CydDC functions as a cytoplasmic cystine reductase to sensitizeEscherichia colito oxidative stress and aminoglycosides

Abstract

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level ofl-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that inEscherichia colithe component of cytochromebd-I terminal oxidase, the CydDC complex, shuttles excessivel-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that thecydDphenotype, unlike that of the bona fidel-cysteine exportereamA, parallels that of thel-cystine importertcyP.Chromosomal induction ofeamA, but not ofcydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellularl-cysteine, whereas induction ofcydDCortcyPcauses the accumulation of cytoplasmicl-cysteine. Congruently, inactivation ofcydDrenders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction ofcydDCsensitizes cells to oxidative stress and aminoglycosides, which can be suppressed byeamAoverexpression. Furthermore, inactivation of the ferric uptake regulator (fur)in Ptet-cydDCor Ptet-tcyPcells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2. These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to anl-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.