## 4 Growth, Metabolism, and ATP Production
tuberculosis_, treatment with three distinct antibiotics (rifampicin, isoniazid, and streptomycin) is known to induce the expression of isocitrate lyase (_icl_), a component of the glyoxylate shunt that converts isocitrate to glyoxylate and succinate [11; 63]. Furthermore, deletion of _icl_ dramatically increases the susceptibility of _M. tuberculosis_ to those drugs [11]. While nutrient starvation decreases the expression of most metabolic genes in _M. tuberculosis_, it has little effect on genes within the glyoxylate shunt, such as _icl_[53]. Utilization of the glyoxylate shunt decreases flux through the TCA cycle and reduces NADH and ATP production [11; 54]. As a result, usage of the glyoxylate shunt is thought to result in reduced levels of reactive oxygen species (ROS), which may contribute to its protective effect [11; 52; 54]. Similar effects were seen in response to aluminum toxicity in _Pseudomonas fluorescens_, suggesting that metabolic tolerance mechanisms are utilized in other types of stress responses [54]. However, defects in the glyoxylate shunt have been shown to increase biofilm formation and tolerance of oxidative stress in _P. aeruginosa_, suggesting that this strategy is not universally employed, even amongst closely related bacteria [64]. Another stark example of metabolic modulation and persistence development is the phosphate metabolism gene _phoU_[65]. _E. coli_ mutants lacking _phoU_ are unable to resume growth following \(\beta\)-lactam exposure and are more susceptible to numerous antibiotics and stress conditions. While wild type persisters remain unsusceptible to all antibiotics tested, _phoU_ mutants that survive initial antibiotic perturbation remain susceptible to \(\beta\)-lactams. Furthermore, loss of _phoU_ sensitizes stationary phase _E. coli_ to ampicillin, which requires active growth for effective killing of wild type cells [65]. Cells lacking _phoU_ upregulate genes involved in energy production; for this reason, it has been suggested that _phoU_ regulates persistence by reducing the expression of metabolic genes in response to stressors such as nutrient limitation or antibiotic exposure [65]. While persister cells, by definition, are not growing during antibiotic challenge, they can and do originate from actively dividing bacteria. Using fluorescent reporters for growth and metabolism, it was estimated that persisters constitute approximately 1 percent of stationary phase cells within an exponentially growing _E. coli_ culture [66]. Within that same culture, only 0.01 percent of persisters originated from actively growing cells. However, because of the high prevalence of growing cells in an exponential culture, as many as 20 percent of persisters may originate from active cells [66]. Within the growing populations, decreased reductase activity was found to be closely associated with persister formation. In fact, growing cells with low reductase activity were 40 times more likely to become persisters [66]. Other studies have shown that bacterial cells with lower rates of protein synthesis were more likely to be persisters [31]. Interestingly, the gene expression profile for these cells more closely resembled exponential- rather than stationary-phase cells. Though energy metabolism was decreased in general, these cells also had increased expression of toxin-antitoxin systems [31]. Therefore, this suggests that although decreased metabolism does greatly increase the likelihood of persister formation, it is not sufficient to explain the phenotype [31; 66]. It is clear that bacterial metabolic state is a major determinant of persister and biofilm formation in vitro. However, the metabolic conditions experienced in vitro are likely to differ dramatically from those encountered within the host. Therefore, it is likely that host metabolism plays a major role in bacterial functional potential [67].