Manganese, Superoxide Dismutase, and Oxygen Tolerance in

JouRNwL OF BACTIFROLOGY, June 1981, p. 928-936 0021-9193/81/060928-09$02.00/0

Vol. 146, No. 3

Manganese, Superoxide Dismutase, and Oxygen Tolerance in Some Lactic Acid Bacteria FREDERICK S. ARCHIBALD AND IRWIN FRIDOVICH* Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received 22 January 1981/Accepted 10 March 1981

A previous study of the aerotolerant bacterium Lactobacillus plantarum, which lacks superoxide dismutase (SOD), demonstrated that it possesses a novel substitute for this defensive enzyme. Thus, L. plantarum contains 20 to 25 mM Mn(II), in a dialyzable form, which is able to scavenge 02 apparently as effectively as do the micromolar levels of SOD present in most other organis. This report describes a survey of the lactic acid bacteria. The substitution of millimolar levels of Mn(II) for micromolar levels of SOD is a common occurrence in this group of microorganisms, which contained either SOD or high levels of Mn(II), but not both. Two strains were found which had neither high levels of Mn(II) nor SOD, and they were, as was expected, very oxygen intolerant. Lactic acid bacteria containing SOD grew better aerobically than anaerobically, whereas the organism containing Mn(II) in place of SOD showed aerobic growth which was, at best, equal to anaerobic growth. Plumbagin (5-hydroxy-2-methyl-1,4naphthoquinone) increases the rate of 02 production in these organisms. Lactobacillus strains containing high intracelluar Mn(II) were more resistant to the oxygen-dependent toxicity of plumbagin than were strains containing lower levels of Mn(II). The results support the conclusion that a high interal level of Mn(II) provides these organisms with an important defense against endogenous 02.-

Superoxide dismutases (SOD), which provide a defense against the toxicity of oxygen by catalytically scavenging 02 (13), have been detected in most organis examined, with the exception of certain obligate anaerobes (27). A few oxygen-tolerant organisms have been found to lack SOD. These are: several mycoplasmnas (25); two disseminating strains of Neisseria gonorrhoeae (30); and Lactobacillus plantarum (17). Apparent anomalies such as these challenge our knowledge of the basis of oxygen toxicity and of the defenses evolved to deal with it and, at the same time, provide an opportunity for extension of that knowledge. Careful study of one of these organisms, L. plantarum, demonstrated that its high intracellular level of Mn(II) takes the place of SOD in scavenging 02 (la). Indeed, L. plantarum has a capacity for scavenging °2 which is comparable to that observed in aerobically grown Escherichia coli. The activity observed in extracts ofE. coli, being due to micromolar levels of SOD, is heat labile, insensitive to EDTA, and nondialyzable. In contrast, the activity of L. plantarum extracts, being due to millimolar levels of Mn(II), is heat stable, dialyzable, and inhibited by EDTA. Mn(ll) deprivation made L. plantarum increasingly intolerant of oxygen, demonstrating the importance

of Mn(ll) as a defense against oxygen toxicity in these organisns (la). The lactic acid bacteria constitute a large group of widely distributed gram-positive rods or cocci which share several properties, including lack of hemes and cyanide-sensitive respiration, production of lactate, frequent accumulation of H202, and the presence of flavin-linked oxidases, catalases, and peroxidases. They are currently clasified into two families, the Streptococcaceae and the Lactobacillaceae (7). Several members of the family Streptococcaceae in the genus Streptococcus have already been examined for their content of SOD. S. lactis, S. faecalis, S. pyogenes (5), and S. sanguis (20) have all been found to possess a single manganesecontaining SOD (MnSOD). None of the other genera in this family, namely Pediococcus, Aerococcus, Leuconostoc, and Gemella, and none of the Lactobacillaceae except for L. plantarum have yet been studied in this way. It seemed important to survey the lactic acid bacteria for their content of Mn(II) and of SOD, as well as for their tolerance towards 02 and 02This would determine how widespread the substitution of high levels of intracellular Mn(ll) for SOD is in this group of microorganisms and might be expected to increase our understanding

