In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.
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PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides
Nature Communications Open Access 01 February 2022
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We thank S.-J. Ahn (University of Florida) for the kind gift of anti-AtlA antibodies, J. Abranches (University of Florida) for providing S. mutans serotype e, f and k strains; J. F. Timoney (University of Kentucky) and J. Huebner (von Hauner Children’s Hospital, LMU) for providing S. equi and E. faecalis, respectively; J. M. Bosken and E. D. Hall (University of Kentucky) for the use of the Thermo Fisher Scientific GC–MS instrument and C. Velez-Ortega (University of Kentucky) for access to a Leica SP8 confocal microscope. This work was supported by National Institutes of Health (NIH) grants (nos. R01 DE028916 from the National Institute of Dental and Craniofacial Research (NIDCR) and R01 AI143690 from the National Institute of Allergy and Infectious Diseases to N.K., R01 GM094363 from the National Institute of General Medical Sciences to A.B.H. and R01 DC014658 from the NIDCD to G.I.F.), Tenovus Scotland large research grant (no. T17/17) and University of Dundee Wellcome Fund (grant no. 105606/Z/14/Z) to S.A.C. and H.C.D., and the Wellcome and Royal Society grant (no. 109357/Z/15/Z) to H.C.D.. SEM was performed at the Electron Microscopy Center, which belongs to the National Science Foundation NNCI Kentucky Multiscale Manufacturing and Nano Integration Node, supported by ECCS-1542174. Carbohydrate composition analysis at the Complex Carbohydrate Research Center was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, US Department of Energy grant (no. DE-FG02-93ER20097). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors declare no competing interests.
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Extended Data Fig. 1 Modifications of SCC control S. mutans cell aggregation and morphology.
a, Sedimentation phenotype of S. mutans strains after overnight growth in THY broth. b, Sedimentation phenotype of sacculi purified from S. mutans strains. Sacculi were resuspended in PBS (OD600 of 8), and allowed to sediment for 72 hours at 4 °C. c, DIC images of S. mutans strains taken from mid-log growth phase. Blue arrows denote small round cells. Scale bar is 1 µm. Representative images from at least three independent experiments are shown in a, b and c.
Extended Data Fig. 2 Fluorescent microscopy of S. mutans intact cells and sacculi labeled by AtlABSP-GFP.
a, Binding of AtlABSP-GFP to the intact WT, ΔsccH, ΔsccH:psccH, ΔsccN and ΔsccN:psccN bacterial cells. b, Binding of AtlABSP-GFP to the sacculi purified from WT, ΔsccH and ΔsccH:psccH. In a and b, yellow and blue arrows show polar and equatorial sites labeled by AtlABSP-GFP, respectively. Scale bar is 1 µm in a and b. The experiments depicted in a and b were performed independently three times and yielded the same results.
Extended Data Fig. 3 Gating Strategy for Flow Cytometry (Fig. 4b).
In this sample gating, E. coli expressing polyrhamnose on the cell surface and its parental strain (E. coli without polyrhamnose) were gated based on the presence of fluorescent signal (AtlABSP-GFP). Bacterial gating occurred at the GFP/SCC density plot omitting signals derived from bacteria stained with GFP alone (GFP). For flow cytometric analysis, at least 10,000 events were collected. Experiments were performed independently three times and yielded the same results. Dot plots of representative results are shown.
Extended Data Fig. 4 S. mutans WT produces SCCs with different degrees of GroP modification.
a, Ion exchange chromatography of SCCs purified from ΔsccH. b, Ion exchange chromatography of SCCs purified from WT S. mutans. SCC material was loaded onto Toyopearl DEAE-650M and eluted with a NaCl gradient (0-0.5 M). Fractions were analyzed for Rha and Glc contents by anthrone assay and total phosphate (Pi) content by malachite green assay following digestion with perchloric acid. c, Composition analysis of minor and major fractions from b. Fractions were pooled, concentrated, and desalted by spin column and analyzed by GC-MS to determine the Rha and Glc concentrations. The concentration of Glc is presented relative to the Rha concentration. Data are means ± S.D., n = 3 biologically independent experiments. P values were calculated by a two-tailed t-test. d, SDS-PAGE analysis of ANDS-labeled SCCs extracted from S. mutans mutants. In a, b and d, the experiments were performed at least three times and yielded the same results. Data from one representative experiment are shown.
Supplementary Figs. 1–18, Supplementary Tables 1–6, Supplementary References.
Source Data Fig. 3b
Uncropped SDS–PAGE gel corresponding to Fig 3b.
Source Data Extended Data Fig. 4d
Uncropped SDS–PAGE gel corresponding to Extended Data Fig 4d.
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Zamakhaeva, S., Chaton, C.T., Rush, J.S. et al. Modification of cell wall polysaccharide guides cell division in Streptococcus mutans. Nat Chem Biol 17, 878–887 (2021). https://doi.org/10.1038/s41589-021-00803-9
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