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Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis

Abstract

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively1,2. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments—forming the Z ring—that undergo treadmilling and recruit later divisome proteins3,4. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division3. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation5,6. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery7,8, which is diverted from the cell periphery to the septum in preparation for division9. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB–DivIC–FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

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Figure 1: Localization of PG synthesis proteins during division of S. aureus.
Figure 2: Hierarchy of the arrival of key PG synthesis proteins to the divisome.
Figure 3: Recruitment of MurJ to the divisome is required for PG incorporation at the septum.
Figure 4: PC190723 impairs FtsZ55–56sGFP treadmilling in S. aureus.
Figure 5: PG synthesis provides force for septum constriction.

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Acknowledgements

We thank R. Sobral for the construction of pSG-murF plasmid; A. Jorge for the construction of the pSG-EzrA plasmid; T. Roemer for providing DMPI and strains AS-022 and AS-185; M. DeLisa for pTRC99a-P7; Richard Novick for pCN51; S. Foster for antibodies against DivIB and DivIC; A. Henriques for critical reading of the manuscript and L. Krippahl for help with image analysis tools. This study was funded by the European Research Council through grant ERC-2012-StG-310987 (to M.G.P.), by Project LISBOA-01-0145-FEDER-007660 Microbiologia Molecular, Estrutural e Celular (to ITQB-NOVA), by the National Institutes of Health through grant NIHGM113172 (to M.V.N.) and FCT fellowships SFRH/BD/71993/2010 (J.M.M.), SFRH/BD/86416/2012 (A.R.P.), SFRH/BPD/95031/2013 (N.T.R.), SFRH/BPD/87374/2012 (H.V.), SFRH/BD/52204/2013 (A.C.T.) and SFRH/BD/77849/2011 (M.T.F.).

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Contributions

J.M.M., A.R.P., N.T.R. and M.G.P. designed the research. J.M.M. and A.R.P. performed all experiments with the exception of the effect of antibiotics on the cell cycle which was performed by P.B.F. B.M.S. developed software for image analysis. J.M.M., A.R.P., N.T.R., P.B.F., H.V., A.C.T, M.S., M.T.F. and V.M. constructed strains. M.S.V. contributed new reagents (HADA). J.M.M., A.R.P., N.T.R., S.R.F. and M.G.P. analysed the overall data. B.M.S. analysed microscopy data quantified by eHooke software. P.B.F. analysed cell-cycle data. J.M.M., A.R.P, N.T.R. and M.G.P. wrote the manuscript.

Corresponding author

Correspondence to Mariana G. Pinho.

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Reviewer Information Nature thanks J. Xiao and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Switching fluorescent tags has no effect on protein co-localization data.

a, b, COL strains that express FtsZ–mCherry and FtsW–sGFP (a) or FtsZ–mCherry and MurJ–sGFP (b) were compared to strains that express FtsZ–CFP and FtsW–mCherry, and FtsZ–CFP and MurJ–mCherry, respectively (described in Fig. 2b, c). Scale bars, 2 μm. c, PCC values between fluorescence channels for each protein fusion pair were calculated for cells that showed septal FtsZ localization. From left to right, n = 138, 136, 133 and 139 cells. Negative PCC values are represented as 0. Data are represented as box-and-whisker plots in which boxes correspond to the first-to-third quartiles, lines inside the boxes indicate the median, and the ends of whiskers and outliers follow a Tukey representation. Statistical analysis was performed using a two-sided Mann–Whitney U test; ns, not significant. Images in a and b are representative of three biological replicates.