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of oxygen toxicity. The results of this survey, tion spectrophotometer equipped with an HGA-2000 which indicate that millimolar levels of intracel- graphite furnace was used to measure [Mn]. 02-scavmeasured by the xanthine oxidaselular Mn(II) are an important component of the enging activityc was with and without 100 ,uM method, cytochrome of the in many defenses against oxygen toxicity In all cases, 1.0 unit of 02--scavenging (26). EDTA lactobacilli, form the body of this report. activity was taken to be that amount causing a 50% decrease of the rate of 02--dependent reduction of MATERIALS AND METHODS cytochrome c. Cytochrome c reduction was assumed Organisms and culture conditions. Lactobacil- to be due to O2- only if it could be inhibited by the lus casei YIT-0001, Lactobacillus fermentum 36, Leu- addition of bovine erythrocyte SOD to the assay mixconostoc mesenteroides 8293, and Streptococcus lactis ML-3 were obtained from the microbiology culture collection of North Carolina State University, Raleigh, through the courtesy of S. Tove. Streptococcus faecalis, used previously (5, 16), was from the culture collection of this laboratory. Lactobacillus acidophilus NCFM was a strain isolated from and used for the commercial production of sweet acidophilus milk. All other strains were obtained as lyophils from the American Type Culture Collection, Rockville, Md. All strains were subcultured onto multiple small APT agar (11) or MRS agar (10) slants and were stored at -70°C until required. Each experiment employed a fresh slant. Cultures were routinely grown on commercial (Difco Laboratories, Detroit, Mich.) MRS or APT broth or agar plates. For survival curves, APT and MRS agars were formulated without added Mn, and glucose was added aseptically after autoclaving. The low-Mn APT medium contained 1.8 yM Mn, and the low-Mn MRS medium contained 0.8 yM Mn, as opposed to their normal Mn concentrations of 710 and 330 uM, respectively. Lactobacillus fermentum, L. acidophilus, L. bulgaricus, and L. ruminis were always cultured on MRS media due to their substantially poorer growth on APT agar. All other organisms were cultured on APT broth and agar, although L. plantarum was grown on MRS agar in experiments in which it was compared with the four strains requiring MRS agar. Cell numbers were determined by relating direct counts to culture absorbance at 600 nm with a Gilford 2000 spectrophotometer, as previously described (la), and by viable counts, employing serial dilutions in sterile APT salts (la) and plating of 10-1l aliquots of the dilutions, in triplicate, on MRS agar

plates. Aerobic broth cultures were shaken at 116 rpm at 37°C. Anaerobic growth was performed in a Coy Chamber (Coy Laboratory Products, Inc., Ann Arbor, Mich.) or in a Brewer-type anaerobic jar. Exposure to hyperbaric oxygen was performed at 37°C in stainlesssteel pressure vessels. All liquid cultures were grown in Erlenmeyer flasks with a vessel volume to fluid volume ratio of 2.5:1. Broth cultures of L. ruminis were placed in an anaerobic jar and were agitated with a magnetic stir bar. Cells were harvested by centrifugation (4,080 x g, 10 min), washed twice in APT salts, pH 6.7, and pelleted at 8,700 x g for 5 min. Crude cell extracts were prepared by resuspending cell pellets in 50 mM potassium phosphate, pH 7.8, if they were to be used for assays of 02 scavenging or in APT salts if they were to be used for diaphorase or other assays. Cells were disrupted by double passage through an Aminco French pressure cell at 20,000 lb/in2, and the resultant extract was clarified for 10 min at 8,700 x g. Assays. A Perkin-Elmer model 107 atomic absorp-

ture. The diaphorase assays, polarographic oxygen consumption measurements, polyacrylamide disk gel electrophoresis, and SOD activity stains were performed as described previously (la). Reagents. Xanthine oxidase was prepared from raw cream (33) and bovine erythrocyte SOD as described previously (26). Culture media were from Difco Laboratories, with the exception of Trypticase peptone (BBL Microbiology Laboratories, Cockeysville, Md.). APT salts buffer contained (per liter) 5.0 g of K2HPO4, 5.0 g of NaCl, 1.25 g of Na2CO3, and 0.8 g of MgSO4 and was adjusted to a pH of 6.7. Organic reagents were from Sigma Chemical Co., St. Louis, Mo.

