Oral Microbiol Ittmiwtol 1992: 1: 309-314
Reduced nicotinamide adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans
M. Higuchi Department of Oral Biochemistry, Tohoku University School of Dentistry, Sendai 980, Japan
Higuchi M. Redticed nicotitiamide adetiine ditiucleotide oxidase involvetnent In defense against oxygeti toxicity of Streptococcus mutans. Oral Microbiol Itntnutwl 1992: 7: 309-314. The growth inhibition of the Streptoeoecus tnutans group, including Streptococcus ttiutans. Streptococcus cricetus. Streptococcus rattits and Streptococcus sobrinus, on glucose by oxygen and the pt-operties of reduced nicotinatnide adenine dinucleotide (NADH) oxidase induced by oxygen, using a t-ept-esentative oxygentolerant strain, were exatnined. The growth response to oxygen varied among strains and correlated with the level of NADH oxidase activity in the cell extract. The induced synthesis of NADH oxidase as well as superoxide distnutase was affected by oxygen tension and enet-gy sources. The induced NADH oxidase involved at least two types, tnajor HiO-fonning NADH oxidase and tninor H^Oifortning NADH oxidase activity. In the presence of a scavenger of HjO,, pyruvate, the growth itihibitioti by oxygen in ati oxygen-sensitive stt-ain (GS5) was protected but not in another oxygen-sensitive strain (MT8148). The high level of induced HnO-fottning NADH oxidase activity protected against oxygen toxicity.
The tnost dominant microbes in plaque fiora at-e streptococci including Streptococcus sanguis. Streptococcus tnitior. Streptococcus tnilleri and the Streptococcus tnutans group involved in the pathogenesis of dental caries (3, 12). Many of these organisms lack sotne tnajor enzymes of oxygen (Oi) tnetabolistn and catalase (3, 7, 12). Although they are considered as facultative anaerobes, Ol affected the sugar tnetabolistn of S. tnutans and S. satigttis (27), the growth of S. .sanguis (6) and the growth and mannitol tnetabolisni of S. tnutans (14). Mot-eovet-, sotne oral streptococci excrete lat-ge atnounts of H^O,, a highly toxic substatice for tnicrobes, in the presence of glucose in a reaction dependent on Ol atid redvtced nicotinatnide adenine dinucleotide (NADH) (4). In our ptevious study, OT affected both tnannitol catabolistn atid the growth of S. tnutans, including representatives of serotypes a, b, c and g, when the organistn was growti on matinitol (14). And the O^ sensitivity correlated with the ability of the strains to induce NADH oxidase and superoxide
distnutase (SOD) under aerobic conditions (14). NADH oxidase, however, appears to play an itnportant role in the tegulation of aerobic metabolistn of tnannitol, because of an additional NADH produced in the reaction convei-ting tnannitol 1phosphate to fructose 6-phosphate (2). The active t-ole of NADH oxidase in defense against Oi toxicity of this bacteriutn has not been elucidated. To clat-ify the function of NADH oxidase, I investigated the Oi sensitivity of S. tnutatis when grown on glucose atid the properties of NADH oxidase induced by Ol. The present study revealed that the growth response to Oi of S. tnutatts varied atnong the strains regardless of whether they were grown on mannitol or on glucose, but correlated with the level of NADH oxidase activity in the cell extracts. Mot-eover, I conclude that the overwhelmingly high level of HiOfortning NADH oxidase in this bacterium and the ratio of H^O-fonning oxidase to H^Oi-foi-tning oxidase are the key to the defense against O; toxicity.