Source data

Extended Data Figure 2 Antisense RNA fragments that target the DivIB–DivIC–FtsL complex increased cell volume and decreased protein expression.

a, Western blot that shows total protein extracts of ColJZpAS-DivIB after 1 h of antisense induction, and control ColJZpEPSA probed with antibodies against either PBP2A (loading control; upper bands) or DivIB (lower bands). b, Western blot that shows total protein extracts of ColJZpAS-DivIC after 1 h of antisense induction, and control ColJZpEPSA probed with antibodies against either PBP2A (loading control; upper bands) or DivIC (lower bands). Images in a and b are representative of three biological replicates. For gel source data, see Supplementary Fig. 1. c, Cell volume of cells that expressed antisense RNA against ftsL, divIB or divIC, or carried vector pEPSA (left to right, n = 421, 379, 279 and 361 cells). Data represented in column graphs in which the height of the column represents the mean and the whiskers are the s.d. Statistical analysis was performed using a two-sided unpaired t-test. ***P < 0.001; first versus second column, P = 3.50 × 10−6; first versus third column, P = 3.80 × 10−8; first versus fourth column, P = 1.27 × 10−29.

Source data

Extended Data Figure 3 FtsW and PBP1 recruitment to the divisome is independent of DivIB–DivIC–FtsL complexes.

The strain ColWZpAS-FtsL, which harboured the FtsZ–CFP and FtsW–mCherry fluorescent fusion pair, and the strain ColP1ZpAS-FtsL, which harboured the FtsZ–mCherry and sGFP–PBP1 fluorescent fusion pair, were depleted of FtsL expression using antisense RNA and imaged by wide field fluorescence microscopy. a, Frequency of FtsZ–CFP and FtsW–mCherry co-occurrence in ColWZpAS-FtsL as compared to control ColWZpEPSA (n = 200 for each). b, Frequency of FtsZ–mCherry and sGFP–PBP1 co-occurrence in ColP1ZpAS-FtsL and in control ColP1ZpEPSA (n = 200 for each). c, d, Very large FtsL-depleted cells were observed either where FtsW–mCherry (c) or where sGFP–PBP1 (d) co-localized with the FtsZ fusion at the septum (arrows). e, f, Inhibition of divisome assembly at an early stage by depletion of FtsA expression in either ColWZpAS-FtsA or ColP1ZpAS-FtsA prevented the recruitment of FtsW–mCherry (e) or sGFP–PBP1 (f) to the septum, concomitant with FtsZ destabilization. Images in cf are representative of three biological replicates. Scale bars, 1 μm.

Source data

Extended Data Figure 4 Inhibition of MurJ activity does not prevent its recruitment to the midcell but impairs PG synthesis.

a, Fluorescence microscopy images of ColMurJ-mCherry cells grown in the presence (left) or absence (right) of the MurJ inhibitor DMPI for 30 min at 2× minimum inhibitory concentration. Scale bar, 2 μm. b, c, Fluorescence microscopy images showing mixed cultures of either DMPI-treated ColDltC-sGFPi cells mixed with COL cells (b) or DMPI-treated COL cells mixed with ColDltC-sGFPi cells (c), after incubation with HADA. The two cultures, which can be easily distinguished owing to GFP expression in ColDltC-sGFPi, were mixed on the same slide to decrease the fluorescence variation of HADA signal that could result from imaging conditions. Data show that HADA incorporation (that is, PG synthesis) is greatly reduced in the presence of DMPI. Phase-contrast–GFP channel overlays are shown on the left, and phase-contrast–HADA channel overlays are shown on the right. Scale bars, 1 μm. d, e, HADA fluorescence signal measured at the midcell (midcell), the periphery (peripheral) or over the entire cell (total) of DMPI-treated ColDltC-sGFPi cells mixed with COL cells (d) or DMPI-treated COL cells mixed with ColDltC-sGFPi cells (e). Images in ac are representative of three biological replicates. Data in d and e are represented as column plots (n = 100 cells for each column) in which the height of the column is the mean and the whiskers indicate s.d. Statistical analysis was performed using two-sided unpaired t-tests. ***P < 0.001. d, P = 2.34 × 10−38 for the midcell; P = 1.81 × 10−29 for the periphery; P = 9.22 × 10−34 for the entire cell. e, P = 1.74 × 10−33 for the midcell; P = 8.77 × 10−25 for the periphery; P = 7.60 × 10−26 for the entire cell.

