Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.
Bibliographical noteFunding Information:
We thank laboratory members and Erkin Kuru (Harvard Med Sch), Yves Brun (Université de Montréal), Seamus Holden (Newcastle Univ), and Jie Xiao and Josh McCausland (Johns Hopkins) for discussions and suggestions; Jim Powers (IUB) for help with microscopy, Luke Lavis (Janelia Lab) for Fluor JF549; Dalia Denapaite (Trento Univ), Reinhold Brückner, and Regine Hakenbeck (Kaiserlautern Univ) for anti‐bPBP2x antibody; and Suzanne Walker and David Z. Rudner (Harvard Med Sch) for supporting preparation of anti‐bPBP2x and anti‐bPBP2b antibodies. This work was supported by NIH Grants RO1GM113172 (to MSV and MEW); RO1GM128439 (to EEC and MEW); R35GM131767 (to MEW); R35GM136365 (to MSV), RO1AI148752 (to S. Walker), RO1AI139083 (To D. Z. Rudner), National Science Foundation Grant MCB1615907 (to S.L.S.); NIH Predoctoral Quantitative and Chemical Biology Training Grant T32 GM109825 (to AJP); NIH Predoctoral Grant F31AI138430 (to MML); and NIH Equipment Grant S10OD024988 to the Indiana University Bloomington (IUB) Light Microscopy Imaging Center.
- FtsX localization
- FtsZ localization
- fluorescent D-amino acids (FDAAs)
- penicillin-binding proteins (PBPs)
- peripheral (elongation) peptidoglycan synthesis
- septal peptidoglycan synthesis