LTTL-AFM datasets of M. smegmatis WT and mutant strains

AFM imaging.

Coverslips were prepared as previously described ⁹. Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) at a ratio of 15:1 (elastomer:curing agent) and cut 1:10 with Hexane to reduce 10-fold the spin-coated layer, while equally increasing the hydrophobicity of the surface. Aliquots of mycobacteria isolated from axenic culture or from infection of macrophages were filtered thourgh a 0.5 µm pore size PVDF filter (Millipore) to remove cell clumps and enrich single cells. Aliquots were deposited on the hydrophobic surface of a PDMS-coated coverslip. 7H9 growth medium was supplied. Where indicated, antibiotic was added to the growth medium. The medium was maintained at 37°C using a custom-made heating element within the sample space and a TC2-80-150 temperature controller (Bioscience tools). Bacteria were imaged by peak force tapping using a Nanoscope 5 controller (Veeco Metrology) at a scan rate of 0.25 – 0.5 Hz and a maximum Z-range of 5 µm. A ScanAsyst fluid cantilever (Bruker) was used. Continuous scanning provided snapshots at 2-30 min intervals. Height, peak force error, adhesion, dissipation, deformation modulus and log modulus were recorded for all scanned images. Peak force error yields a fine representation of the height on the order of 10 nm in the Z-axis; this is computed as the difference between the peak force setpoint and the actual value. Images were processed using Gwyddion (Department of Nanometrology, Czech Metrology Institute). ImageJ was used for extracting bacterial cell surface height and modulus values and generating dynamic and quantitative mean values of the mechanical properties of individual cells.

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Mean cell surface stiffness. Mycobacterial cell surface stiffness is averaged over the relatively flat surface along the longitudinal midline of the cell surface, which is probed by AFM imaging in peak force tapping mode and interpreted as the Young’s modulus as per the Derjaguin-Muller-Toporov (DMT) model. The absolute values can vary from one experiment to the next, as a function of variations in the biophysical properties of the fluid medium (temperature and fluid density). Therefore, the mean cell surface rigidity is interpreted as a relative measure comparing the bacterium to the modulus of the PDMS-coated coverslip sample surface (Supplementary Figure 14).

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