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Coordinate induction of peroxidase and H2O2-forming nucleoside oxidase in Flavobacterium meningosepticum
FEMS Microbiology Letters 175 (1999) 113^117
Coordinate induction of peroxidase and H2 O2 -forming nucleoside oxidase in Flavobacterium meningosepticum Shinji Koga, Jun Ogawa, Yang-Mun Choi, Sakayu Shimizu * Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan Received 16 December 1998; received in revised form 31 March 1999; accepted 31 March 1999
Abstract Flavobacterium meningosepticum produced a peroxidase coordinately with a H2 O2 -forming nucleoside oxidase under culture conditions where the bacterium inducibly produced the nucleoside oxidase. The nucleoside oxidase and peroxidase both required Fe2 or Fe3 for their production, and Cu2 enhanced the production of both enzymes. The addition of nucleoside analogs to the medium also enhanced the production of both enzymes. The time course of peroxidase production coincided with that of the nucleoside oxidase production. These results suggest that the peroxidase has a specific function related to the nucleoside oxidase reaction, in breaking down the H2 O2 formed, which would otherwise cause oxidative cell damage. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Coordinate induction; Peroxidase; Nucleoside oxidase; Flavobacterium meningosepticum
1. Introduction Nucleoside oxidase has been reported to be one of the enzymes responsible for the oxidative degradation of nucleosides. The Pseudomonas maltophilia enzyme [1^4] was shown to catalyze the oxidation of nucleosides to the corresponding nucleoside-5P-carboxylic acids via nucleoside-5P-aldehydes using molecular O2 as a primary electron acceptor without the formation of H2 O2 . Recently, we found another type of nucleoside oxidase in Flavobacterium meningosepticum which produces H2 O2 in addition to the reaction products, nucleoside-5P-aldehyde and nucleoside-5P-carboxylic acid [5]. * Corresponding author: Tel.: +81 (75) 7536115; Fax: +81 (75) 7536128; E-mail: [email protected]
Peroxidase (EC 1.11.1.7) occurs in a wide variety of organisms including plants, animals and microorganisms. The speci¢city and biological function of peroxidase vary with the source of the enzyme, one of its important functions being protection against H2 O2 -caused cell damage. In this study, the production of peroxidase by F. meningosepticum was investigated to elucidate its physiological function in relation to the H2 O2 -forming nucleoside oxidase. 2. Materials and methods 2.1. Microorganism, medium and cultivation F. meningosepticum T-2799 (AKU 160, Faculty of Agriculture, Kyoto University), which was isolated
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 8 2 - 2
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from soil, was grown in a basal medium comprising 10 g sucrose, 1 g K2 HPO4 , 0.5 g MgSO4 W7H2 O, and 10 g of suitable nitrogen source listed in Table 1 in 1 l of deionized water, pH 7.0. Polypepton, yeast extract, and NZ-amine were obtained from Daigo Nutritional (Tokyo), Oriental Yeast (Osaka), and Wako Pure Chemical Industries, (Osaka), respectively. The cultivations (50 ml medium in 500-ml £asks) were carried out at 28³C with shaking (120 rpm) for 28 h. Cells were harvested by centrifugation (10 000Ug, 10 min, 4³C) and washed with physiological saline (0.85% (w/v) NaCl). Cell growth was determined by measuring the turbidity (OD at 660 nm) of the culture broth (1 mg dry weight per ml = 0.65). 2.2. Enzyme assay Nucleoside oxidase, peroxidase and catalase activities were assayed with soluble cell-free extracts of F. meningosepticum prepared by ultrasonic disruption (9 kHz, 180 W, 10 min, 4³C) of washed cells and subsequent centrifugation (14 000Ug, 30 min, 4³C). The activity of nucleoside oxidase was assayed by measuring the formation of H2 O2 with horseradish peroxidase (Boehringer, Mannheim Germany) in the presence of inosine as a substrate, as described previously [5]. One unit (U) of the nucleoside oxidase was de¢ned as the amount catalyzing the formation of 1 Wmol H2 O2 per min under the assay conditions. The peroxidase assay mixture comprised, in 1 ml, 50 Wmol acetate bu¡er (pH 5.5), 1 Wmol H2 O2 , 0.6