RESULTS Superoxide scavenging activity. Lactobacilli representing subgenus groupings IA, IB, and II (7) were selected. These correspond to the former division of the genus Lactobacillus into thermobacteria (L. bulgaricus, L. acidophilus, L. ruminis), streptobacteria (L. plantarum, L. casei), and betabacteria (L. fermentum), respectively. Four genera of the family Streptococcaceae were also examined. Table 1 lists the organisms examined and for each organism gives the 02-scavenging activity observed when extracts were assayed with or without EDTA and both before and after dialysis. Dialyzable, EDTA-sensitive 02--scavenging activity, presumably due to Mn(II), was observed in all L. plantarum strains, L. casei, L. fermentum, L. ruminis, two strains of L. mesenteroides and Pediococcus pentosaceus. All of these, possessing an alternative to SOD, were devoid of this enzyme. Conversely, those organisms which contained true SOD did not contain the EDTA-sensitive and dialyzable activity. In this category were Aerococcus viridans, S. lactis, S. faecalis, S. sanguis, and E. coli. Two organisms, L. bulgaricus and L. acidophilus, were extraordinary in that they were poor in 02--scavenging activity of any kind. L. bulgaricus had no detectable activity, whereas L. acidophilus had a weak activity which was dialyzable, yet insensitive to EDTA. Catechols are known to react with 02 (15) and were considered, but the Arnow test for catechols and phenolic acids (2), when applied to extracts of L. acidophilus, gave a negative result. A 2-,ug/ml

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ARCHIBALD AND FRIDOVICH

TABLE 1. 02 -scavenging activities of ceU extractsa Undialyzedb

Dialyzed

Organiism +EDTA -EDTA +EDTA -EDTA Lactobacillus plantarum 14917 (APT)

a I.

0.01

0

1

3 2 Hours at 37 OC

4

FIG. 2. The toxicity of aerated plumbagin. Organisms were grown for 12 h at 370C in MRS broth (330 WM Mn). In the case of L. ruminis, this growth was under anaerobic conditions, whereas the other organisms were grown aerobically. Aliquots of these cultures were then diluted approximately 500-fold into duplicate 50-ml flasks containing 20-ml portions of MRS broth with 0.8 pM Mn(II) and 0.5 mg of puromycin per ml to prevent growth. Plumbagin was then added, in 10 gl of ethanol, to a final concentration of 25 uM to one set offlasks. The control set received 10 p1 of ethanol. The flasks were then incubated aerobically in a water bath shaker at 370C, and serial dilutions and plating for viable counts were performed at 0, 1, 2, and 4 h after plumbagin addition. Lines 1 and 3 were obtained with L. plantarum in the absence and in the presence of plumbagin, respectively. Similarly, lines 5 and 8 were obtained with L. bulgaricus, lines 6 and 7 with L. acidophilus, and lines 2 and 4 with L. ruminis. Points in the figure represent the number of viable colony-forming units present as a percent of the number present in the same flask approximately 5 min after plumbagin addition.

grown in broth containing only 1.6,uM Mn(II) (lines 2 and 4).

DISCUSSION Those lactic acid bacteria which contained high intracellular levels of Mn(II), in a dialyzable form, were devoid of true SOD. Conversely, those which possessed true SOD did not contain high levels of Mn(II). This suggests that high [Mn(II)] represents a substitution for SOD and, like the enzyme it replaces, serves to scavenge

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J. BACTERIOL.

1.0

0.1

0.01_ 0.001I 0 1

2 3 Hours at 370C

4

FIG. 3. Effects of intracellular Mn on the toxicity of aerated plumbagin. L. plantarum 14917 was grown aerobically for 12 h at 37°C in APT broth containing 1.6 or 710 Mn II). These cultures were then diluted to a final ceU concentration of 2 x 106 to 6 x 106 cells/ml in duplicate 50-ml flasks containing 20-ml portions of APT broth [1.6 Mn(II)l plus 0.5 mg of puromycin per ml. Duplicate sets were prejared with and without 180 pM plumbagin. The flasks were incubated aerobicaly in a water bath shaker at 37°C and at intervals samples were taken for enumeration of viable cells, as in Fig. 2. Lines 2 and 3 were obtained in the absence and in the presence ofplumbagin, respectively, with L. plantarum grown in the Mn(II)-rich medium. In a comparable way, lines 1 and 4 were obtained with cells grown in the MnaII)deficient medium. Data points represent viable colony-forming units as in Fig. 2.

