Contribution of NADH Oxidase to Aerobic Metabolism of Streptococcus pyogenes

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Abstract
An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O 2 -rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O 2 to H 2 O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O 2 conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O 2 conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions 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 2 O 2 in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H 2 O 2 and rescued the growth defect of the mutants under high-O 2 conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O 2 with carbohydrate limitation.

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