We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H 2 O 2 , implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc r mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc r mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc r ahpC::Em r ::nox-1 triple mutant nor the dpr::Spc r sod::Em r double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.Bacteria contain certain enzymes capable of reacting with oxygen, and they cannot avoid confronting harmful reactive oxygen species, including superoxide anion (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and organic hydroperoxide, if they are exposed to air. To live in the presence of oxygen, they have to convert these reactive oxygen species to nontoxic molecules. Therefore, enzymes such as superoxide dismutases (SOD), catalases, and peroxidases are ubiquitously distributed in aerotolerant bacteria.Lactic acid bacteria, including Streptococcus mutans, lack cytochromes and other heme-containing proteins. Most lactic acid bacteria, except several lactobacilli which acquire catalase activity if a source of heme is added to their growth medium (42), also lack catalase. However, they can grow in the presence of air. In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is inconsistent with their aerotolerance. Mechanisms by which lactic acid bacteria cope with peroxide-mediated stress are therefore an area under active investigation. We previously identified H 2 O 2 -forming NADH oxidase (Nox-1) in S. mutans and found that Nox-1 is homologous with a flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC (16,17,30). We also identified ahpC, which is homologous with ahpC of S. typhimurium, upstream of nox-1 in S. mutans (30). Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a bicomponent peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H 2 O 2 to their respective alcohols and/or H 2 ...

Two distinct NADH oxidases, corresponding to H202-forming and H20-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H202-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6 6 and exhibited optimal activity at pH 6-0. The H20-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7-5. Both enzymes oxidized NADH (K, 0.05 and 0.025 mM for the H202-and H20-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271,383 and 449 nm for the H,O,-forming enzyme and 271, 375 and 447 nm for the H,O-forming enzyme. Antibodies raised against the H,O,-forming enzyme or the H20-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.

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Inhibition of tumor promoter-induced Epstein-Barr virus (EBV) activation was screened using tissue culture and thallus extracts of lichens. Usnea longissima ACH. thallus and Cetraria ornata MULL. ARG. tissue culture showed strong inhibitory activity. We identified (+)-usnic acid (1), barbatic acid (2), diffractaic acid (3), 4-O-demethylbarbatic acid (4), and evernic acid (5) as inhibitors of EBV activation from the U. longissima thallus. Of these compounds, (+)-usnic acid exhibited the highest inhibitory activity (IC50 = 1.0 microM).

We have previously identified two distinct NADH oxidases corresponding to H2O2-forming oxidase (Nox-1) and H2O-forming oxidase (Nox-2) induced inStreptococcus mutans. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component ofSalmonella typhimurium alkyl hydroperoxide reductase; an open reading frame upstream of nox-1 also showed homology to AhpC, the direct peroxide-reducing component of S. typhimurium alkyl hydroperoxide reductase. To determine their physiological functions in S. mutans, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD+ but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of Escherichia coli TA4315 (ahpCF-defective mutant) transformed with pAN119 containing both nox-1 and ahpC genes was not only restored but enhanced relative to that of E. coli K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of S. mutans to cumene hydroperoxide and H2O2, implying that the existence of some other antioxidant system(s) independent of Nox-1 in S. mutanscompensates for the deficiency.