Wmol (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 1.5 Wmol 4-aminoantipyrine and a cell-free extract. After 1 min preincubation without the cell-free extract at 37³C, the reaction was initiated by the addition of the cell-free extract. After 10 min incubation at 37³C, the reaction was stopped by adding 2 ml of 0.5% (w/v) SDS. Then the increase in the absorbance at 555 nm depending on the formation of quinoneimine dye was measured. A blank without H2 O2 was included if necessary. A molar extinction coe¤cient of 392 000 M31 cm31 for the quinoneimine dye formed was used for calculation of the activity. One unit of the peroxidase was de¢ned as the amount catalyzing the oxidation of 1 Wmol of TOOS (equal to consumption of 2 Wmol H2 O2 ) per min under the assay conditions mentioned above. The catalase assay was performed by the method of Hildebrandt and Roots [6]. The catalase assay mixture comprised, in 1 ml, 100 Wmol potassium phosphate bu¡er (pH 6.3), 20 Wmol H2 O2 and a cell-free extract. After 1 min preincubation without the cell-free extract at 30³C, the reaction was initiated by the addition of the cell-free extract and then the decrease in absorbance at 240 nm was monitored at 30³C. A molar extinction coe¤cient of 43.6 M31 cm31 for H2 O2 was used for calculation of the enzyme activity [6]. One unit of the catalase was de¢ned as the amount catalyzing the degradation of 1 Wmol of H2 O2 per min under the assay conditions mentioned above.
Table 1 E¡ects of nitrogen sources on nucleoside oxidase, peroxidase and catalase production by F. meningosepticum Nitrogen source
Cell density (mg dry weight ml31 )
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
Catalase (U mg31 )
Polypepton Yeast extract NZ-amine Meat extract Casamino acids Peptone Tryptone Corn steep liquor Amino acid mixture
15 16 14 3.2 3.2 13 14 9.5 14
3.9 3.3 2.6 n.d. n.d. n.d. n.d. n.d. n.d.
3.9 3.1 2.9 n.d. n.d. n.d. n.d. n.d. n.d.
19 20 18 1.6 1.6 7.5 8.5 13 4.7
The bacterium was cultivated in basal medium supplemented with 1.0% (w/v) of each nitrogen source under the conditions given in Section 2. Activity is expressed as U mg31 dry weight. Results are averages of three separate determinations that were reproducible within þ 10%. n.d.: not detected (less than 1035 U mg31 ).
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115
Table 2 E¡ects of metal ions on nucleoside oxidase and peroxidase production by F. meningosepticum Metal ion
None FeCl3 W6H2 O FeSO4 W7H2 O CuSO4 W5H2 O ZnSO4 W7H2 O SnCl2 W2H2 O NiCl2 W6H2 O MnCl2 W6H2 O CaSO4 W2H2 O FeCl3 W6H2 O+CuSO4 W5H2 O
Polypepton medium
Peptone medium
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
3.9 5.3 5.2 5.2 3.3 2.8 2.5 3.1 3.3 6.0
3.9 5.9 5.9 6.0 3.7 2.8 3.0 3.2 3.6 7.2
n.d. 4.9 4.3 n.d. n.d. n.d. n.d. n.d. n.d. 6.5
n.d. 4.9 4.1 n.d. n.d. n.d. n.d. n.d. n.d. 6.9
The bacterium was cultivated in basal medium supplemented with 1.0% (w/v) of Polypepton and 0.001% (w/v) of each metal ion under the conditions given in Section 2. Activity is expressed as U mg31 dry weight. Results are averages of three separate determinations that were reproducible within þ 10%. n.d. : not detected (less than 1035 U mg31 ).