02- and thus to protect the cell against the direct and indirect toxic effects of this radical. Mn(II) is known to be oxidized by O2-. In the presence of pyrophosphate the trivalent manganese is stabilized and can accumulate (21). In the absence of such stabilizing chelating agents MnO2+ is formed and promptly decays (4), possibly to MnO2 plus Mn(II) plus 02. Cell interiors are rich in pyrophosphates, such as nucleoside triphosphates and the dinucleotide coenzymes, which should lend stability to Mn(III). Nevertheless,

Mn(llI) is a strong oxidant and can spontaneously oxidize NAD(P)H (9, 28). We therefore expect that Mn(II) would scavenge 02 much as peroxidases scavenge H202, i.e., at the expense of some reductant, such as NAD(P)H. This is in contrast to the action of SOD on 02 which, like the action of catalase on H202, requires no other reducing substrate. In the xanthine oxidase-cytochrome c assay for O2--scavenging activity, micromolar levels of Mn(II) exerted an activity comparable to that of nanomolar levels of SOD (la). Similarly, in the lactic acid bacteria, millimolar levels of Mn(II) can replace micromolar levels of SOD. There are several indications that high intracellular levels of Mn(II) do serve as a defense against endogenous 02 and against oxygen toxicity. Thus, decreasing intracellular [Mn(ll)] in L. plantarum, by growth in Mn-deficient media, imposed an oxygen intolerance (la). Similarly, Mn(II)-deficient L. plantarum cells are now seen to be much more susceptible to the lethality of aerobic plumbagin than are Mn(II)-replete cells. Since this naphthoquinone derivative has been shown to impose a markedly increased flux of 02 upon these cells, it is clear that a high intracellular level of Mn(II) does protect the cells against this radical much as SOD was previously shown to protect E. coli against the same insult (18). Finally, when lactobacilli containing low intracellular levels of Mn(ll) (L. bulgaricus and L. acidophilus) are compared with strains containing high intracellular levels of Mn(II) (L. plantarum and L. ruminis), the former are vastly more sensitive to the lethality of aerobic plumbagin than are the latter. It is instructive to consider L. ruminis in some detail. As a rumen inhibitant it ordinarily grows in an Mn-rich and anaerobic environment. It is an obligate anaerobe in that it will not grow in the presence of oxygen. However, it does contain high intracellular [Mn(II)], is very resistant to aerobic plumbagin, and survives for days on the surface of agar plates. Since transmission of L. ruminis from one ruminant to another will almost certainly involve contact with air, incorporation of high intracellular levels of Mn to render 02 merely bacteriostatic may well be of selective advantage. Its inability to grow may be due to one or more essential Orsensitive enzymes, coenzymes, or redox couples. For example, sulflhydryl to disulfide oxidation, although it may inactivate the enzyme or coenzyme affected, is readily reversible when anaerobic reducing conditions are again imposed. In contrast, 02-, because of other radicals it can engender, is capable of irreversibly damaging informational macromolecules, such as the nucleic acids (4a, 8, 22, 23, 29, 32); this remains to be explored.

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Although it is attractive to propose that high intracellular Mn is ancestral to MnSOD, there are reasons for considering this not to be a primitive characteristic. Lactic acid bacteria with high internal levels of Mn and no SOD require numerous amino acids and other growth factors (19) and, although unable to synthesize hematin, some strains can synthesize a hemecontaining catalase, if supplied with hematin (34). There are, in fact, reports that hematin in the medium will permit the synthesis of cytochromes and allow limited oxidative phosphorylation in S. faecalis 10C1 (6, 31). All of this, plus the evolutionary relationships of various microbial groups as deduced from the sequence of oligonucleotide fragments of 16S RNA (12), suggests that the apparent simplicity of the lactic acid bacteria is an adaptation or divergence and not primordial. This is consistent with the finding of SOD enzymes in such apparently truly primitive groups as the Archaebacteria (methanogens) (T. Kirby, J. R. Lancaster, Jr., and I. Fridovich, Arch. Biochem. Biophys., in press). Therefore, it follows that substitution of high levels of Mn(II) for SOD in lactic acid bacteria is an adaptation, possibly to life in Mn-rich media, and it may not be coincidental that the two lactobacilli lacking high levels of Mn were dairy organisms, milk being very low in Mn, whereas those containing high levels of Mn are all usually found associated with plant material generally high in Mn from the chloroplasts (1). P. pentosaceus and L. mesenteroides are presently classified with streptococci. The Streptococcoceae and Lactobacillaceae are closely related biochemically and are distinguished primarily on the basis of morphology, i.e., cocci as opposed to bacilli. Yet, there is a continuum of shapes from long rods, such as L. bulgaricus, to very short rods or ovoids, such as L. coryneformis, to true cocci (7). P. pentosaceus and L. mesenteroides were similar to the Lactobacillaceae in that they contained high levels of Mn(II), instead of SOD, whereas the streptococci studied contained SOD. Accumulation of millimolar levels of intracellular Mn(II) must be accompanied by many adaptations, including mechanisms for Mn uptake and changes in the many enzymes ordinarily capable of binding and being affected by Mg(II) and Mn(II). All of this suggests that P. pentosaceus and L. mesenteroides may be more closely related to lactobacilli than to streptococci. The conclusions reached here concerning the distribution of high Mn and SOD are based on examination of relatively few strains. A broader survey for the employment of high Mn(II) or of SOD, including facultative and anaerobic strains of Gemella, sporolactobacilli, bifidobacteria, coryneforms, and other silage and

