM HEPES pH 8.0, 10 mM MgCl₂, 200 mM KCl). Reactions were incubated at room temperature for 5 minutes before being spotted onto grids and negatively stained with 1% uranyl acetate for visualization by electron microscopy. B) Stable FtsZ filaments are bundled by SlmA/SBS. Reactions were carried out as in (A) except that GTP was replaced with GMPCPP. C) Comparison of FtsZ bundles and SBS-SlmA-FtsZ bundles obtained in the presence of GMPCPP. At 5 µM FtsZ assembles into bundles, which contain nicely aligned FtsZ proto-filaments. In contrast, the FtsZ bundles induced by SBS-SlmA have a rougher appearance and seen at lower FtsZ concentrations. Reactions were performed as in B).</p

FtsZ-K190V and FtsZ-D86N bind SlmA.

<p>A) SBS17-30mer bound SlmA co-sediments with stable FtsZ polymers formed with GMPCPP. Polymerization assays were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004460#pgen-1004460-g003" target="_blank">Fig. 3A</a> except that the protein concentration was 5 µM and GTP was replaced by GMPCPP. After the reactions were incubated at room temperature for 5 minutes, FtsZ polymers were sedimented by ultracentrifugation. Proteins in the supernatant and pellet fractions were separated by SDS-PAGE. B) Biolayer interferometry assay to assess FtsZ binding to SlmA bound to DNA. Streptavidin biosensor tips loaded with biotin conjugated SBS17-30mer and SlmA were incubated with FtsZ or the FtsZ mutants (4 µM) and the association monitored.</p

FtsZ tail mutants resistant to MinC/MinD display differential sensitivity to de-localized SlmA.

<p>Plasmids pSD133 (P<i>tac::slmA</i>) and p2SBSK (pUC18 with 2SBS sites) were introduced into the <i>ftsZ⁻</i> strain DU11 (<i>ftsZ⁰ slmAfrt<>recA::Tn10</i>) complemented with pBANG112 or its derivatives containing different <i>ftsZ</i> alleles. One colony of each resultant strain was resuspended in 1 ml LB medium, serially diluted by 10 and 3 µl from each dilution was spotted on LB plates containing various concentrations of IPTG and supplemented with antibiotics. The plates were incubated at 30°C for 30 hours before being photographed.</p