Quantifying the organization and dynamics of M. smegmatis morphology from Long-Term Time-Lapse Atomic Force Microscopy

The rod shaped Mycobacterium smegmatis displays complex cell surface morphology, characterized by wave-form cell surface features and driven by asymmetric growth dynamics. To systematically analyze these morphological variations, we developed a comprehensive computational pipeline for automated processing of Long-Term Time-Lapse Atomic Force Microscopy (LTTL-AFM) images of M. smegmatis cells cultured in axenic conditions of growth and stress. Upon running the pipeline to produce large enough datasets of single cell height profiles, we identify and statistically study key features that govern cell surface morphology: We confirm that M. smegmatis cells undergo bi-phasic, asymmetric pole growth with constant elongation rate at the old pole and a shift in the rate of elongation after a lag phase at the new pole. Stable wave-form cell surface peaks and troughs propagate along the long axis of the cell, which emerge as a result of polar elongation. Backtracking in time from cell division, we detect that division-site selection occurs at the wave-trough nearest mid-cell. To reproduce the fundamental cell features observed, we introduced a reaction-diffusion mathematical model on an evolving one-dimensional surface. Our simulations indicate that the dynamic manifestation of wave-form cell surface morphology in M. smegmatis can be explained by the interaction of as few as two "chemical species", providing a plausible theoretical basis for how molecular determinants may functionally control wave-form morphology in pole-growing bacteria.