F. meningosepticum coordinately produced peroxidase with nucleoside oxidase when it was cultivated in the basal medium supplemented with Polypepton, yeast extract, or NZ-amine as a nitrogen source (Table 1). The cells showed neither enzyme activity with peptone, tryptone, corn steep liquor or amino acid mixture as a nitrogen source, even though they supported cell growth. Catalase activity was also higher in a medium containing Polypepton, yeast extract or NZ-amine than in a medium containing other nitrogen sources. However, the cells produced catalase with all the other nitrogen sources unlike peroxidase and nucleoside oxidase, suggesting that the peroxidase induction is more closely related to the induction of the nucleoside oxidase than that of the catalase.
tive) (Table 2). In both media, cell growth did not change with the addition of metal ions. In the Polypepton medium, both peroxidase and nucleoside oxidase production were enhanced by the addition of FeCl3 W6H2 O, FeSO4 W7H2 O or CuSO4 W5H2 O. In the peptone medium, in which neither peroxidase nor nucleoside oxidase activity was found without a metal ion, both enzymes were produced only in the presence of FeCl3 W6H2 O or FeSO4 W7H2 O. The addition of CuSO4 W5H2 O to the FeCl3 W6H2 O-containing Polypepton- or peptone-containing medium further enhanced the production of both enzymes. The e¡ects of nucleoside analogs (0.1% (w/v) in the medium) on the coordinate production of peroxidase and nucleoside oxidase were investigated with the basal medium supplemented with Polypepton, FeCl3 W6H2 O, and CuSO4 W5H2 O (Fig. 1). Among the 25 compounds tested, 2P-deoxyadenosine, 2P-deoxyguanosine, 1-methyladenosine and N6 -methyladenosine enhanced the production of peroxidase as well as that of nucleoside oxidase.
3.2. E¡ective compounds for coordinate production of peroxidase and nucleoside oxidase
3.3. Time courses of peroxidase and nucleoside oxidase production
The e¡ects of various metal ions on the coordinate production of peroxidase and nucleoside oxidase were investigated with the basal medium containing Polypepton (e¡ective inducer) or peptone (not e¡ec-
F. meningosepticum was cultivated in the optimized medium (basal medium supplemented with Polypepton, FeCl3 W6H2 O, CuSO4 W5H2 O, and N6 methyladenosine), and then cell growth, and peroxi-
3. Results 3.1. Induction of peroxidase and nucleoside oxidase during growth on di¡erent nitrogen sources
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Fig. 1. E¡ects of nucleoside analogs on the production of nucleoside oxidase and peroxidase. F. meningosepticum was cultivated in basal medium supplemented with 1.0% (w/v) of Polypepton, 0.001% (w/v) of FeCl3 W6H2 O, 0.001% (w/v) of CuSO4 W5H2 O, and 0.1% (w/v) of each nucleoside analog under the conditions given in Section 2. Activity is expressed as U per mg dry cells. Open bars: nucleoside oxidase activity ; solid bars: peroxidase activity.
H2 O2 appeared not to be involved in the induction, because some substrates of nucleoside oxidase which are oxidized with the formation of H2 O2 , such as adenosine, guanosine and inosine [5], enhanced neither the peroxidase production nor the nucleoside oxidase production. The time course of peroxidase production coincided with that of nucleoside oxidase production. These results suggest that the peroxidase was functionally related to the nucleoside oxidase. The peroxidase gene might be located on the same operon as that of the nucleoside oxidase, with the peroxidase playing a role in scavenging local H2 O2 speci¢cally produced by nucleoside oxidase. F. meningosepticum also produced the H2 O2 -decomposing enzyme, catalase. However, catalase was produced even under conditions where the bacterium showed no nucleoside oxidase activity, and was produced independently from the nucleoside oxidase throughout the course of cultivation. Catalase activity increased in media containing nitrogen sources which induced the nucleoside oxidase, and the catalase activity was much higher than the peroxidase activity, so the catalase might share some function in H2 O2 decomposition with the peroxidase. However, the evident coordinate induction of the peroxidase with the nucleoside oxidase suggests that the peroxidase has a more speci¢c function related to the nucleoside oxidase reaction.
dase, nucleoside oxidase and catalase activities were followed (Fig. 2). Bacterial growth reached a stationary phase after 18 h. Catalase activity also increased up to 18 h and decreased thereafter. In contrast, peroxidase and nucleoside oxidase activities both increased up to 36 h and decreased thereafter.
4. Discussion F. meningosepticum produced peroxidase only under conditions where H2 O2 -forming nucleoside oxidase was induced. Both enzymes appeared to be coordinately induced by speci¢c nitrogen sources and required Fe2 or Fe3 for their expression. Peroxidase production was enhanced by some nucleoside analogs (2P-deoxyadenosine, 2P-deoxyguanosine, 1methyladenosine and N6 -methyladenosine), which also enhanced the production of nucleoside oxidase.
