DOI: 10.5061/dryad.1vhhmgr1w
title: Confocal images for Mycobacteria biofilm from: Lipoarabinomannan regulates septation in Mycobacterium smegmatis

description: 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.
description: 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.
description: # Confocal images for Mycobacteria biofilm from: Lipoarabinomannan
 regulates septation in *Mycobacterium smegmatis*
 [https://doi.org/10.5061/dryad.1vhhmgr1w](https://doi.org/10.5061/dryad.1vhhmgr1w) This data set contains the original confocal images for mycobacteria biofilm in the manuscript titled "Lipoarabinomannan regulates septation in *Mycobacterium smegmatis*". ## Description of the data and file structure Included are two data types: 1. Original data (nd2) files. They can be opened with Nikon Element Software or FIJI. 2. Multi-page Tifs can be opened directly or in FIJI.