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Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

Abstract

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

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Figure 1: Schematic of the cell wall synthetic pathways in B. subtilis.
Figure 2: Inhibition of WTA export with targocil leads to dissolving of the MreB cytoskeleton.
Figure 3: PG synthesis is mislocalized after WTA inhibition.
Figure 4: Availability of the carrier lipid, but not cell surface WTA, is required for MreB filament formation.
Figure 5: Prolonged treatment with cell wall inhibitors leads to dissolution of MreB into the cytoplasm.
Figure 6: Presence of lipid II is required for MreB filament formation and motion.

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Acknowledgements

We thank A. Meeske and D. Rudner (Harvard Medical School) for strains bEG275 and bKM424, K. Asai (Saitama University) for strain BSU2007, and P. Stoddard (Harvard University) for strain bPS01. We also thank S. Ringgaard and T. Bernhardt for critical reading of the manuscript. We are also grateful to Y. Brun for the fluorescent D-amino acids, and to T. Böttcher (Harvard Medical School) and H. Elliott (Harvard Image and Data Analysis Core) for the temporal variance algorithm. This work was funded by US National Institutes of Health (NIH) grant P01AI083214 (to S.W.), NIH grant GM-047446 (to J.D.H.), NIH grant GM073831 (to D. Rudner for initial support of E.C.G.). E.C.G. was also supported by the Smith Family Award and a Searle Scholar Fellowship. K.S. gratefully acknowledges the DFG for a Research Fellowship.

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Authors and Affiliations

Authors

Contributions

K.S., Y.-J.E., E.C.G., J.D.H. and S.W. designed the experiments; K.S. and E.C.G. took images for targocil treatment and TarGH depletion; Y.-J.E. and K.S. did the experiments for repletion and for imaging with various TIRF angles; M.D. provided various strains, helped with the microscopy and provided preliminary results; K.S. prepared the samples for, and Y.L. performed and analyzed the microarray; other experiments and all image analysis were done by K.S.; K.S. and S.W. wrote the paper.

Corresponding authors

Correspondence to Ethan C Garner or Suzanne Walker.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figures 1–8. (PDF 4854 kb)

MreB-GFP in targocil-sensitive B. subtilis cells.

Cells shown with or with out addition of targocil after the third frame (1 min) of the time lapse, or after washing out of targocil as indicated. Images were acquired every 30 sec over 30 min on a spinning disk confocal microscope. This movie corresponds to images shown in Fig. 2. (MOV 4035 kb)

MreBGFP in a targocil-insensitive strain, with addition of targocil after the third frame (1 min) of the time lapse.

Images were acquired every 30 sec over 30 min on a spinning disk confocal microscope. This movie corresponds to images shown in Supplementary Fig. 1b. (MOV 434 kb)

MreBGFP during TagGH depletion.

5 min time lapse, frame rate 10 sec, spinning disk confocal microscope. Movies were acquired either in the presence of inducer or 2 h after depletion as indicated. This movie corresponds to images shown in Supplementary Fig. 1c. (MOV 650 kb)

Pbp2A-GFP in a targocil-sensitive strain, with or without 1 h targocil treatment as indicated.

Images were acquired with a frame rate of 1 sec, streaming acquisition over 100 sec on a TIRF microscope. This movie corresponds to images shown in Fig. 3a. (MOV 581 kb)

MreB-GFP in tagF depletion strain, grown with inducer or after depleting for 2 h as indicated.

5 min time-lapse, frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 4a. (MOV 193 kb)

MreB-GFP in tagO null mutant strain lacking WTAs.

5 min time-lapse, frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 4b. (MOV 22 kb)

MreB-GFP in uppS depletion strain, in the presence and 4 h after removal of the inducer.

5 min time lapse, frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 4c. (MOV 39 kb)

MreB-GFP in a strain untreated or treated with a cell wall inhibitor (vancomycin) or with a protein synthesis inhibitor (tetracycline) as indicated.

5 min time lapse, frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 5a. (MOV 229 kb)

MreBGFP in an otherwise wild-type background strain without or with treatment for 1 h with the antibiotics as indicated.

Images were acquired with a 10 s frame rate for 5 min on a TIRF microscope. This movie corresponds to images shown in Supplementary Fig. 4. (MOV 480 kb)

GFP-Mbl at its native locus under control of the native promoter; untreated or treated with vancomycin for 1 h as indicated.

5 min time lapse, frame rate 10 s, TIRF. This movie corresponds to images of GFP-Mbl shown in Supplementary Fig. 5b. (MOV 118 kb)

MreB-GFP at its native locus under control of the native promoter; untreated or treated with vancomycin for 1 h as indicated.

5 min time lapse, frame rate 10 s, TIRF. This movie corresponds to images of MreB shown in Supplementary Fig. 5b. (MOV 118 kb)

MreBGFP after washing out bacitracin after 1 h treatment at the time points indicated.

Images were acquired every 1 min for 30 min, TIRF. This movie corresponds to images shown in Supplementary Fig. 6c. (MOV 591 kb)

MreB-GFP in strain without ECF sigma factors, either untreated or after 1 h vancomycin treatment.

5 min time lapse, frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 6b. (MOV 206 kb)

MreB-GFP in MurG depletion strain. Shown are 5-min time-lapse series after 4 h of depletion, and 15, 30 and 45 min after re-addition of inducer.

Frame rate 10 s, TIRF. This movie corresponds to images shown in Fig. 6c. (MOV 95 kb)

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Schirner, K., Eun, YJ., Dion, M. et al. Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB. Nat Chem Biol 11, 38–45 (2015). https://doi.org/10.1038/nchembio.1689

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