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Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division

Abstract

Mechanisms to control cell division are essential for cell proliferation and survival1. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes2,3,4 to maintain mechanical integrity of the cell wall5. Recent studies suggest that cell separation is governed by mechanical forces6,7. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here, we use a combination of atomic force microscope imaging, nanomechanical mapping and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall, concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with an atomic force microscope. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division.

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Fig. 1: The PCF is a nanoscale cell-surface feature that marks the future division site in M. smegmatis.
Fig. 2: Daughter cell cleavage occurs with millisecond kinetics by rupture around the PCF.
Fig. 3: Turgor pressure drives cell cleavage through stress concentration at the PCF.
Fig. 4: AFM-applied local stress on the PCF can substitute for an essential cell division enzyme (RipA).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank E. J. Rubin for generously providing the RipA conditional depletion strain of M. smegmatis. This research was supported in part by grants to G.E.F. from the Swiss National Science Foundation (205321_134786 and 205320_152675), from the European Union FP7/2007-2013/ERC under grant agreement no. 307338-NaMic, H2020 - UE Framework Programme for Research & Innovation (2014-2020); ERC-2017-CoG; InCell; Project number 773091, from the Commission for Technology and Innovation under CTI no. 18330.1 PFNM-NM, and by a grant to J.D.M. from the Swiss National Science Foundation (310030B_176397). H.A.E. was supported by an EMBO Long Term Fellowship (191-2014) and an EMBO Advanced Long Term Fellowship (750-2016).

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Contributions

P.D.O., G.E.F. and J.D.M. conceptualized the study. P.D.O., M.T.M.H. and H.A.E. developed imaging protocols. P.D.O., M.T.M.H., G.E.F. and J.D.M. designed experiments. P.D.O. and M.T.M.H. performed the experiments and analysed the data. A.P.N. participated in building the instrument and simulations. P.D.O., M.T.M.H., G.E.F. and J.D.M. wrote the manuscript.

Corresponding authors

Correspondence to John D. McKinney or Georg E. Fantner.

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The authors declare no competing interests.

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Peer review information Nature Physics thanks Yves Dufrene and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Table 1 and refs. 1 and 2.

Reporting Summary

Supplementary Video 1

A time sequence of M. smegmatis cells undergoing abrupt cleavage imaged in QNM with a low force setpoint. Frame rate: 5 min.

Supplementary Video 2

A time sequence of stiffness increase at the PCF imaged in QNM with a higher force setpoint. Frame rate: 5 min.

Supplementary Video 3

A time sequence of an alternative mechanism of separation of sibling cells when one sibling cell is deflated by piercing with the AFM cantilever tip. Sibling cells do not undergo rapid cell cleavage; instead, the surviving sibling gradually sheds the deflated sibling.

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Odermatt, P.D., Hannebelle, M.T.M., Eskandarian, H.A. et al. Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nat. Phys. 16, 57–62 (2020). https://doi.org/10.1038/s41567-019-0679-1

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