Source data

Extended Data Figure 5 Effect of antibiotics on cell-cycle progression of S. aureus.

SIM images of Nile-Red-stained COL cells treated with DMPI, PC190723, oxacillin (Oxa), DMSO or TSB (mock-treated controls) for the duration of one cell cycle (30 min). Images are representative of three biological replicates. Scale bars, 1 μm.

Extended Data Figure 6 Kymographs that show the constriction of FtsZ55–56sGFP and MurJ–sGFP rings.

a, Kymographs of 10 cells per column that show the constriction of FtsZ55–56sGFP rings, obtained by imaging ColFtsZ55–56sGFP cells every 5 min for a total of 60 min (laser power 50%), in the absence (control) or presence of either PC190723 (+PC) or DMPI (+DMPI). Because S. aureus cells are not synchronised, cells at all stages of cytokinesis can be observed. Larger FtsZ55–56sGFP rings had a biphasic behaviour (no or slow constriction, followed by fast constriction) whereas smaller rings that are further ahead in the cell cycle were only observed undergoing the fast constriction step. The addition of PC190723 inhibited the constriction of larger rings (top two kymographs) but not of smaller rings, which were able to complete cytokinesis. The addition of DMPI completely blocked constriction of FtsZ55–56sGFP rings of all sizes. b, Kymographs showing constriction of MurJ–sGFP rings in ColMurJ-sGFP cells imaged every 5 min for a total of 60 min (laser power 100%). Cells in which a MurJ–sGFP signal appeared on the second frame were chosen for analysis to ensure that the entire constriction process was imaged. Fast constriction started immediately on the arrival of MurJ–sGFP at the division septum and therefore rings did not show biphasic behaviour. Data are representative of three biological replicates. Scale bars, 0.5 μm.

Extended Data Figure 7 The Z ring protein EzrA shows impaired treadmilling in the presence of PC190723 and biphasic ring constriction.

a, ColpSGEzrA-GFP cells that express a functional EzrA fusion to GFP were imaged by SIM every 5 s in the absence (EzrA control) or presence (EzrA + PC) of PC190723. Kymographs were obtained by extracting fluorescence intensity values along the red line indicated in cells in the left panels. Similar to the observations for FtsZ55–56sGFP, the addition of PC190723 abolished EzrA movement (vertical lines in the kymographs). b, ColpSGEzrA-GFP cells were imaged by SIM every 5 min in the absence (left) or presence (right) of PC190723, and kymographs that show the constriction of EzrA–GFP rings were plotted. Under control conditions, the larger EzrA rings showed biphasic constriction behaviour whereas in the presence of PC190723 only rings in the second stage of cytokinesis were able to constrict. Data in a and b are representative of two biological replicates. Scale bars, 1 μm. c, To test the functionality of the EzrA–GFP construct, the strains COL, ColpSGEzrA-GFP and COLΔEzrA (which lacks ezrA) were imaged by phase contrast and cell areas were measured. The lack of EzrA in COLΔEzrA (n = 959) resulted in cell enlargement, whereas the size distribution of ColpSGEzrA-GFP (n = 957) cells mimicked that of parental strain COL (n = 851), which indicates that the EzrA fluorescent fusion is functional.

Source data

Extended Data Figure 8 Kymographs that show the constriction of FtsZ55–56sGFP, FtsW–sGFP and MurJ–sGFP rings during cell division.

The strains ColFtsZ55–56sGFP, ColFtsW-sGFP and ColMurJ-sGFP were imaged every 10 min in the absence (control) or presence (+PC) of PC190723 for a total of 60 min. MurJ–sGFP control kymographs were performed on cells in which the MurJ–sGFP signal appeared on the second frame to ensure that the entire constriction process was observed (that is, to confirm that the absence of a biphasic behaviour was not the result of only imaging cells in the later stages of cell division). FtsZ55–56sGFP and FtsW–sGFP rings showed biphasic constriction behaviour (no or slow constriction, followed by fast constriction). The addition of PC190723 inhibited the constriction of larger FtsZ55–56sGFP and FtsW–sGFP rings (top kymographs) but not of smaller rings that were undergoing fast constriction. MurJ–sGFP rings displayed only fast constriction, and therefore were always able to constrict in the presence of PC190723. Data are representative of three biological replicates. Scale bars, 0.5 μm.

