Aerobic-Type Ribonucleotide Reductase in the Anaerobe Bacteroides fragilis

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Abstract
Bacteroides fragilis , a component of the normal intestinal flora, is an obligate anaerobe capable of long-term survival in the presence of air. Survival is attributed to an elaborate oxidative stress response that controls the induction of more than 28 peptides, but there is limited knowledge concerning the identities of these peptides. In this report, RNA fingerprinting by arbitrarily primed PCR identified five new genes whose expression increased following exposure to O 2 . Nucleotide sequence analysis of the cloned genes indicated that they encoded an outer membrane protein, an aspartate decarboxylase, an efflux pump, heat shock protein HtpG, and an NrdA ortholog constituting the large subunit of a class Ia ribonucleotide reductase (RRase). Attention was focused on the nrdA gene since class I RRases are obligate aerobic enzymes catalyzing the reduction of ribonucleoside 5′-diphosphates by a mechanism that requires molecular oxygen for activity. Sequence analysis of the nrd locus showed that two genes, nrdA and nrdB , are located in the same orientation in a 4.5-kb region. Northern hybridization and primer extension experiments confirmed induction of the genes by O 2 and suggested they are an operon. The B. fragilis nrdA and nrdB genes were overexpressed in Escherichia coli , and CDP reductase assays confirmed that they encoded an active enzyme. The enzyme activity was inhibited by hydroxyurea, and ATP was shown to be a positive effector of CDP reductase activity, while dATP was an inhibitor, indicating that the enzyme was a class Ia RRase. A nrdA mutant was viable under anaerobic conditions but had decreased survival following exposure to O 2 , and it could not rapidly resume growth after O 2 treatment. The results presented indicate that during aerobic conditions B. fragilis NrdAB may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery.

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