Genetic and biochemical characterization of the MinC-FtsZ interaction in Bacillus subtilis.

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species

FtsZ polymers by SAXS and cryo-EM

32 p.-6 fig.+ 26 p. mat.supl.FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM). We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5⁰ - 5⁰ curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16 subunit - 220 Å diameter mini-rings, here observed by cryo-EM. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (~50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the inter-filament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.This work was supported by grants MINECO BFU2014-51823-R (JMA), SAF2014-52301-R (OL), BFU2016-75319-R (JFD), EMBO fellowship ALTF-171-2005 (MAO), FAPESP grant 13/26897-7 (BEPE) and 10/51870-7 (PC), doctoral contracts CSIC-JAE (ERA) and FPI (AV).Peer reviewe

FtsZ mutant measurements.

<p>FtsZ mutant measurements.</p

Cell division phenotype of FtsZ mutants.

<p>Strains containing an IPTG inducible allele of <i>minD</i> (<i>thrC</i>::P_spac-hy-<i>minD</i>, <i>erm</i>) plus either wild-type or Min-resistant <i>ftsZ</i> alleles were grown on LB-agar chambers with or without 1 mM IPTG for 2 hours and imaged by microscopy. Membranes were stained with FM 5–95. Arrowheads point to a typical minicell (yellow) and an abnormally sized minicell (red). Strain numbers: FtsZ wild-type (AB164), T111A (AB70), K243R (AB168), D287V (AB174), R376T (AB177). The scale bar corresponds to 5 µm.</p

The binding site for MinC differs in <i>B. subtilis</i> and <i>E. coli</i> FtsZ.

<p>A. Comparison between mutations that promote MinC resistance in <i>B. subtilis</i> (red residues) and <i>E. coli</i> (blue residues) mapped onto the 2VAM FtsZ structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060690#pone.0060690-Oliva2" target="_blank">[75]</a>. <i>E. coli</i> residue N280 corresponds to <i>B. subtilis</i> D280; E276 corresponds to Q276; R271 corresponds to S271. Two other important landmarks are highlighted in the structure: catalytic residue D213, shown in yellow, defines the center of the FtsZ-FtsZ interface (the polymerization axis follows a vertical line through this residue); and the point where the CTP should emerge from the structure (residue F315) is shown in orange. B. Surface electrostatic potential of <i>B. subtilis</i> FtsZ (2VAM) obtained with the “protein contact potential” tool of PyMOL. Note that the MinC binding site corresponds to a highly negative region of the FtsZ molecule.</p

Electron microscopy of FtsZ polymers formed in the presence or absence of MinC.

<p>Purified wild-type and mutant FtsZ were polymerized in reactions containing 5 µM of FtsZ, Mes/NaOH 50 mM, MgCl₂ 10 mM, KCl 133 mM, pH 6.5, and either no (top row) or 20 µM MinC (bottom row). Polymerization reactions were spotted on carbon coated 400 mesh copper grids and stained with uranyl acetate for transmission electron microscopy. Scale bars equal 200 nm.</p

Polymerization of FtsZ mutants in vitro is not affected by MinC.

<p>A. Representative light scattering trace of an experiment performed with wild-type FtsZ in the absence of inhibitor and in the presence of MinC or MinC19. Reactions contained 7 µM of FtsZ and 20 µM of MinC or MinC19 in Mes/NaOH 50 mM, MgCl₂ 10 mM, KCl 133 mM, DEAE-dextran 0.6 mg/mL, pH 6.5. B. Experiments similar to A were performed and the inhibition of the polymerization of each FtsZ mutant by MinC was quantified relative to the maximum light scattering signal in the absence of MinC. The original light scattering traces corresponding to this graph can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060690#pone.0060690.s004" target="_blank">Fig. S4</a>. Measurements were repeated at least three times for each FtsZ mutant with similar results.</p

Trp fluorescence measurements of MinC binding to mutant FtsZ.

<p>Wild-type or double-mutant (K243R,D287V) FtsZ <b>(</b>2 µM) were mixed with 1 µM MinC Y44W in buffer Tris/HCl 20 mM, KCl 100 mM, EDTA 5 mM, pH 7,5 and fluorescence emission at 320–360 nm was recorded. A. Representative trace of one experiment. B. Averaged quantitation of 3 experiments, with standard deviations.</p