CydDC functions as a cytoplasmic cystine reductase to sensitize <i>Escherichia coli</i> to oxidative stress and aminoglycosides.

Abstract

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in <i>Escherichia coli</i> the component of cytochrome <i>bd</i>-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the <i>cydD</i> phenotype, unlike that of the bona fide l-cysteine exporter <i>eamA</i>, parallels that of the l-cystine importer <i>tcyP.</i> Chromosomal induction of <i>eamA</i>, but not of <i>cydDC</i>, from a strong pLtetO-1 promoter (P_tet) leads to the increased level of extracellular l-cysteine, whereas induction of <i>cydDC</i> or <i>tcyP</i> causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of <i>cydD</i> renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of <i>cydDC</i> sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by <i>eamA</i> overexpression. Furthermore, inactivation of the ferric uptake regulator (<i>fur)</i> in P_tet-<i>cydDC</i> or P_tet-<i>tcyP</i> cells results in dramatic loss of survival, whereas catalase (<i>katG</i>) overexpression suppresses the hypersensitivity of both strains to H₂O₂ These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.