Key words: NADH oxidase: oxygen toxicity: Streptococcus mutans M. Higuchi, Research Center, Nippon Paint Co., Neyagawa 572, Japan Accepted for publication March 3, 1992
Materials and methods iWicroorganisms
The 8 strains of the 5. tnutatis group used in this study including 5. tnutatis. Streptococcus ericetus. Streptococcus rattus and Streptococcus sobrinus, are listed in Table 1. They were maintained monthly by transferring thetn on blood agar atid tnitis-salivarius agar and were kept at 4 C. The organistns were grown in a broth containing (%, w/v): tt-ypticase peptone (BBL Microbiology Systetns, Cockeysville, MD), 1.0; yeast extract (Daigo Chetnical, Osaka, Japan), 0.2; NaCl, 0.2; K,HP04, 0.3; KH,PO4, 0.2; K,CO,, 0.1; MgSO4-4HA 0.01; MnS04 - 4H:0, 0.002; mannitol or glucose, 1.0 (TYM or TYG tnedium, pH 7.0). The mediutn was sterilized at 121 C for 15 tnin. Pyruvate was separately sterilized by ftltration. For aerobic growth, the organistns were cultut-ed in a 500-tnl flask containing 100 ml medium under air with vigorous shaking (120 t-ptn). For strictly anaerobic growth, the organistns were cultured in a Pyrex glass bottle (500 tnl) or
310
Higuchi
Table I. Study strains of the S. mutans group Strain HS6 FAl NCTC 10449 NCIB11723 (JC2) Ingbritt MT8148 GS5 6715
Serotype a b c e c c c d
a test tube (12 x 100 mm) with a screw cap in an anaerobic glove box (Hirasawa Woi-ks, Tokyo, Japan) under an attnosphere of m'/« Nj, 10% H, and 10% CO2. All anaerobic media were prereduced for at least 24 h before incubation to ensure strict anaerobiosis. All cultures were preadapted in TYM or TYG medium, inoculated with approxitnately 10'' cells/ml and maintained at 35 °C. Enzyme induction
Anaerobically grown organistns frotn the mid-exponential phase (0.45-0.48 A660) were harvested by centrifugation (21,000 xg-, 10 tnin) and resuspended in 600 tnl of TYM tnedium. The effect of aeration on enzyme induction was determined as follows; the cell suspension was divided into 6 portions, one of 50 tnl, 4 of 100 tnl or one of 150 ml volumes anaerobically. One 100-ml bottle containing 100 ml cell suspension was kept for 2 h at 35 "C in an anaerobic glove box, after which the cells were seditnented by centrifugation (2l,000xg-, 10 min), washed 3 times with 50 mM potassium phosphate buffer (pH 7.0) and kept in ice-water under atiaerobic conditions. Another 100-ml bottle containing 100 tnl of cell suspension was placed aerobically without shaking and 3 500-tnl flasks containing 50 ml (condition a), 100 ml (condition b) and 150 ml (condition c) of cell suspension, respectively, were stoppered with cotton plugs atid incubated utider air with shaking (120 rpm). These aerobic incubations were performed for 2 h at 35°C, after which the cells wet-e sedimented by cetitrifugation (21,000 x g, 10 tnin), washed 3 times with 50 mM potassiutn phosphate buffer (pH 7.0) and kept in ice-water under aerobic conditions. The retnaining 100-tnl cell suspension was placed in ice-water to arrest enzytne induction, and then sedimented and washed as described above. The pellets were kept aerobically in ice-water. The effect of chloratnphenicol on en-
Species 5. S. S. S. S. S. S. S.
