In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at mid-cell1. Over the past decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ ring2,3,4,5. However, the human pathogen Streptococcus pneumoniae, in common with many other organisms, is devoid of these canonical systems and the mechanisms of positioning the division machinery remain unknown4,6. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in pneumococcus. Mid-cell-anchored protein Z (MapZ) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ ring through direct protein–protein interactions. MapZ-mediated control differs from previously described systems mostly on the basis of negative regulation of FtsZ assembly. Furthermore, MapZ is an endogenous target of the Ser/Thr kinase StkP, which was recently shown to have a central role in cytokinesis and morphogenesis of S. pneumoniae7,8,9. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades.
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This work was supported by grants from the CNRS, the Université Claude Bernard Lyon 1, the FINOVI foundation, the Agence National de la Recherche (ANR-12-BSV3-0008-01) and the Region Rhône-Alpes (ARC1 and financial support for A.F. (Cluster 10) and C.Z. (CMIRA)). C.L. was supported by a Wellcome Trust Programme Grant to D.S. (WT083469MA), advanced microscopy at Micron Oxford was supported by a Wellcome Trust Strategic Award (091911), and Y.V.B. was supported by a National Institutes of Health grant (GM051986). We acknowledge the contribution of the PLATIM and the Protein Science facilities of the SFR Biosciences Gerland-Lyon Sud (UMS344/US8) and the Centre Technologique des Microstructures de l’Université Lyon 1. We thank C. Chamot for technical assistance in microscopy, S. Uphoff and J. Wiktor for providing help with Matlab and A.-M. Di Guilmi for providing us with the antibody against pneumococcal FtsZ. We thank N. Campo, N. Dubarry and J.-P. Claverys for stimulating discussions and critical reading of the manuscript. N. Campo is also thanked for her help with time-lapse microscopy.
The authors declare no competing financial interests.
Extended data figures and tables
a, Cell length distribution of wild-type, ΔmapZ, mapZ-2TA, mapZ-2TE, mapZΔcyto, mapZΔextra and mapZΔ(1–41) strains, as well as for ΔmapZ/PZn-mapZ in the presence of 0, 0.1 or 0.2 mM of ZnCl2 inducer. Average cell length (L) and width (W) are given with standard deviations for a total of n cells analysed from three independent experiments. P value < 1.59 × 10−2, two-tailed t distribution determined using a non-parametric statistical test for a critical value of 0.05. b, Phase contrast microscopy and FM4-64 membrane staining imaging of mapZ+ cells (mapZ is restored at the chromosomal locus in ΔmapZ), ΔmapZ/PZn-mapZ (ΔmapZ cells complemented ectopically with PZn-mapZ), mapZΔcyto and mapZΔextra cells. Images are representative of experiments made in triplicate.
a, MapZ topology (that is, specification of the membrane spanning segments and their in/out orientation relative to the membrane) was predicted by five different topology algorithms (SCAMPI-seq, SACAMPI-msa, PRODIV-TMHMM, PRO-TMHMM and OCTOPUS) using TOPCONS (http://topcons.net). ZPRED (green line) predicts the distance to the membrane centre of each amino acid and ΔG scale (light blue) shows the predicted free energy of membrane insertion for a window of 21 amino acids centred around each position in the sequence. The transmembrane span is indicated in grey. Predictions of cytoplasmic and extracellular localizations are shown in red and dark blue, respectively. b, Wide-field microscopy images of cells producing the C-terminal fusion of MapZ with GFP. GFP fluorescence (right panel) and phase contrast images (left panel). Images are representative of experiments made in triplicate.
a, Microscopy images of GFP–MapZ and FtsZ–RFP in wild-type cells. Insert images show 3D-SIM orthogonal views of MapZ and FtsZ rings. b, Localization dotplots of MapZ-ring and FtsZ-ring positions along the cell length in wild-type cells. c, Ratio of cells with single or multiple MapZ rings and FtsZ rings as a function of cell length. b, c, Data are derived from analysis of 1,036 cells (n indicates the number of cells analysed in each panel). d, Distance between MapZ outer rings compared with distance between FtsZ outer rings as a function of cell length. e, Same as Fig. 2b but after swapping the GFP and RFP fluorescent protein labels. Indicative images showing MapZ, FtsZ, or both MapZ and FtsZ signals are shown for rfp-mapZ ftsZ-gfp cells at four different cell cycle stages. A wide-field view is also shown. Images are representative of experiments made in triplicate.