Extended Data Figure 9 Graphs of FtsZ, MurJ and FtsW ring diameter during constriction.

a, Graphs of ring diameters of FtsZ–mCherry (red) and MurJ–sGFP (blue) in strain ColJgZm. SIM images were taken every 10 min and measurements of ring diameter of FtsZ–mCherry and MurJ–sGFP were performed in cells in which a MurJ–sGFP signal first appeared after 20 min. b, Graphs of ring diameters of FtsZ–mCherry (red) and FtsW–sGFP (blue) in the strain ColWgZm. SIM images were taken every 10 min and measurements of ring diameter of FtsZ–mCherry and FtsW–sGFP were performed in cells in which FtsZ–mCherry ring diameter was constant for at least the first 20 min.

Source data

Extended Data Figure 10 Model for cytokinesis in S. aureus.

a, Cells in phase 1 of the cell cycle (before septum synthesis is initiated) synthesize peptidoglycan at the cell periphery. b, In preparation for division, FtsZ and early components of the divisome assemble at the midcell. c, At this stage, cells initiate the first, slow step of cytokinesis (which is dependent on FtsZ treadmilling) and FtsZ may act as the driving force for the initial invagination of the membrane. d, MurJ then arrives at the divisome, in a process dependent on the presence of the sub-complex DivIB–DivIC–FtsL, bringing the PG precursor (lipid II) flippase activity to the midcell. The major S. aureus PG synthase (PBP2) is recruited to the midcell through substrate (translocated lipid II) recognition, and massive PG synthesis is initiated to synthesize the septum. From this point on, cytokinesis no longer depends on FtsZ treadmilling and is most probably driven by PG synthesis.

Supplementary information

Life Sciences Reporting Summary (PDF 1364 kb)

Supplementary Tables 1-5

This file contains Supplementary Table 1 – Strains used to study localisation of peptidoglycan synthesis in Staphylococcus aureus, Supplementary Table 2 – Sequences encoding RNA antisense fragments targeting divisome genes, Supplementary Table 3 – Strains and plasmids used in this study, Supplementary Table 4 – Oligonucleotides used in this study, and Supplementary Table 5 – Cloning strategies for the construction of strains using pMAD vector. (PDF 779 kb)

Supplementary Figure 1

This file contains source data for western blots displayed in Extended Data Figure 2. (PDF 93 kb)

FtsZ55-56sGFP movement SR-SIM videos (left) followed by 15 min interval timelapse (right) in the absence (M9) or presence (M9+PC) of PC190723 5 µg/mL.

FtsZ55-56sGFP movement SR-SIM videos (left) followed by 15 min interval timelapse (right) in the absence (M9) or presence (M9+PC) of PC190723 5 µg/mL. Scale bar 0.5 µm. (MOV 46 kb)

FtsZ55-56sGFP rings imaged every 5 min by SR-SIM.

FtsZ55-56sGFP rings imaged every 5 min by SR-SIM. Scale bar 0.5 µm. (MOV 126 kb)

FtsZ55-56sGFP rings imaged every 5 min by SR-SIM in the presence of PC190723 5 µg/mL. The video shows a cell which constricts (1), a cell which constricts with defects (2) and a cell which does not constrict (3).

FtsZ55-56sGFP rings imaged every 5 min by SR-SIM in the presence of PC190723 5 µg/mL. The video shows a cell which constricts (1), a cell which constricts with defects (2) and a cell which does not constrict (3). Scale bar 0.5 µm. (MOV 112 kb)

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Monteiro, J., Pereira, A., Reichmann, N. et al. Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis. Nature 554, 528–532 (2018). https://doi.org/10.1038/nature25506

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