Measuring membrane fluidity in live mycobacteria reveals subcellular lateral variation and pole-selective responses to mycomembrane perturbation

The cell envelope is an oft-cited factor in the ability of mycobacteria to tolerate antibiotics, host immunity, and environmental stress. In vitro studies have led to a prevailing model in which the mycobacterial envelope exhibits low fluidity that hinders the entry of antibiotics and other stressors. Although fluidity affects essential processes and is dynamically regulated across all domains of life, few studies have measured membrane fluidity in live mycobacteria. To address this gap, we used the environmentally-sensitive probe C-Laurdan to develop an imaging- and flow cytometry-based method for measuring cell envelope fluidity directly in live cells. Our approach enables cell envelope labeling across diverse mycobacterial species, including M. smegmatis and M. tuberculosis . We characterized fluidity as a function of subcellular localization, antibiotic treatment, and genetic perturbation. The unusual growth characteristics of mycobacteria, including polar growth and asymmetric growth and division, contribute to intercellular heterogeneity that is thought to enhance survival under stress. Indeed, we observed that the poles are more fluid than sidewalls, and that the old pole is more fluid than the new pole. Further, daughter cells have unequal membrane fluidity upon division and this asymmetry is reduced in a mutant with decreased asymmetric polar growth. Chemical or genetic disruption of the mycomembrane led to a shared alteration of the fluidity pattern and susceptibility to two antibiotics, suggesting that GP signatures may predict antibiotic susceptibility. This approach expands the toolkit for assessing fluidity in mycobacteria and enables deeper investigation into how biophysical properties influence bacterial physiology and antibiotic susceptibility.