Reference
cricetus rattus mutans mulans tnutatis mutans mutans sobrinus
9 9 7 3 17 13 11 16
zyme induction was determined as follows; anaerobically prepared cell suspensions were divided into 100-tnl volutnes in 6 500-ml flasks. One flask was placed anaerobically in ice-water to arrest enzytne induction. The other 5 flasks wet-e stoppered with cotton plugs and incubated at 35°C under air with vigorous shaking (120 rptn). At 0, 0.5, 1.0 and 2.0 h after initiating the aeration, chloratnphenicol (CM: 250 figl ml) was added in 4 of the 5 flasks. After 2 h aeration, the flasks wet-e placed in ice-water and then the cells were sedimented by centrifugation and washed as described above. The pellets were kept aerobically in ice-water. Preparation of extracts
All seditnented cells were disrupted by sonification within 2 h of the harvesting. The pellets were suspended in 50 mM potassium phosphate buffer (pH 7.0; 4 tnl per g wet weight) and disrupted by sonification using a Kubota Insonater (Model 200 M, Kubota, Tokyo, Japan) at 200 W for 15 tnin, at 0 C. Following centrifugation (25,000 xg, 30 min), the supernatant fluids were dialyzed against 4-1 50 tnM potassiutn phosphate buffer containing 0.2 niM-EDTA for 18 h at 4 C under air and used as crude extracts. • Assay for NAD(P)H oxidase
The activity was tneasured at 30 °C spectrophotometrically by tnonitoring the oxidation of NAD(P)H in the reaction tnixture (1 tnl) at 340 nm. One unit of enzyme activity was defined as the amount of enzyme (mg protein) that catalyzed the oxidation of LO /anol NAD(P)H/tnin. The reaction mixture contained 50 lnM potassium phosphate buffer (pH 7.2), 0.17 mM NADH atid extracts (0.1 to 50 fig protein/ml). The reaction was started by adding extracts. The reduction of 2,6-dichloro-indoplienol (DCIP), cytochrotne c and
K,Fe(CN)f, by extracts were assayed by tneasuring the changes in A^JQ, A550 atid A42,,, respectively. Assay for NADH peroxidase (EC 1.11.1.1)
The activity was tneasured by tnonitoring the oxidation of NADH using a spectt-ophototneter equipped in an anaerobic glove box (under an atmosphere of 80%. N, and 20% H,) at 30 "C in an identical medium as that used for assaying NADH oxidase except for the addition of 0.3 mM H1O,. Estimation of O^ uptaite and H2O2 production
The Ol uptake and H,O2 production by the extracts were estitnated polarographically at 30 C with an oxygeti monitor (model 53; Yellow Springs Instrument Cotnpany, Yellow Springs, OH) (14). The reaction mixture contained 50 niM potassium phosphate buffer (pH 7.2), 0.17 mM NADH with or without 0.02 tnM FMN or FAD, and extracts. The reactioti was started by the addition of NADH. When OT uptake had ceased or NADH had been consumed, 20 /(I of catalase (320 units/tnl) was added to the t-eaction tnixture. The amount of H^Oi produced in the reaction tnixture was detertnined from O2 concentration increased by adding catalase. The electrode was calibrated by the phenyl hydrazine/ferricyanide tnethods (21).
Assay for superoxide dismutase (SOD) (EC 1.15.1.1.)
The activity was assayed by the xanthine oxidase/cytochrome c tnethod (26). One unit of SOD activity was defined as the amount (mg protein) that gave a 50'^!) decrease in the reduction rate of cytochrotne c. Anothier anaiyticai metiiod
Protein concentration was detertnined by the method of Lowry et al. (19) with crystalline bovine serum albutnin as the standard. Chemicais
NADH, NADPH and xanthine oxidase were obtained from Boehringer, London, UK. Ferricytochrotne c, flavin mononucleotide (FMN), flavin adenitie dinucleotide (FAD), catalase and
NADH oxidase of S. mutatis phenazineinethosulfate were purchased from Sigma Chemical Co., St. Louis, MO. Xanthine from cow milk was purchased fi-otn Nakarai Chemicals, Tokyo, Japan. Chloramphenicol (CM) was purchased frotn Wako Pure Chetnical Industries, Tokyo, Japan. Results Effect of O2 on fhe growth of S. mutans on giucose
The growth patterns of S. ttiutans when grown on glucose under static aerobic, vigorously shaking aerobic or strictly anaerobic conditions indicated significant differences of oxygen sensitivity among the strains as previously observed with tnannitol cultures (14). Almost all strains tested grew as well under aerobic conditions without shaking as under strictly anaerobic conditions. In contrast, under vigorously shaking aerobic conditions, one group of strains iticluding MT8I48, 6715, and GS5, which were designated as oxygensensitive strains, grew with a severely i-educed growth rate (4.8, 3.8 and near 18 h doublitig titne, t-espectively). However, another group including NCTC10449, NCIB11723, Ingbritt and FAl, which wet-e designated as oxygentolerant strains, adapted to grow with sotne variations (1.2 h tnean doubling time).
