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.
Highlights
Bacteria in their terrestrial, aquatic or host environments constantly encounter nutrient deprivation and spend most of their life in non-growing resting states
To determine mycobacterial survival under different levels of nutrient starvation we transferred exponentially growing M. smegmatis from rich 7H9 medium either to phosphate buffered saline (PBS) alone, or to PBS containing 0.025% Tween80, a fatty acid ester which is metabolized by mycobacteria (Tomioka, 1983; Lofthouse et al, 2013), and determined the number of viable cells by CFU measurement over time
Light microscopic inspection of acid-fast stained culture samples was carried out to detect any differences in cell morphology under the two different starvation culture conditions
Summary
Aquatic or host environments constantly encounter nutrient deprivation and spend most of their life in non-growing resting states. Exponential growth under rich nutrient conditions is rare outside the laboratory (Matin et al, 1989; Rittershaus et al, 2013; Lenaerts et al, 2015) To endure these unfavorable conditions, survival responses have evolved into elaborate schemes of either physiological adaptation accompanied by distinct morphological changes or physiological adaptation only (Roszak and Colwell, 1987). Bacteria employing the former strategy are categorized as ‘differentiating’ or ‘sporulating’ bacteria, such as the endospore-forming Bacillus subtilis or the exospore-forming filamentous Streptomyces coelicolor. The ‘Loebel bacilli’ are regarded as persister bacilli and may represent a bacterial subpopulation in the host responsible for the difficulties in eradicating infections with antibiotics (Betts et al, 2002; Xie et al, 2005)
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