Mild Nutrient Starvation Triggers the Development of a Small-Cell Survival Morphotype in Mycobacteria

Abstract

Mycobacteria, generally believed to be non-sporulating, are well known to survive shock starvation in saline for extended periods of time in a non-replicating state without any apparent morphological changes. Here, we uncover that mycobacteria can undergo cellular differentiation by exposing Mycobacterium smegmatis to mild starvation conditions. Traces of various carbon sources in saline triggered the development of a novel small resting cell (SMRC) morphotype. Development of SMRCs could also be observed for other mycobacteria, suggesting evolutionary conservation of this differentiation pathway. Fluorescence microscopic analyses showed that development of SMRCs progresses via septated, multi-nucleoided cell intermediates, which divide to generate mono-nucleoided SMRCs. Intriguingly, saline shock-starved large resting cells (LARCs), which did not show cell size or surface changes when observed by scanning electron microscopy, remodeled their internal structure to septated, multi-nucleoided cells, similar to the intermediates seen during differentiation to SMRCs. Our results suggest that mycobacteria harbor a starvation-induced differentiation program in which at first septated, multi-nucleoided cells are generated. Under zero-nutrient conditions bacteria terminate development at this stage as LARCs. In the presence of traces of a carbon source, these multi-nucleoided cells continue differentiation into mono-nucleoided SMRCs. Both SMRCs and LARCs exhibited extreme antibiotic tolerance. SMRCs showed increased long-term starvation survival, which was associated with the presence of lipid inclusion bodies.

Keywords: Mycobacterium smegmatis; bacterial differentiation; mycobacteria; non-replicating bacteria; quiescence; starvation.

FIGURE 1

Survival and cell shape of shock (PBS) and gently (PBS-Tween80) starved M. smegmatis. (A) CFU of bacilli subjected to shock and gentle starvation over 14 days. Experiments were performed three times in triplicates and representative results are shown with means and standard deviations. (B) Light microscopy images of acid-fast stained bacilli exposed to shock and gentle starvation over 14 days. (C) Scanning electron microscopy images of log-phase and 14-day-old starved bacilli. White scale bars correspond to 2 μm. Representative fields are shown for (B,C). Addition of Tween80 to 14-day-old PBS starved cultures did not result in the formation of small cells.

FIGURE 2

Cell length distribution in (A) log-phase cultures, (B) 14-day-old cultures starved in PBS-Tween80, (C) 14-day-old cultures starved in PBS. Eighty-five percentage of log phase cells had a length of 5.2 ± 0.9 μm. Ninety-four percentage of PBS-Tween80-starved cells had a length of 1.4 ± 0.3 μm. Seventy-five percentage of PBS-starved cells had a length of 4.1 ± 1.1 μm. One hundred and twenty cells were measured for each culture.

FIGURE 3

Growth and cell shape of 14-day-old PBS-Tween80 (small resting cells, SMRCs) or PBS (large resting cells, LARCs) starved M. smegmatis after transfer to rich medium. (A) CFU of starved bacilli after transfer to 7H9 medium over time. The experiment was performed three times in triplicates and a representative example is depicted showing means with standard deviations. (B) Light microscopic images of acid-fast stained bacilli sampled at indicated time points. Representative fields are shown.

FIGURE 4

DNA, membrane and fat body visualization of gently (PBS-Tween80) and shock (PBS) starved M. smegmatis. (A,B) Log-phase cultures were subjected to gentle and shock starvation. Samples were collected over time and stained with DAPI (blue) to visualize DNA and FM4-64 (red) to visualize membranes. (A) Fluorescence microscopy imaging of samples taken during the first 24 h of starvation. (B) Confocal microscopy imaging of DAPI and FM4-64 stained log-phase and 14-day-old starved bacilli, and the corresponding DIC images. Arrows indicate apparent septa. (C) Confocal microscopy imaging of Nile red (green) stained log-phase bacilli and 14-day-old starved bacilli to visualize fat bodies. Corresponding DIC images are shown in the lower panel. Fields shown are representative.

FIGURE 5

Stress tolerance and long-term survival of shock (PBS) starved and gently (PBS-Tween80) starved M. smegmatis. (A,B) CFU determinations after exposure of starved 14-day-old cultures to acid (pH 1.5, 20 min) or detergent (0.5% SDS, 30 min) stress. (C) Survival curves of starved cultures exposed to anaerobiosis using anaerobic jars. LARCs, 14-day-old PBS starved cultures. SMRCs, 14-day-old PBS-Tween80 starved cultures. Log-phase, log-phase cultures. (D) Long-term viability of log-phase bacilli exposed to PBS-Tween80 and PBS starvation. Data shown are means and standard deviations from three independent biological replicates.

FIGURE 6

Nutrient starvation-induced differentiation in M. smegmatis. Model depicting starvation-induced differentiation of log-phase cells first into LARCs. Under zero-nutrient starvation (PBS), development stops here. In the presence of traces of a carbon source (PBS-Tween80), LARCs undergo cell division and separate into SMRCs. Blue: DNA, red: septa, black: cell envelope. Arrows indicate polar growth of log-phase cells.