Correlation between the growth rate and NADiH oxidase in extracts
To elucidate the nature of growth inhibition by O2, NADH oxidase activity of both the Oj-tolerant and the 02-sensitive strains in the late-exponential phase of gt-owth wei-e exatnined. The results (Fig. 1) show a clear correlation between NADH oxidase activity in cell extracts from aerobically grown cultures with vigorous shaking and their growth rate. The enzyme activity in extracts from 02-sensitive strains was lower than that from O2tolerant strains. A significantly higher activity of NADH oxidase was detected in extt-acts from strains NCTC 10449, NCIB11723, Itigbt-itt and FAl. This corresponded to their faster growth rate under vigot-ous shaking and aerobic conditions. These 02-tolerant strains had a maximutn activity of NADH oxidase in the tnid- to the iate-exponential phase of gt-owth. Accordingly, the NADH oxidase activity of NCIBl 1723 was lower than the 3 other O,-tolerant
Aclivily (U/mg protein)
Fig. 1. Correlation between growth and NADH oxidase activity in cell extracts. Extracts were prepared from cells grown to lateexponential phase on glucose under vigorously shaking (o) and static ( • ) aerobic conditions. NADH oxidase activity of each strain was plotted against the reciprocal of the generation time. 1: HS6, 2: FAl, 3: NCTC10449. 4: NCIBl 1723. 5: Ingbritt, 6: MT8148, 7: GS5, 8: 6715.
strains, though this strain grew with the highest growth rate atnong the stt-ains tested so fat-. All strains exhibited low levels of enzytne activity (
Reduced nicotinamide adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans
M. Higuchi Department of Oral Biochemistry, Tohoku University School of Dentistry, Sendai 980, Japan
Higuchi M. Redticed nicotitiamide adetiine ditiucleotide oxidase involvetnent In defense against oxygeti toxicity of Streptococcus mutans. Oral Microbiol Itntnutwl 1992: 7: 309-314. The growth inhibition of the Streptoeoecus tnutans group, including Streptococcus ttiutans. Streptococcus cricetus. Streptococcus rattits and Streptococcus sobrinus, on glucose by oxygen and the pt-operties of reduced nicotinatnide adenine dinucleotide (NADH) oxidase induced by oxygen, using a t-ept-esentative oxygentolerant strain, were exatnined. The growth response to oxygen varied among strains and correlated with the level of NADH oxidase activity in the cell extract. The induced synthesis of NADH oxidase as well as superoxide distnutase was affected by oxygen tension and enet-gy sources. The induced NADH oxidase involved at least two types, tnajor HiO-fonning NADH oxidase and tninor H^Oifortning NADH oxidase activity. In the presence of a scavenger of HjO,, pyruvate, the growth itihibitioti by oxygen in ati oxygen-sensitive stt-ain (GS5) was protected but not in another oxygen-sensitive strain (MT8148). The high level of induced HnO-fottning NADH oxidase activity protected against oxygen toxicity.
