Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes

An understanding of how the heme-deficient gram-positive bacterium Streptoc occus pyogenes establishes infections in O-2-rich environments requires car eful analysis of the gene products important in aerobic metabolism. NADH ox idase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic aci d bacteria which directly catalyzes the four-electron reduction of O-2 to H 2O. To elucidate a putative rule for this enzyme in aerobic metabolism, NOX ase-deficient mutants were constructed by insertional inactivation of the g ene that encodes NOXase. Characterization of the resulting mutants related that growth in rich medium under low-O-2 conditions was indistinguishable f rom that of the wild type. However, the mutants were unable to grow under h igh-O-2 conditions and demonstrated enhanced sensitivity to the superoxide- generating agent paraquat. Mutants cultured in liquid medium under conditio ns of carbohydrate limitation and high O-2 tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H 2O2 in the growth medium compared to the field-type strain. All of these mu tant phenotypes could be overcome by the addition of glucose. Either the ad dition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumula tion of H2O2 and rescued the growth defect of the mutants under high-O-2 co nditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in envir onments high in O-2 with carbohydrate limitation.