Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)-spin trapping showed that superoxide production and generat ion of derivative hydroxyl radical were dependent on membrane-associated de methylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellula r superoxide production was inhibited by exogenous haematin, an essential c ofactor for cytochrome bd, and by fumarate, a substrate for fumarate reduct ase. As integral membrane quinol oxidases, cytochrome bd and fumarate reduc tase redox cycle demethylmenaquinone, and are necessary for aerobic and ana erobic respiration respectively. A rat model of intestinal colonization dem onstrated that conditions exist in the mammalian intestinal tract that perm it a mode of respiration for E. faecalis that results in the formation of h ydroxyl radical. These results identify and characterize the mechanism by w hich E. faecalis generates extracellular free radicals.