The tnost dominant microbes in plaque fiora at-e streptococci including Streptococcus sanguis. Streptococcus tnitior. Streptococcus tnilleri and the Streptococcus tnutans group involved in the pathogenesis of dental caries (3, 12). Many of these organisms lack sotne tnajor enzymes of oxygen (Oi) tnetabolistn and catalase (3, 7, 12). Although they are considered as facultative anaerobes, Ol affected the sugar tnetabolistn of S. tnutans and S. satigttis (27), the growth of S. .sanguis (6) and the growth and mannitol tnetabolisni of S. tnutans (14). Mot-eovet-, sotne oral streptococci excrete lat-ge atnounts of H^O,, a highly toxic substatice for tnicrobes, in the presence of glucose in a reaction dependent on Ol atid redvtced nicotinatnide adenine dinucleotide (NADH) (4). In our ptevious study, OT affected both tnannitol catabolistn atid the growth of S. tnutans, including representatives of serotypes a, b, c and g, when the organistn was growti on matinitol (14). And the O^ sensitivity correlated with the ability of the strains to induce NADH oxidase and superoxide
distnutase (SOD) under aerobic conditions (14). NADH oxidase, however, appears to play an itnportant role in the tegulation of aerobic metabolistn of tnannitol, because of an additional NADH produced in the reaction convei-ting tnannitol 1phosphate to fructose 6-phosphate (2). The active t-ole of NADH oxidase in defense against Oi toxicity of this bacteriutn has not been elucidated. To clat-ify the function of NADH oxidase, I investigated the Oi sensitivity of S. tnutatis when grown on glucose atid the properties of NADH oxidase induced by Ol. The present study revealed that the growth response to Oi of S. tnutatts varied atnong the strains regardless of whether they were grown on mannitol or on glucose, but correlated with the level of NADH oxidase activity in the cell extracts. Mot-eover, I conclude that the overwhelmingly high level of HiOfortning NADH oxidase in this bacterium and the ratio of H^O-fonning oxidase to H^Oi-foi-tning oxidase are the key to the defense against O; toxicity.
Key words: NADH oxidase: oxygen toxicity: Streptococcus mutans M. Higuchi, Research Center, Nippon Paint Co., Neyagawa 572, Japan Accepted for publication March 3, 1992
Materials and methods iWicroorganisms
The 8 strains of the 5. tnutatis group used in this study including 5. tnutatis. Streptococcus ericetus. Streptococcus rattus and Streptococcus sobrinus, are listed in Table 1. They were maintained monthly by transferring thetn on blood agar atid tnitis-salivarius agar and were kept at 4 C. The organistns were grown in a broth containing (%, w/v): tt-ypticase peptone (BBL Microbiology Systetns, Cockeysville, MD), 1.0; yeast extract (Daigo Chetnical, Osaka, Japan), 0.2; NaCl, 0.2; K,HP04, 0.3; KH,PO4, 0.2; K,CO,, 0.1; MgSO4-4HA 0.01; MnS04 - 4H:0, 0.002; mannitol or glucose, 1.0 (TYM or TYG tnedium, pH 7.0). The mediutn was sterilized at 121 C for 15 tnin. Pyruvate was separately sterilized by ftltration. For aerobic growth, the organistns were cultut-ed in a 500-tnl flask containing 100 ml medium under air with vigorous shaking (120 t-ptn). For strictly anaerobic growth, the organistns were cultured in a Pyrex glass bottle (500 tnl) or
310
Higuchi
Table I. Study strains of the S. mutans group Strain HS6 FAl NCTC 10449 NCIB11723 (JC2) Ingbritt MT8148 GS5 6715
Serotype a b c e c c c d
a test tube (12 x 100 mm) with a screw cap in an anaerobic glove box (Hirasawa Woi-ks, Tokyo, Japan) under an attnosphere of m'/« Nj, 10% H, and 10% CO2. All anaerobic media were prereduced for at least 24 h before incubation to ensure strict anaerobiosis. All cultures were preadapted in TYM or TYG medium, inoculated with approxitnately 10'' cells/ml and maintained at 35 °C. Enzyme induction
