The growth and division of mycobacteria, which include several clinically relevant pathogens, deviate significantly from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. In addition to being structurally distinct, the molecular components of the mycobacterial envelope are also evolutionarily unique, including the phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM). LM and LAM modulate host immunity during infection, but their role outside of intracellular survival remains poorly understood, despite their widespread conservation among non-pathogenic and opportunistically pathogenic mycobacteria. Previously, Mycobacterium smegmatis and Mycobacterium tuberculosis mutants producing structurally altered LM and LAM were shown to grow slowly under certain conditions and to be more sensitive to antibiotics, suggesting that mycobacterial lipoglycans may support cellular integrity or growth. To test this, we constructed multiple biosynthetic lipoglycan mutants of M. smegmatis and determined the effect of each mutation on cell wall biosynthesis, envelope integrity, and division. We found that mutants deficient in LAM, but not LM, fail to maintain cell wall integrity in a medium-dependent manner, with envelope deformations specifically associated with septa and new poles. Conversely, a mutant producing abnormally large LAM formed multiseptated cells in way distinct from that observed in a septal hydrolase mutant. These results show that LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell envelope integrity and septal placement.

For microcolony growth, cells from frozen stock were incubated for 48-72 hours in liquid LB 10 media supplemented with 0.05% Tween-80 under shaken conditions at 37°C. The culture was 11 diluted 100x in fresh LB media (without Tween-80) and deposited in the center of a glass-12 bottomed well in a 96-well plate (MatTek P96G-1.5-5-F). The culture was then grown at 37°C 13 under static conditions for approximately 48 hours. The medium was replaced with fresh LB 14 media supplemented with 0.1% SYTO9 and 0.1% propidium iodide (Invitrogen) and incubated 15 for 1 hour. The microcolonies were imaged using a confocal spinning disk unit (Yokogawa CSU-16 W1), mounted on a Nikon Eclipse Ti2 microscope with a 100x silicone oil immersion objective 17 (N.A. = 1.35), and captured by a Photometrics Prime BSI CMOS camera.