Fig. 2. Enzyme production by F. meningosepticum. The bacterium was cultivated under the same conditions as given in the legend to Fig. 1 except for the cultivation time (54 h). N6 -Methyladenosine was used as an inducer. Activity is expressed as U mg31 dry cells. a : nucleoside oxidase activity; O: peroxidase activity; E : catalase activity ; b: cell density.
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Metabolic activation of molecular O2 very often results in the production of H2 O2 , which has been shown to be deleterious to most cellular components [7,8]. Bacteria produce H2 O2 -decomposing enzymes, namely catalase, catalase-peroxidase and peroxidase, to protect themselves from H2 O2 -caused cell damage, and in some cases peroxidase plays an important role in H2 O2 decomposition. For example, in a microaerophile, Spirillum voltans, a mutant that had NADH peroxidase activity was much more resistant to H2 O2 than the parent strain, which had no NADH peroxidase activity [9]. In some anaerobic bacteria, only peroxidase activity but not catalase and superoxide dismutase activity showed correlation with oxygen tolerance [10]. Rhodobacter capsulatus produces catalase-peroxidase and peroxidase, and a catalase-peroxidase negative mutant showed only a slight di¡erence from the parent strain as to the resistance to oxidative stress, suggesting that the peroxidase has an important function in eliminating activated O2 molecules in the bacterium [11]. The present ¢nding of coordinate induction of the peroxidase with the H2 O2 -forming nucleoside oxidase could indicate a similar speci¢c function of peroxidase. Investigation of the endogenous hydrogen donor for the peroxidase reaction would be of interest. NADH, NADPH, ascorbate, glutathione and reduced cytochrome c were tested with partially puri¢ed peroxidase, and only NADH was found as a potential electron donor. However, this should be further con¢rmed with pure peroxidase. Puri¢cation of the peroxidase and the protein-level interaction of the peroxidase and nucleoside oxidase are of further interest.
117
References [1] Isono, Y., Hoshino, M. and Sudo, T. (1988) Some characteristics of a new enzyme, nucleoside oxidase. Agric. Biol. Chem. 52, 2135^2136. [2] Isono, Y., Sudo, T. and Hoshino, M. (1989) Puri¢cation and reaction of a new enzyme, nucleoside oxidase. Agric. Biol. Chem. 53, 1663^1669. [3] Isono, Y., Sudo, T. and Hoshino, M. (1989) Properties of a new enzyme, nucleoside oxidase, from Pseudomonas maltophilia LB-86. Agric. Biol. Chem. 53, 1671^1677. [4] Isono, Y. and Hoshino, M. (1989) Laccase-like activity of nucleoside oxidase in the presence of nucleosides. Agric. Biol. Chem. 53, 2197^2203. [5] Koga, S., Ogawa, J., Cheng, L-Y., Yamada, H. and Shimizu, S. (1997) Nucleoside oxidase, a hydrogen peroxide-forming oxidase, from Flavobacterium meningosepticum. Appl. Environ. Microbiol. 63, 4282^4286. [6] Hildebrandt, A.G. and Roots, I. (1975) Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch. Biochem. Biophys. 171, 385^397. [7] Elstner, E.F. (1982) Oxygen activation and oxygen toxicity. Annu. Rev. Plant Physiol. 33, 73^96. [8] Malmstrom, B.G. (1982) Enzymology of oxygen. Annu. Rev. Biochem. 51, 21^59. [9] Patrick, S. and Krieg, N.R. (1998) A hydrogen peroxide resistant mutant of Spirillum voltans has NADH peroxidase activity but no increased oxygen tolerance. Can. J. Microbiol. 44, 87^91. [10] Rolfe, R.D., Hentges, D.J., Campbell, B.J. and Barrett, J.T. (1978) Factors related to the oxygen tolerance of anaerobic bacteria. Appl. Environ. Microbiol. 36, 306^313. [11] Hochman, A., Figueredo, A. and Wall, J.D. (1992) Physiological functions of hydroperoxidases in Rhodobacter capsulatus. J. Bacteriol. 174, 3386^3392.
