Asymmetry and Aging of Mycobacterial Cells Lead to Variable Growth and Antibiotic Susceptibility

Abstract

Cells use both deterministic and stochastic mechanisms to generate cell–to-cell heterogeneity, which enables the population to better withstand environmental stress. Here we show that, within a clonal population of mycobacteria, there is significant deterministic heterogeneity in elongation rate that arises because mycobacteria grow in an unusual, unipolar fashion. Division of the asymmetrically growing mother cell gives rise to daughter cells that differ in elongation rate and size. Because the mycobacterial cell division cycle is governed by time not cell size, rapidly elongating cells do not divide more frequently than slowly elongating cells. Importantly, the physiologically distinct subpopulations of cells that arise through asymmetric growth and division are differentially susceptible to clinically important classes of antibiotics.

Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

“…Recently, it was reported that Mycobacterium smegmatis grows in an unusual unipolar manner by preferential extension of the old cell pole, whereas the new cell pole remains quiescent until the next cell division has been completed 12,15,16 . Unipolar growth was hypothesized to give rise to asymmetric cell division, resulting in sibling cells of unequal sizes and growth velocities, with larger old-pole cells growing faster than their smaller newpole siblings 15 .…”

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

“…At the appropriate time 7H9 media with 1 μg/ml calcein was added automatically by the CellASIC system. For the experiment shown in Figure 1b, imaging and microfluidics was done as described in ref 2.…”

“…To assess polar growth an amine-reactive dye was used for pulse-chase experiments as described in 2 . The analysis of these images was done in a semi-automated fashion: First single cells were identified manually on the phase image and marked using a segmented line that was drawn consistently from the new pole to the old pole.…”

“…2 the amount of growth at the new and old poles is measured over the duration of the cell cycle. left panel: The ratio of growth (new pole/old pole) for single cells (n=137 cell for WT; n=125 cells for Δ lamA ; dark black lines represent medians; the absolute values are shown in Extended Data Fig 6a).…”

“…Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1 . In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3 . The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown.…”