UNLABELLED: We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na(+)-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 μmol min(-1) mg(-1)). Overexpression of plasmid-encoded Na(+)-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min(-1) mg(-1)). By analyzing a variant of Na(+)-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae The impact of superoxide formation by the Na(+)-NQR on the virulence of V. cholerae is discussed.
IMPORTANCE: In several studies, it was demonstrated that the Na(+)-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na(+)-NQR as the site of superoxide formation in the cytoplasm of V. cholerae Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na(+)-NQR as the sole electrogenic NADH dehydrogenase.