FEMSLE 8777 19-5-99
Coordinate induction of peroxidase and H2 O2 -forming nucleoside oxidase in Flavobacterium meningosepticum Shinji Koga, Jun Ogawa, Yang-Mun Choi, Sakayu Shimizu * Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan Received 16 December 1998; received in revised form 31 March 1999; accepted 31 March 1999
Abstract Flavobacterium meningosepticum produced a peroxidase coordinately with a H2 O2 -forming nucleoside oxidase under culture conditions where the bacterium inducibly produced the nucleoside oxidase. The nucleoside oxidase and peroxidase both required Fe2 or Fe3 for their production, and Cu2 enhanced the production of both enzymes. The addition of nucleoside analogs to the medium also enhanced the production of both enzymes. The time course of peroxidase production coincided with that of the nucleoside oxidase production. These results suggest that the peroxidase has a specific function related to the nucleoside oxidase reaction, in breaking down the H2 O2 formed, which would otherwise cause oxidative cell damage. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Coordinate induction; Peroxidase; Nucleoside oxidase; Flavobacterium meningosepticum
1. Introduction Nucleoside oxidase has been reported to be one of the enzymes responsible for the oxidative degradation of nucleosides. The Pseudomonas maltophilia enzyme [1^4] was shown to catalyze the oxidation of nucleosides to the corresponding nucleoside-5P-carboxylic acids via nucleoside-5P-aldehydes using molecular O2 as a primary electron acceptor without the formation of H2 O2 . Recently, we found another type of nucleoside oxidase in Flavobacterium meningosepticum which produces H2 O2 in addition to the reaction products, nucleoside-5P-aldehyde and nucleoside-5P-carboxylic acid [5]. * Corresponding author: Tel.: +81 (75) 7536115; Fax: +81 (75) 7536128; E-mail: [email protected]
Peroxidase (EC 1.11.1.7) occurs in a wide variety of organisms including plants, animals and microorganisms. The speci¢city and biological function of peroxidase vary with the source of the enzyme, one of its important functions being protection against H2 O2 -caused cell damage. In this study, the production of peroxidase by F. meningosepticum was investigated to elucidate its physiological function in relation to the H2 O2 -forming nucleoside oxidase. 2. Materials and methods 2.1. Microorganism, medium and cultivation F. meningosepticum T-2799 (AKU 160, Faculty of Agriculture, Kyoto University), which was isolated
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 8 2 - 2
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from soil, was grown in a basal medium comprising 10 g sucrose, 1 g K2 HPO4 , 0.5 g MgSO4 W7H2 O, and 10 g of suitable nitrogen source listed in Table 1 in 1 l of deionized water, pH 7.0. Polypepton, yeast extract, and NZ-amine were obtained from Daigo Nutritional (Tokyo), Oriental Yeast (Osaka), and Wako Pure Chemical Industries, (Osaka), respectively. The cultivations (50 ml medium in 500-ml £asks) were carried out at 28³C with shaking (120 rpm) for 28 h. Cells were harvested by centrifugation (10 000Ug, 10 min, 4³C) and washed with physiological saline (0.85% (w/v) NaCl). Cell growth was determined by measuring the turbidity (OD at 660 nm) of the culture broth (1 mg dry weight per ml = 0.65). 2.2. Enzyme assay Nucleoside oxidase, peroxidase and catalase activities were assayed with soluble cell-free extracts of F. meningosepticum prepared by ultrasonic disruption (9 kHz, 180 W, 10 min, 4³C) of washed cells and subsequent centrifugation (14 000Ug, 30 min, 4³C). The activity of nucleoside oxidase was assayed by measuring the formation of H2 O2 with horseradish peroxidase (Boehringer, Mannheim Germany) in the presence of inosine as a substrate, as described previously [5]. One unit (U) of the nucleoside oxidase was de¢ned as the amount catalyzing the formation of 1 Wmol H2 O2 per min under the assay conditions. The peroxidase assay mixture comprised, in 1 ml, 50 Wmol acetate bu¡er (pH 5.5), 1 Wmol H2 O2 , 0.6
