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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA
Nature Microbiology Open Access 09 August 2021
Nature Communications Open Access 04 June 2021
Scientific Reports Open Access 04 March 2020
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Adams, D. W. & Errington, J. Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nature Rev. Microbiol. 7, 642–653 (2009)
Bramkamp, M. & van Baarle, S. Division site selection in rod-shaped bacteria. Curr. Opin. Microbiol. 12, 683–688 (2009)
Wu, L. J. & Errington, J. Nucleoid occlusion and bacterial cell division. Nature Rev. Microbiol. 10, 8–12 (2012)
Pinho, M. G., Kjos, M. & Veening, J. W. How to get (a)round: mechanisms controlling growth and division of coccoid bacteria. Nature Rev. Microbiol. 11, 601–614 (2013)
Monahan, L. G. & Harry, E. J. Identifying how bacterial cells find their middle: a new perspective. Mol. Microbiol. 87, 231–234 (2013)
Monahan, L. G., Liew, A. T. F., Bottomley, A. L. & Harry, E. J. Division site positioning in bacteria: one size does not fit all. Front. Microbiol. 5, 19 (2014)
Beilharz, K. et al. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. Proc. Natl Acad. Sci. USA 109, E905–E913 (2012)
Fleurie, A. et al. Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae. Mol. Microbiol. 83, 746–758 (2012)
Fleurie, A. et al. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet. 10, e1004275 (2014)
Hanks, S. K., Quinn, A. M. & Hunter, T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52 (1988)
Kannan, N., Taylor, S. S., Zhai, Y., Venter, J. C. & Manning, G. Structural and functional diversity of the microbial kinome. PLoS Biol. 5, e17 (2007)
Pereira, S. F., Goss, L. & Dworkin, J. Eukaryote-like serine/threonine kinases and phosphatases in bacteria. Microbiol. Mol. Biol. Rev. 75, 192–212 (2011)
Nováková, L. et al. Identification of multiple substrates of the StkP Ser/Thr protein kinase in Streptococcus pneumoniae. J. Bacteriol. 192, 3629–3638 (2010)
Bernsel, A., Viklund, H., Hennerdal, A. & Elofsson, A. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res. 37, W465–W468 (2009)
Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew. Chem. Int. Edn Engl. 51, 12519–12523 (2012)
Daniel, R. A. & Errington, J. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 113, 767–776 (2003)
Ma, X. & Margolin, W. Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ. J. Bacteriol. 181, 7531–7544 (1999)
Singh, J. K., Makde, R. D., Kumar, V. & Panda, D. A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ. Biochemistry 46, 11013–11022 (2007)
Haney, S. A. et al. Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA. J. Biol. Chem. 276, 11980–11987 (2001)
Król, E. et al. Bacillus subtilis SepF binds to the C-terminus of FtsZ. PLoS ONE 7, e43293 (2012)
Wheeler, R., Mesnage, S., Boneca, I. G., Hobbs, J. K. & Foster, S. J. Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria. Mol. Microbiol. 82, 1096–1109 (2011)
Tomasz, A., Jamieson, J. D. & Ottolenghi, E. The fine structure of Diplococcus pneumoniae. J. Cell Biol. 22, 453–467 (1964)
Higgins, M. L. & Shockman, G. D. Model for cell wall growth of Streptococcus faecalis. J. Bacteriol. 101, 643–648 (1970)
Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. An rpsL cassette, Janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl. Environ. Microbiol. 67, 5190–5196 (2001)
Martin, B., Prudhomme, M., Alloing, G., Granadel, C. & Claverys, J. P. Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae. Mol. Microbiol. 38, 867–878 (2000)
Cortay, J. C. et al. In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis. J. Biol. Chem. 269, 14885–14891 (1994)
Canova, M. J., Kremer, L. & Molle, V. pETPhos: a customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates. Plasmid 60, 149–153 (2008)
Martin, B. et al. Expression and maintenance of ComD–ComE, the two-component signal-transduction system that controls competence of Streptococcus pneumoniae. Mol. Microbiol. 75, 1513–1528 (2010)
Thanbichler, M. & Shapiro, L. MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 126, 147–162 (2006)
de Jong, I. G., Beilharz, K., Kuipers, O. P. & Veening, J. W. Live cell imaging of Bacillus subtilis and Streptococcus pneumoniae using automated time-lapse microscopy. J. Vis. Exp. 53, 3145 (2011)
Lesterlin, C., Ball, G., Schermelleh, L. & Sherratt, D. J. RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506, 249–253 (2014)
Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008)
Gustafsson, M. G. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008)
Sliusarenko, O., Heinritz, J., Emonet, T. & Jacobs-Wagner, C. High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol. Microbiol. 80, 612–627 (2011)
Krzywinski, M. & Altman, N. Points of significance: significance, P values and t-tests. Nature Methods 10, 1041–1042 (2013)
Krzywinski, M. & Altman, N. Points of significance: importance of being uncertain. Nature Methods 10, 809–810 (2013)
Król, E. & Scheffers, D. J. FtsZ polymerization assays: simple protocols and considerations. J. Vis. Exp. 81, e50844 (2013)
Morlot, C. et al. Interaction of penicillin-binding protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis. Mol. Microbiol. 90, 88–102 (2013)
Morlot, C., Zapun, A., Dideberg, O. & Vernet, T. Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle. Mol. Microbiol. 50, 845–855 (2003)
Borchert, N. et al. Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. Genome Res. 20, 837–846 (2010)
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols 2, 1896–1906 (2007)
Olsen, J. V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021 (2005)
Franz-Wachtel, M. et al. Global detection of protein kinase D-dependent phosphorylation events in nocodazole-treated human cells. Mol. Cell. Proteomics 11, 160–170 (2012)
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)
Combet, C., Blanchet, C., Geourjon, C. & Deleage, G. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147–150 (2000)
UniProt Consortium Update on activities at the Universal Protein Resource (UniProt) in 2013. Nucleic Acids Res. 41, D43–D47 (2013)
Pearson, W. R. & Lipman, D. J. Improved tools for biological sequence comparison. Proc. Natl Acad. Sci. USA 85, 2444–2448 (1988)
Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004)
Eddy, S. R. Accelerated Profile HMM Searches. PLOS Comput. Biol. 7, e1002195 (2011)
Flicek, P. et al. Ensembl 2013. Nucleic Acids Res. 41, D48–D55 (2013)
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)
About this article
Cite this article
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
This article is cited by
Nature Chemical Biology (2021)
Nature Chemical Biology (2021)
CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA
Nature Microbiology (2021)
Nature Communications (2021)
Scientific Reports (2020)