# Cellular Self-Digestion and Persistence in Bacteria
## 5 Links between Metabolism and Cellular Self-Digestion  
Bacteria must produce a significant amount of energy molecules and building blocks to meet their high metabolic demands. This may lead to metabolic stress that promotes cellular self-digestion. For example, metabolic activity can result in free radical formation via the respiratory chain [228], which can damage proteins, RNAs, DNAs, and lipids, thereby initiating their cellular degradation [229; 230]. Although the synthesis of glycolytic enzymes, as well as pyruvate formate lyase, phospho-transacetylase, and acetate kinase, and the subsequent downregulation of enzymes associated with anabolic pathways, has been observed during self-digestion in stationary phase [101; 231], persister cells may have unique metabolic mechanisms (Figure 1). In one previous study, we explored the relationship between metabolic activity and persistence using fluorescence-activated cell sorting (FACS) and a redox sensor green dye that measures cytochrome and oxidoreductase rates in the electron transport chain (ETC). With this system, we detected a positive correlation between ETC activity and persistence in stationary-phase cultures [55]. To further determine whether metabolic activity in stationary phase is involved in persister formation, we treated cells with metabolic inhibitors or transferred them to anaerobic conditions in early-stationary phase and measured persister cell levels in late-stationary phase [55; 232]. We found that these treatments significantly reduce persister cell levels, confirming an essential role for metabolism in persister cell formation. Given that the metabolism of non-growing cells primarily derives from the digestion of endogenous cellular components, such as phospholipids, ribosomes, and proteins, during stationary phase [55], we further measured cell size, protein levels, and rRNA integrity in cell cultures with increased (i.e., untreated late-stationary phase) or decreased (i.e., metabolic inhibitor-treated or anaerobic-transferred late-stationary phase) levels of persister cells [55]. We found that untreated late-stationary-phase cells contain significantly more degraded rRNAs and proteins and are markedly smaller than metabolic inhibitor-treated or anaerobically transferred stationary-phase cells [55]. We also determined that deletion of metabolic genes encoding the citric acid (TCA) cycle and ETC enzymes reducespersister levels by preventing digestion of intracellular components, yielding cells that are more vulnerable to cell death when exposed to antibiotics in fresh medium [55; 232] (Figure 1). Although persister cell metabolism is significantly reduced compared to that of exponentially growing cells [13; 37; 102; 103; 233], persister cells must still undergo active energy metabolism in order to maintain their adenylate energy charge. Notably, there is evidence that self-digestion mediates the metabolism of persister cells, particularly those formed throughout the stationary phase (Figure 1). In fact, numerous independent studies have shown that persisters can harbor ETC activities [55; 234], catabolize certain substrates to generate proton motive force (PMF) [234; 235; 236], produce energy molecules [237; 238], and drive the futile production and degradation of RNA, leading to energy generation and dissipation [239]. Persister cells must also be able to repair antibiotic-induced damage to survive [16; 17], and most repair mechanisms (e.g., DNA repair) are strongly ATP-dependent [240; 241; 242; 243]. Further, a number of independent groups have shown that deletion of enzymes associated with the TCA cycle and ETC (e.g., _sdhA_, _sucA_, _mdh_) drastically reduce persister levels, indicating the importance of energy metabolism in persister cell formation and/or survival [244; 21; 55; 245].