Wmol (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 1.5 Wmol 4-aminoantipyrine and a cell-free extract. After 1 min preincubation without the cell-free extract at 37³C, the reaction was initiated by the addition of the cell-free extract. After 10 min incubation at 37³C, the reaction was stopped by adding 2 ml of 0.5% (w/v) SDS. Then the increase in the absorbance at 555 nm depending on the formation of quinoneimine dye was measured. A blank without H2 O2 was included if necessary. A molar extinction coe¤cient of 392 000 M31 cm31 for the quinoneimine dye formed was used for calculation of the activity. One unit of the peroxidase was de¢ned as the amount catalyzing the oxidation of 1 Wmol of TOOS (equal to consumption of 2 Wmol H2 O2 ) per min under the assay conditions mentioned above. The catalase assay was performed by the method of Hildebrandt and Roots [6]. The catalase assay mixture comprised, in 1 ml, 100 Wmol potassium phosphate bu¡er (pH 6.3), 20 Wmol H2 O2 and a cell-free extract. After 1 min preincubation without the cell-free extract at 30³C, the reaction was initiated by the addition of the cell-free extract and then the decrease in absorbance at 240 nm was monitored at 30³C. A molar extinction coe¤cient of 43.6 M31 cm31 for H2 O2 was used for calculation of the enzyme activity [6]. One unit of the catalase was de¢ned as the amount catalyzing the degradation of 1 Wmol of H2 O2 per min under the assay conditions mentioned above.
Table 1 E¡ects of nitrogen sources on nucleoside oxidase, peroxidase and catalase production by F. meningosepticum Nitrogen source
Cell density (mg dry weight ml31 )
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
Catalase (U mg31 )
Polypepton Yeast extract NZ-amine Meat extract Casamino acids Peptone Tryptone Corn steep liquor Amino acid mixture
15 16 14 3.2 3.2 13 14 9.5 14
3.9 3.3 2.6 n.d. n.d. n.d. n.d. n.d. n.d.
3.9 3.1 2.9 n.d. n.d. n.d. n.d. n.d. n.d.
19 20 18 1.6 1.6 7.5 8.5 13 4.7
The bacterium was cultivated in basal medium supplemented with 1.0% (w/v) of each nitrogen source under the conditions given in Section 2. Activity is expressed as U mg31 dry weight. Results are averages of three separate determinations that were reproducible within þ 10%. n.d.: not detected (less than 1035 U mg31 ).
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Table 2 E¡ects of metal ions on nucleoside oxidase and peroxidase production by F. meningosepticum Metal ion
None FeCl3 W6H2 O FeSO4 W7H2 O CuSO4 W5H2 O ZnSO4 W7H2 O SnCl2 W2H2 O NiCl2 W6H2 O MnCl2 W6H2 O CaSO4 W2H2 O FeCl3 W6H2 O+CuSO4 W5H2 O
Polypepton medium
Peptone medium
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
Nucleoside oxidase (U1033 U mg31 )
Peroxidase (U1033 U mg31 )
3.9 5.3 5.2 5.2 3.3 2.8 2.5 3.1 3.3 6.0
3.9 5.9 5.9 6.0 3.7 2.8 3.0 3.2 3.6 7.2
n.d. 4.9 4.3 n.d. n.d. n.d. n.d. n.d. n.d. 6.5
n.d. 4.9 4.1 n.d. n.d. n.d. n.d. n.d. n.d. 6.9
The bacterium was cultivated in basal medium supplemented with 1.0% (w/v) of Polypepton and 0.001% (w/v) of each metal ion under the conditions given in Section 2. Activity is expressed as U mg31 dry weight. Results are averages of three separate determinations that were reproducible within þ 10%. n.d. : not detected (less than 1035 U mg31 ).
F. meningosepticum coordinately produced peroxidase with nucleoside oxidase when it was cultivated in the basal medium supplemented with Polypepton, yeast extract, or NZ-amine as a nitrogen source (Table 1). The cells showed neither enzyme activity with peptone, tryptone, corn steep liquor or amino acid mixture as a nitrogen source, even though they supported cell growth. Catalase activity was also higher in a medium containing Polypepton, yeast extract or NZ-amine than in a medium containing other nitrogen sources. However, the cells produced catalase with all the other nitrogen sources unlike peroxidase and nucleoside oxidase, suggesting that the peroxidase induction is more closely related to the induction of the nucleoside oxidase than that of the catalase.