a, Time-lapse images of GFP–MapZ dynamics during cell growth and division showing progressive separation of the outer rings (green arrow) and appearance of a 3rd mid-cell ring (red arrow) (similar to Supplementary Video 1). Time is given in minutes. b, 3D-SIM images showing the very early stages of MapZ separation in the first stages of cell elongation. The numbers correspond to the inter-ring distance (IRD) in nm. c, Cell size distribution of cells with two MapZ rings reveals splitting of MapZ in the early stages of cells elongation. d, Distribution of IRD in cells with two MapZ rings (error bars show s.d. from three experiments). c, d, Data are derived from analysis of 280 cells (n indicates the number of cells analysed in each panel). Images are representative of experiments made in triplicate.
Extended Data Figure 5 MapZ position depends on PG synthesis and FtsZ position depends on MapZ functionality.
a, Localization of MapZ after inhibition of PG synthesis in wild-type pneumococcus. Microscopy images of GFP–MapZ in wild-type cells before (top), and 15 min after addition of vancomycin (middle) or norfloxacin (bottom). Vancomycin, which inhibits PG synthesis, impairs localization of GFP–MapZ, whereas norfloxacin, which inhibits topoisomerases, has no effect on MapZ septal localization. b, c, Localization of FtsZ–GFP in mapZΔcyto (b) and corresponding FtsZ–GFP ring positioning along the cell length normalized to 1 (c). d, e, Localization of FtsZ–GFP in mapZΔextra (d) and corresponding FtsZ–GFP ring positioning along the cell length normalized to 1 (e). Images are representative of experiments made in triplicate.
a, Time-lapse images of FtsZ–GFP (green) dynamics in ΔmapZ cells. FtsZ polymers fail to position correctly even in cells with normal shape (arrow 1), resulting in asymmetric cell division or cell lysis (arrow 2) (stills correspond to Supplementary Video 5). b, Microscopy images showing colocalization of PG synthesis revealed by pulse labelling with TDL (red) and mispositioned FtsZ–GFP structures (green) in ΔmapZ cells. Three fields of view from three independent experiments are shown. c, 3D-SIM and schematic of FtsZ dumbbells with histograms of the cell ratios with 1, 2 or 3 rings. The average number of rings per cell is shown. d, 3D-SIM image of DAPI-stained DNA and FtsZ–GFP in ΔmapZ cells. e, Localization of GFP–MapZΔ(1–41) in gfp-mapZΔ(1–41). f, Localization of FtsZ–GFP in mapZΔ(1–41). g, Corresponding FtsZ–GFP ring positioning along the cell length normalized to 1. h, i, Time-lapse images of FtsZ–GFP (green) dynamics in mapZΔ(1–41) (h) and mapZΔcyto (i) cells. FtsZ mispositioning, even in cells with normal shape leads to asymmetric cell division (arrow 1) or cell lysis (arrow 2). Images are representative of experiments made in triplicate.
a, Purification of proteins used in surface plasmon resonance experiments. The MapZ cytoplasmic domain, MapZ-2TE cytoplasmic domain, FtsZ, MapZ extracellular domain, FtsZ(1–407) fragment (FtsZ deleted from the C-terminal α-helix), StkP cytoplasmic domain, PhpP, MapZ N-terminal peptide from Met 1 to Gly 41, MapZ peptide from Val 42 to Ser 98 and MapZ peptide from Val 42 to Lys 158 were overproduced in E. coli BL21 and analysed by SDS–PAGE. b, Schematic model of MapZ and secondary structure prediction of the cytoplasmic domain of MapZ. Secondary structure codes ‘e’, ‘c’ and ‘h’ indicate predicted α-helices (blue), random coils (orange) and extended strands (green), respectively. c–e, Surface plasmon resonance analyses of interaction between FtsZ and MapZ. c–i, Full-length FtsZ (c, d, e, f, g, i) or FtsZ1-407 (h) was covalently coupled to the surface of a CM5 sensorchip. Increasing amounts of either MapZ cytoplasmic domain (c, h), MapZ extracellular domain (g), MapZ-2TE (i) cytoplasmic domain, MapZ(1–41) (d), MapZ(42–98) (e) and MapZ(42–158) (f) peptides were injected onto the FtsZ- or FtsZ(1–407)-coupled sensorship. RU, resonance units. The measurements were made in triplicate. The affinity (KD), association (Ka) and dissociation constants (Kd) are indicated. Images are representative of experiments made in triplicate.
