Masako Higuchi

Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans

Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-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 H2O-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 (Km 0.05 and 0.025 mM for the H2O2- and H2O-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 H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-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.

Role of the dpr Product in Oxygen Tolerance in Streptococcus mutans

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.

Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein

Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase. A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.

Using Lichen Tissue Cultures in Modern Biology

We conducted physiological, pharmacological, and chemical studies using tissue cul- tures derived from natural lichens. Cultured tissues of about 200 lichen species were initiated from vegetative thalli collected in various regions of the world. The cultures grew on both complex and defined media. Growth was significantly affected by temperature, as well as by carbon and nitrogen sources. Some tissue cultures produced secondary substances (i.e., depsidones and usnic acid), but most acetone extracts did not show the same patterns of TLC spots as the corresponding natural thalli. Some lichen tissue cultures produced more usnic acid than natural thalli. Several pharma- cological activities were screened for both tissue cultures and natural lichens.

Molecular Biology of Oxygen Tolerance in Lactic Acid Bacteria: Functions of NADH Oxidases and Dpr in Oxidative Stress.

Lactic acid bacteria including Streptococcus mutans lack cytochromes and heme-containing proteins. Most lactic acid bacteria 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 not always consistent with its aerotolerance. Mechanisms, by which lactic acid bacteria establish their growth in air, are therefore an active area of investigation. We identified two kinds of NADH oxidase genes, nox-1 and nox-2 for H2O2-forming NADH oxidase (Nox-1) and H2O-forming NADH oxidase (Nox-2), respectively, in S. mutans and found that Nox-1 is homologous with flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC. We also identified ahpC which is homologous with ahpC of S. typhimurium, upstream of nox-1 in S. mutans. In the first and second parts of this article, we will refer to the role of Nox-1 which acts together with AhpC as bi-component peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohol and/or H2O. We will also refer to the role of Nox-2 in carbohydrate metabolism of S. mutans in its aerobic life. Nox-2 was found to be involved in regenerating NAD+, which is required for glycolysis in S. mutans. While studying nox-1 and ahpC double deletion mutant of S. mutans, we found that the mutant still showed the same level peroxide tolerance as did the wild-type strain. The finding suggested the existence of another antioxidant system in addition of Nox-1 and AhpC in S. mutans. We identified a new gene, dpr (for Dps-like Peroxide Resistance gene) and its product, Dpr, as an iron-binding protein which is responsible for oxygen tolerance in S. mutans. In the third part of this article, we will refer to the current status of knowledge of molecular cloning of dpr, the characteristics of dpr-disruption mutants, and a mechanism by which Dpr confers aerotolerance to S. mutans.

The Effect of Oxygen on the Growth and Mannitol Fermentation of Streptococcus mutans

The effects of oxygen on growth and mannitol fermentation of eight strains of Streptococcus mutans were compared under aerobic and strictly anaerobic conditions. The growth of three strains was severely inhibited by oxygen, whereas the others were oxygen-tolerant. The growth of two of the oxygen-tolerant strains was significantly enhanced by oxygen. The activities of superoxide dismutase and NADH oxidase in extracts from aerobically grown bacteria showed a positive correlation with the growth rate under aerobic conditions. The activities of these enzymes in oxygen-sensitive strains grown aerobically were as small as those in anaerobically grown cultures. Moreover, the enzyme activities increased during aeration of anaerobically grown oxygen-tolerant strains, but not in oxygen-sensitive strains. In all strains, oxygen changed mannitol catabolism from heterolactic to homolactic fermentation. It was concluded that oxygen-tolerance of S. mutans is dependent on the ability of strains to induce NADH oxidase and superoxide dismutase.

Preferential Induction of Rough Variants in Streptococcus mutans by Ethidium Bromide

Different colonial variants of Streptococcus mutans were studied. Rough variants were induced frequently from an original mucoid-type strain in cultures to which ethidium bromide was added. Rough variants had less insoluble dextran like polysaccharides than the original mucoid strain. Mitomycin C caused cell lysis in mucoid cells but not in rough cells.

Plasmid DNA Satellite Bands Seen in Lysates of Streptococcus mutans that Form Insoluble Extracellular Polysaccharides

When two different strains of Streptococcus mutans, PK-1 and JC-2, were used to prepare cell lysates, a satellite band of plasmid deoxyribonucleic acid (DNA) was seen. The mutants of PK-1 and JC-2 were defective in their ability to synthesize insoluble extracellular polysaccharides and had no detectable satellite band of DNA. These mutants were induced by treatment with ethidium bromide, acridine orange, or sodium dodecyl sulfate.