tive) (Table 2). In both media, cell growth did not change with the addition of metal ions. In the Polypepton medium, both peroxidase and nucleoside oxidase production were enhanced by the addition of FeCl3 W6H2 O, FeSO4 W7H2 O or CuSO4 W5H2 O. In the peptone medium, in which neither peroxidase nor nucleoside oxidase activity was found without a metal ion, both enzymes were produced only in the presence of FeCl3 W6H2 O or FeSO4 W7H2 O. The addition of CuSO4 W5H2 O to the FeCl3 W6H2 O-containing Polypepton- or peptone-containing medium further enhanced the production of both enzymes. The e¡ects of nucleoside analogs (0.1% (w/v) in the medium) on the coordinate production of peroxidase and nucleoside oxidase were investigated with the basal medium supplemented with Polypepton, FeCl3 W6H2 O, and CuSO4 W5H2 O (Fig. 1). Among the 25 compounds tested, 2P-deoxyadenosine, 2P-deoxyguanosine, 1-methyladenosine and N6 -methyladenosine enhanced the production of peroxidase as well as that of nucleoside oxidase.
3.2. E¡ective compounds for coordinate production of peroxidase and nucleoside oxidase
3.3. Time courses of peroxidase and nucleoside oxidase production
The e¡ects of various metal ions on the coordinate production of peroxidase and nucleoside oxidase were investigated with the basal medium containing Polypepton (e¡ective inducer) or peptone (not e¡ec-
F. meningosepticum was cultivated in the optimized medium (basal medium supplemented with Polypepton, FeCl3 W6H2 O, CuSO4 W5H2 O, and N6 methyladenosine), and then cell growth, and peroxi-
3. Results 3.1. Induction of peroxidase and nucleoside oxidase during growth on di¡erent nitrogen sources
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Fig. 1. E¡ects of nucleoside analogs on the production of nucleoside oxidase and peroxidase. F. meningosepticum was cultivated in basal medium supplemented with 1.0% (w/v) of Polypepton, 0.001% (w/v) of FeCl3 W6H2 O, 0.001% (w/v) of CuSO4 W5H2 O, and 0.1% (w/v) of each nucleoside analog under the conditions given in Section 2. Activity is expressed as U per mg dry cells. Open bars: nucleoside oxidase activity ; solid bars: peroxidase activity.
H2 O2 appeared not to be involved in the induction, because some substrates of nucleoside oxidase which are oxidized with the formation of H2 O2 , such as adenosine, guanosine and inosine [5], enhanced neither the peroxidase production nor the nucleoside oxidase production. The time course of peroxidase production coincided with that of nucleoside oxidase production. These results suggest that the peroxidase was functionally related to the nucleoside oxidase. The peroxidase gene might be located on the same operon as that of the nucleoside oxidase, with the peroxidase playing a role in scavenging local H2 O2 speci¢cally produced by nucleoside oxidase. F. meningosepticum also produced the H2 O2 -decomposing enzyme, catalase. However, catalase was produced even under conditions where the bacterium showed no nucleoside oxidase activity, and was produced independently from the nucleoside oxidase throughout the course of cultivation. Catalase activity increased in media containing nitrogen sources which induced the nucleoside oxidase, and the catalase activity was much higher than the peroxidase activity, so the catalase might share some function in H2 O2 decomposition with the peroxidase. However, the evident coordinate induction of the peroxidase with the nucleoside oxidase suggests that the peroxidase has a more speci¢c function related to the nucleoside oxidase reaction.
dase, nucleoside oxidase and catalase activities were followed (Fig. 2). Bacterial growth reached a stationary phase after 18 h. Catalase activity also increased up to 18 h and decreased thereafter. In contrast, peroxidase and nucleoside oxidase activities both increased up to 36 h and decreased thereafter.
4. Discussion F. meningosepticum produced peroxidase only under conditions where H2 O2 -forming nucleoside oxidase was induced. Both enzymes appeared to be coordinately induced by speci¢c nitrogen sources and required Fe2 or Fe3 for their expression. Peroxidase production was enhanced by some nucleoside analogs (2P-deoxyadenosine, 2P-deoxyguanosine, 1methyladenosine and N6 -methyladenosine), which also enhanced the production of nucleoside oxidase.