Anaerobically grown organistns frotn the mid-exponential phase (0.45-0.48 A660) were harvested by centrifugation (21,000 xg-, 10 tnin) and resuspended in 600 tnl of TYM tnedium. The effect of aeration on enzyme induction was determined as follows; the cell suspension was divided into 6 portions, one of 50 tnl, 4 of 100 tnl or one of 150 ml volumes anaerobically. One 100-ml bottle containing 100 ml cell suspension was kept for 2 h at 35 "C in an anaerobic glove box, after which the cells were seditnented by centrifugation (2l,000xg-, 10 min), washed 3 times with 50 mM potassium phosphate buffer (pH 7.0) and kept in ice-water under atiaerobic conditions. Another 100-ml bottle containing 100 tnl of cell suspension was placed aerobically without shaking and 3 500-tnl flasks containing 50 ml (condition a), 100 ml (condition b) and 150 ml (condition c) of cell suspension, respectively, were stoppered with cotton plugs atid incubated utider air with shaking (120 rpm). These aerobic incubations were performed for 2 h at 35°C, after which the cells wet-e sedimented by cetitrifugation (21,000 x g, 10 tnin), washed 3 times with 50 mM potassiutn phosphate buffer (pH 7.0) and kept in ice-water under aerobic conditions. The retnaining 100-tnl cell suspension was placed in ice-water to arrest enzytne induction, and then sedimented and washed as described above. The pellets were kept aerobically in ice-water. The effect of chloratnphenicol on en-
Species 5. S. S. S. S. S. S. S.
Reference
cricetus rattus mutans mulans tnutatis mutans mutans sobrinus
9 9 7 3 17 13 11 16
zyme induction was determined as follows; anaerobically prepared cell suspensions were divided into 100-tnl volutnes in 6 500-ml flasks. One flask was placed anaerobically in ice-water to arrest enzytne induction. The other 5 flasks wet-e stoppered with cotton plugs and incubated at 35°C under air with vigorous shaking (120 rptn). At 0, 0.5, 1.0 and 2.0 h after initiating the aeration, chloratnphenicol (CM: 250 figl ml) was added in 4 of the 5 flasks. After 2 h aeration, the flasks wet-e placed in ice-water and then the cells were sedimented by centrifugation and washed as described above. The pellets were kept aerobically in ice-water. Preparation of extracts
All seditnented cells were disrupted by sonification within 2 h of the harvesting. The pellets were suspended in 50 mM potassium phosphate buffer (pH 7.0; 4 tnl per g wet weight) and disrupted by sonification using a Kubota Insonater (Model 200 M, Kubota, Tokyo, Japan) at 200 W for 15 tnin, at 0 C. Following centrifugation (25,000 xg, 30 min), the supernatant fluids were dialyzed against 4-1 50 tnM potassiutn phosphate buffer containing 0.2 niM-EDTA for 18 h at 4 C under air and used as crude extracts. • Assay for NAD(P)H oxidase
The activity was tneasured at 30 °C spectrophotometrically by tnonitoring the oxidation of NAD(P)H in the reaction tnixture (1 tnl) at 340 nm. One unit of enzyme activity was defined as the amount of enzyme (mg protein) that catalyzed the oxidation of LO /anol NAD(P)H/tnin. The reaction mixture contained 50 lnM potassium phosphate buffer (pH 7.2), 0.17 mM NADH atid extracts (0.1 to 50 fig protein/ml). The reaction was started by adding extracts. The reduction of 2,6-dichloro-indoplienol (DCIP), cytochrotne c and
K,Fe(CN)f, by extracts were assayed by tneasuring the changes in A^JQ, A550 atid A42,,, respectively. Assay for NADH peroxidase (EC 1.11.1.1)
The activity was tneasured by tnonitoring the oxidation of NADH using a spectt-ophototneter equipped in an anaerobic glove box (under an atmosphere of 80%. N, and 20% H,) at 30 "C in an identical medium as that used for assaying NADH oxidase except for the addition of 0.3 mM H1O,. Estimation of O^ uptaite and H2O2 production
The Ol uptake and H,O2 production by the extracts were estitnated polarographically at 30 C with an oxygeti monitor (model 53; Yellow Springs Instrument Cotnpany, Yellow Springs, OH) (14). The reaction mixture contained 50 niM potassium phosphate buffer (pH 7.2), 0.17 mM NADH with or without 0.02 tnM FMN or FAD, and extracts. The reactioti was started by the addition of NADH. When OT uptake had ceased or NADH had been consumed, 20 /(I of catalase (320 units/tnl) was added to the t-eaction tnixture. The amount of H^Oi produced in the reaction tnixture was detertnined from O2 concentration increased by adding catalase. The electrode was calibrated by the phenyl hydrazine/ferricyanide tnethods (21).
