Abstract
Cell division is tightly controlled in space and time to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation1. It is unknown how division site selection is controlled in bacteria that lack homologues of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections2. Here, we use correlated optical and atomic-force microscopy3,4 to demonstrate that morphological landmarks (waveform troughs) on the undulating surface of mycobacterial cells correspond to future sites of cell division. Newborn cells inherit wave troughs from the (grand)mother cell and ultimately divide at the centre-most wave trough, making these morphological features the earliest known landmark of future division sites. In cells lacking the chromosome partitioning (Par) system, missegregation of chromosomes is accompanied by asymmetric cell division at off-centre wave troughs, resulting in the formation of anucleate cells. These results demonstrate that inherited morphological landmarks and chromosome positioning together restrict mycobacterial division to the midcell position.
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Acknowledgements
The authors thank S. Kondo, I. Andrews, M. Malier and O. Venzin for help in data processing and visualization. P.D.O. and G.E.F. thank A. Radenovic for discussions and the use of research equipment. G.E.F. thanks C. Su and P. McPhail from Bruker Nano Surfaces Division for technical assistance. This work was funded in part by grants to J.D.M. from the Swiss National Science Foundation (310030_156945) and the Innovative Medicines Initiative (115337), a joint undertaking of the European Union Seventh Framework Programme and EFPIA (http://www.imi.europa.eu/), as well as by grants to G.E.F. from the Swiss National Science Foundation (205321_134786 and 205320_152675), the European Union FP7/2007-2013/ERC under grant agreement no. 307338-NaMic, and EU-FP7/Eurostars under award E!8213. H.A.E. was supported by a European Molecular Biology Organization Long Term Fellowship (EMBO ALTF 191-2014 and aALTF 750-2016).
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H.A.E., J.D.M. and G.E.F. conceived the study and designed the experiments. H.A.E., P.D.O., J.X.Y.V., M.T.M.H., A.P.N. and N.D. conducted the work. H.A.E., J.D.M. and G.E.F. wrote the manuscript. All authors reviewed, edited and approved the final version of the manuscript.
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Supplementary information
Supplementary Information
Supplementary Figures 1-18. (PDF 1337 kb)
Supplementary Data 1
Mycobacterial cell surface physiology. (XLSX 88 kb)
Supplementary Video 1
Time-lapse AFM of bacteria exposed to isoniazid (INH). Representative time-lapse AFM experiment in which M. smegmatis was immobilized on a PDMS-coated coverslip, cultured under a constant flow of 7H9 medium, and imaged for 36 hours at 12-minute intervals. INH (50 µg/ml, corresponding to 10× MIC) was added to the medium at t= 700 minutes. (MOV 6437 kb)
Supplementary Video 2
Time-lapse AFM of bacterial old pole growth. The old cell pole depicted in Supplementary Fig. 4 was imaged, following cell growth from the establishment of one wave trough to division taking place within that wave trough. The image is a representation of the mechanical properties of the cell pole imaged with better than 10 nm resolution. To probe mechanical properties beneath the cell surface, a peak force set-point 1.5-fold higher than in time-lapse experiments was used. Filament-like structures are apparent along the length of the cell pole, as described in Supplementary Fig. 8. (MOV 112223 kb)
Supplementary Video 3
Time-lapse AFM of a growing chain of RipA-depleted cells. A conditional RipA knockdown strain of M. smegmatis is defective in cell cleavage under conditions of RipA depletion, forming long chains of cells that fail to separate from each other. AFM imaging was initiated at the beginning of RipA depletion and continued through the course of multiple cell divisions. The video shows a time-lapse image series depicting the cell height from the peak force error channel and the sites at which division takes place. A kymograph representation of this time-lapse experiment is depicted in Supplementary Fig. 5. Scale bar, 2 µm. Time stamp in the upper left corner is represented as the month (10), day (14 to 15), hour, minute. (AVI 20205 kb)
Supplementary Video 4
Dual time-lapse AFM and fluorescence microscopy of bacteria expressing FtsZ-GFP. M. smegmatis expressing FtsZ–GFP was imaged at 15-minute intervals by AFM and fluorescence microscopy. Fluorescence images were recorded on the green (FtsZ–GFP) channel, merged and stacked with the AFM height images to generate the video. Selected snapshots from this video are shown in Fig. 3a, represented as a three-dimensional representation of the cell surface. (AVI 1317 kb)
Supplementary Video 5
Time-lapse AFM and fluorescence microscopy of bacteria expressing FtsZ–GFP and stained with FM4-64. M. smegmatis expressing FtsZ–GFP was stained with the fluorescent membrane dye FM4-64 and imaged at 15-minute intervals by AFM and fluorescence microscopy. AFM peak force error images (left) and fluorescence images recorded on the green (FtsZ–GFP) and red (FM4-64) channels (right) are depicted. Selected snapshots from this movie are shown in Supplementary Fig. 13, represented as fluorescence intensity and cell height profile along the cell length. Scale bar, 2 µm. (MOV 43190 kb)
Supplementary Video 6
Time-lapse AFM and fluorescence microscopy of bacteria stained with FM4-64. M. smegmatis was stained with the fluorescent membrane stain FM4-64 and imaged at 15-minute intervals by AFM and fluorescence microscopy. Fluorescence images were recorded on the red (FM4-64) channel, merged with the AFM height images, and stacked to generate the movie. (AVI 107 kb)
Supplementary Video 7
Time-lapse AFM and fluorescence microscopy of bacteria expressing Wag31–GFP. M. smegmatis expressing Wag31–GFP was imaged at 15-minute intervals by AFM and fluorescence microscopy. Fluorescence images were recorded on the green (Wag31–GFP) channel, merged with the AFM height images, and stacked to generate the movie. (AVI 1219 kb)
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Eskandarian, H., Odermatt, P., Ven, J. et al. Division site selection linked to inherited cell surface wave troughs in mycobacteria. Nat Microbiol 2, 17094 (2017). https://doi.org/10.1038/nmicrobiol.2017.94
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DOI: https://doi.org/10.1038/nmicrobiol.2017.94
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