Fig. 2. Enzyme production by F. meningosepticum. The bacterium was cultivated under the same conditions as given in the legend to Fig. 1 except for the cultivation time (54 h). N6 -Methyladenosine was used as an inducer. Activity is expressed as U mg31 dry cells. a : nucleoside oxidase activity; O: peroxidase activity; E : catalase activity ; b: cell density.
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S. Koga et al. / FEMS Microbiology Letters 175 (1999) 113^117
Metabolic activation of molecular O2 very often results in the production of H2 O2 , which has been shown to be deleterious to most cellular components [7,8]. Bacteria produce H2 O2 -decomposing enzymes, namely catalase, catalase-peroxidase and peroxidase, to protect themselves from H2 O2 -caused cell damage, and in some cases peroxidase plays an important role in H2 O2 decomposition. For example, in a microaerophile, Spirillum voltans, a mutant that had NADH peroxidase activity was much more resistant to H2 O2 than the parent strain, which had no NADH peroxidase activity [9]. In some anaerobic bacteria, only peroxidase activity but not catalase and superoxide dismutase activity showed correlation with oxygen tolerance [10]. Rhodobacter capsulatus produces catalase-peroxidase and peroxidase, and a catalase-peroxidase negative mutant showed only a slight di¡erence from the parent strain as to the resistance to oxidative stress, suggesting that the peroxidase has an important function in eliminating activated O2 molecules in the bacterium [11]. The present ¢nding of coordinate induction of the peroxidase with the H2 O2 -forming nucleoside oxidase could indicate a similar speci¢c function of peroxidase. Investigation of the endogenous hydrogen donor for the peroxidase reaction would be of interest. NADH, NADPH, ascorbate, glutathione and reduced cytochrome c were tested with partially puri¢ed peroxidase, and only NADH was found as a potential electron donor. However, this should be further con¢rmed with pure peroxidase. Puri¢cation of the peroxidase and the protein-level interaction of the peroxidase and nucleoside oxidase are of further interest.
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References [1] Isono, Y., Hoshino, M. and Sudo, T. (1988) Some characteristics of a new enzyme, nucleoside oxidase. Agric. Biol. Chem. 52, 2135^2136. [2] Isono, Y., Sudo, T. and Hoshino, M. (1989) Puri¢cation and reaction of a new enzyme, nucleoside oxidase. Agric. Biol. Chem. 53, 1663^1669. [3] Isono, Y., Sudo, T. and Hoshino, M. (1989) Properties of a new enzyme, nucleoside oxidase, from Pseudomonas maltophilia LB-86. Agric. Biol. Chem. 53, 1671^1677. [4] Isono, Y. and Hoshino, M. (1989) Laccase-like activity of nucleoside oxidase in the presence of nucleosides. Agric. Biol. Chem. 53, 2197^2203. [5] Koga, S., Ogawa, J., Cheng, L-Y., Yamada, H. and Shimizu, S. (1997) Nucleoside oxidase, a hydrogen peroxide-forming oxidase, from Flavobacterium meningosepticum. Appl. Environ. Microbiol. 63, 4282^4286. [6] Hildebrandt, A.G. and Roots, I. (1975) Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch. Biochem. Biophys. 171, 385^397. [7] Elstner, E.F. (1982) Oxygen activation and oxygen toxicity. Annu. Rev. Plant Physiol. 33, 73^96. [8] Malmstrom, B.G. (1982) Enzymology of oxygen. Annu. Rev. Biochem. 51, 21^59. [9] Patrick, S. and Krieg, N.R. (1998) A hydrogen peroxide resistant mutant of Spirillum voltans has NADH peroxidase activity but no increased oxygen tolerance. Can. J. Microbiol. 44, 87^91. [10] Rolfe, R.D., Hentges, D.J., Campbell, B.J. and Barrett, J.T. (1978) Factors related to the oxygen tolerance of anaerobic bacteria. Appl. Environ. Microbiol. 36, 306^313. [11] Hochman, A., Figueredo, A. and Wall, J.D. (1992) Physiological functions of hydroperoxidases in Rhodobacter capsulatus. J. Bacteriol. 174, 3386^3392.
FEMSLE 8777 19-5-99