Assay for superoxide dismutase (SOD) (EC 1.15.1.1.)
The activity was assayed by the xanthine oxidase/cytochrome c tnethod (26). One unit of SOD activity was defined as the amount (mg protein) that gave a 50'^!) decrease in the reduction rate of cytochrotne c. Anothier anaiyticai metiiod
Protein concentration was detertnined by the method of Lowry et al. (19) with crystalline bovine serum albutnin as the standard. Chemicais
NADH, NADPH and xanthine oxidase were obtained from Boehringer, London, UK. Ferricytochrotne c, flavin mononucleotide (FMN), flavin adenitie dinucleotide (FAD), catalase and
NADH oxidase of S. mutatis phenazineinethosulfate were purchased from Sigma Chemical Co., St. Louis, MO. Xanthine from cow milk was purchased fi-otn Nakarai Chemicals, Tokyo, Japan. Chloramphenicol (CM) was purchased frotn Wako Pure Chetnical Industries, Tokyo, Japan. Results Effect of O2 on fhe growth of S. mutans on giucose
The growth patterns of S. ttiutans when grown on glucose under static aerobic, vigorously shaking aerobic or strictly anaerobic conditions indicated significant differences of oxygen sensitivity among the strains as previously observed with tnannitol cultures (14). Almost all strains tested grew as well under aerobic conditions without shaking as under strictly anaerobic conditions. In contrast, under vigorously shaking aerobic conditions, one group of strains iticluding MT8I48, 6715, and GS5, which were designated as oxygensensitive strains, grew with a severely i-educed growth rate (4.8, 3.8 and near 18 h doublitig titne, t-espectively). However, another group including NCTC10449, NCIB11723, Ingbritt and FAl, which wet-e designated as oxygentolerant strains, adapted to grow with sotne variations (1.2 h tnean doubling time).
Correlation between the growth rate and NADiH oxidase in extracts
To elucidate the nature of growth inhibition by O2, NADH oxidase activity of both the Oj-tolerant and the 02-sensitive strains in the late-exponential phase of gt-owth wei-e exatnined. The results (Fig. 1) show a clear correlation between NADH oxidase activity in cell extracts from aerobically grown cultures with vigorous shaking and their growth rate. The enzyme activity in extracts from 02-sensitive strains was lower than that from O2tolerant strains. A significantly higher activity of NADH oxidase was detected in extt-acts from strains NCTC 10449, NCIB11723, Itigbt-itt and FAl. This corresponded to their faster growth rate under vigot-ous shaking and aerobic conditions. These 02-tolerant strains had a maximutn activity of NADH oxidase in the tnid- to the iate-exponential phase of gt-owth. Accordingly, the NADH oxidase activity of NCIBl 1723 was lower than the 3 other O,-tolerant
Aclivily (U/mg protein)
Fig. 1. Correlation between growth and NADH oxidase activity in cell extracts. Extracts were prepared from cells grown to lateexponential phase on glucose under vigorously shaking (o) and static ( • ) aerobic conditions. NADH oxidase activity of each strain was plotted against the reciprocal of the generation time. 1: HS6, 2: FAl, 3: NCTC10449. 4: NCIBl 1723. 5: Ingbritt, 6: MT8148, 7: GS5, 8: 6715.
strains, though this strain grew with the highest growth rate atnong the stt-ains tested so fat-. All strains exhibited low levels of enzytne activity (