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rumen organisms should therefore provide much information on the distribution of high intracellular Mn and its relationship to oxygen toxicity. A number of surveys for SOD in anaerobes have been published to date, and all used the xanthine oxidase-cytochrome c assay containing 0.1 mM EDTA. These would therefore have missed the EDTA-sensitive activity due to Mn(II) and should be redone. ACKNOWVLEDGENTS This work was supported by research grants from the Institute of General Medical Sciences of the National Institutes of Health, Bethesda, Md.; from the U.S. Army Research Office, Research Triangle Park, N.C.; and from Merck & Co., Inc., Rahway, N.J. F.S.A. is the recipient of a Medical Research Council of Canada postodoctoral fellowship. LITERATURE CITED 1. Altman, P. L, and D. S. Dittmer (ed). 1968. Metabolism, p. 80-88. Federation of American Societies of Experimental Biology, Bethesda, Md. la.Archibald, F. S., and L. Fridovich. 1981. Manganese and defenses against oxygen toxicity ir. Lactobacillus plantarum. J. Bacteriol. 145:442-451. 2. Arnow, L. E. 1937. Colorimetric determination of the components of 3,4-dihydroxy phenyilaanine-tyrosine mixtures. J. Biol. Chem. 118:531-537. 3. Beauchamp, C., and L. Fridovich. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276-287. 4. Bielski, B. H. J., and P. C. Chan. 1978. Products of reactions of superoxide and hydroxyl radicals with Mn2+ cation. J. Am. Chem. Soc. 100:1920-1921. 4a.Brawn, K., and L. Fridovich. 1981. DNA strand scission by enzymically-generated oxygen radicals. Arch. Biochem. Biophys. 206:414-419. 5. Britton, L., D. P. Malinowski, and I. Fridovich. 1978. Superoxide dismutase and oxygen metabolism in Streptocococcus faecalis and comparisons with other organisms. J. Bacteriol. 134:229-236. 6. Bryan-Jones, D. G., and R. Wittenbury. 1969. Hematin-dependent oxidative phosphorylation in Streptococcus faecalis. J. Gen. Microbiol. 58:247-260. 7. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 8. Cone, R., S. K. Hasan, J. W. Lown, and A. R. Morgan. 1976. The mechanism of the degradation of DNA by streptonigrin. Can. J. Biochem. 54:219-223. 9. Curnutte, J. T., M. L. Karnovsky, and B. M. Babior. 1976. Manganese dependent NADPH oxidation by granulocyte particles. J. Clin. Invest. 57:1059-1067. 10. DeMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135. 11. Evans, J. B., and C. F. Niven. 1951. Nutrition of the heterofermentative lactobacilli that cause greening of cured meat products. J. Bacteriol. 62:599-603. 12. Fox, G. E., E. Stackebrandt, R. B. Hespell, J. Gibson, J. Maniloff, T. A. Dyer, R. S. Wolfe, W. E. Balch, R. S. Tanner, L. J. Magrum, L. B. Zahlen, R. Blakemore, R. Gupta, L. Bonen, B. J. Lewis, D. A. Stahl, K. R. Luehrsen, K. N. Chen, and C. R. Walse. 1980. The phylogeny of procaryotes. Science 209:457-463. 13. Fridovich, I. 1979. Superoxide and superoxide dismutases, p. 67-90. In G. L. Eichhorn and L. G. Marzilli (ed.), Advances in inorganic biochemistry. Elsevier/ North-Holland Publishing Co., New York.

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