Extended Data Figure 8 Analysis of MapZ in vivo phosphorylation and impact on FtsZ GTPase activity and polymerization.
a, b, MapZ is phosphorylated on threonine 67 (a) and threonine 78 (b). The spectra show the fragmentation pattern of the phosphopeptides DEIEADKFAT(ph)R corresponding to amino acids 58–68 and KEEFVET(ph)QSLDDLIQEM(ox)R corresponding to amino acids 72–89. c, Influence of MapZ and MapZ-2TE cytoplasmic domains on FtsZ GTPase activity. Purified FtsZ was incubated with GTP either alone or in the presence of MapZ or MapZ-2TE cytoplasmic domains and free phosphate was revealed using malachite green colour development. Data are shown with s.d. for three independent experiments. d, FtsZ polymerization in the presence of MapZcyto, wild-type or mutated, cytoplasmic domains. FtsZ was incubated in the presence or absence of GTP and either MapZ or MapZ-2TE. The samples were then processed as described in Methods. Images are representative of experiments made in triplicate.
Extended Data Figure 9 Interplay between MapZ and StkP and PhpP and conservation of MaZ in bacterial genomes.
a, Simultaneous localization of GFP–StkP and RFP–MapZ in wild-type cells. Overlays between GFP (green), RFP (red) and phase contrast show that StkP locates at mid-cell while MapZ ring separation proceeds, as depicted in the summary diagram below. b, Dephosphorylation of MapZ by PhpP. MapZ cytoplasmic domain was phosphorylated by StkPKD and then incubated for various times (30 s to 10 min) with the protein phosphatase PhpP. MapZ dephosphorylation was analysed by autoradiography. c, Conservation analysis of mapZ homologues in 6,305 bacterial genomes. The left panel shows the taxonomy of the bacterial superkingdom. The right panel indicates the number of genera, the number of sequenced genomes, the number of genomes coding for MapZ homologous proteins and the percentage of genomes coding for MapZ homologous proteins. Images are representative of experiments made in triplicate.
This file contains Supplementary Tables 1 and 2 and Supplementary References. (PDF 329 kb)
Time-lapse analysis of GFP-MapZ in wild-type cells. (MOV 685 kb)
3D-SIM of FtsZ-GFP in wild-type cells. (MPG 9078 kb)
3D-SIM of FtsZ-GFP in ΔmapZ cells. (MPG 6644 kb)
The video shows an overlay of GFP (green) and phase-contrast (gray) images. (MOV 642 kb)
The video shows an overlay of GFP (green) and phase-contrast (gray) images. (MOV 165 kb)
3D-SIM of DAPI-stained nucleoids in ΔmapZ cells showing chromosome pinching (MOV 6233 kb)
3D-SIM of FtsZ-GFP in DAPI-stained ΔmapZ cells showing aberrant chromosome associated with aberrant FtsZ structures
3D-SIM of FtsZ-GFP in DAPI-stained ΔmapZ cells showing aberrant chromosome associated with aberrant FtsZ structures. (MPG 8928 kb)
The video shows an overlay of GFP (green) and phase-contrast (gray) images. (MOV 208 kb)
The video shows an overlay of GFP (green) and phase-contrast (gray) images. (MOV 320 kb)
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Fleurie, A., Lesterlin, C., Manuse, S. et al. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature 516, 259–262 (2014). https://doi.org/10.1038/nature13966
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