Survival of non-sporulating Bacillus subtilis upon energy limitation

Abstract

Bacillus subtilis has the capacity to undergo cell differentiation into distinct phenotypes. The most studied phenotype is the dormant cell type named endospore which persists in unfavourable conditions for extended periods of time. Nevertheless sporulation is not adopted by the entire cell population. During nutrient exhaustion a subpopulation of cells survives while the remaining cells either sporulate or lyse. How these surviving cells endure adverse environmental conditions is currently unclear. Incubation of non-sporulating B. subtilis under nutrient starvation revealed their capacity to remain viable for extended periods of time, over 100 days. During the first 14 days sporulation did not provide a survival benefit over the non-sporulating cells. Further characterisation of the non-sporulating cells showed that the cells are metabolically active and undergoing cell growth, albeit at a slow rate. Extracellular proteases, most likely releasing utilisable nutrients from extracellular proteins were shown to play an important role in starvation survival. At last, the cells appeared to be more tolerant to antibiotics and oxidative stress. The spread of antibiotic resistance and the existence of antibiotic tolerant phenotypes have prompted investigation into new antibiotics and specific modes of action. Dissipating membrane potential, a mode of action of most membrane-targeting antibiotics triggers killing of non-growing B. subtilis cells. Deletions of genes involved in oxidative damage prevention and repair (sodA, spx, recA and perR) increases the killing, indicating ROS contribute to cell death. Moreover a superoxide scavenger, tiron, resulted in an increase in survival. The two analysed membrane potential dissipating compounds valinomycin and CCCP turned out to trigger cell killing by a different mode of action. The cytochrome bc1 complex appears to have a key role in cell death triggered by valinomycin, possibly as a source of superoxide. This was indicated by an increase in survival observed for a qcrA deletion. This effect was not seen for CCCP. Instead, the majority of the electron transport chain (ETC) mutants examined increased sensitivity to CCCP.