Production of superoxide dismutase in Streptococcus lactis by a combination of use of hyperbaric oxygen and fermentation with cross-flow filtration

The production conditions of superoxide dismutase (SOD) in the cells of Streptococcus lactis by using hyperbaric oxygen (O(2)) are described. The SOD activity of anaerobically grown cells was 5-6 U/mg protein. When the culture broth was pressurized by O(2) at 6 atm, the SOD activity was more than twice as high as that under anaerobic culture conditions. However, there is little or no significant increase in SOD activity by exogenous addition of catalase for detoxifying hydrogen peroxide accumulated in the broth and/or controlling the pH of the broth at 6.8 during the pressurization by O(2). The increase in SOD activity by hyperbaric O(2) was possible not only at the late-logarithmic growth phase but also at the initial time for the stationary growth phase. For improvement of SOD productivity, we tried a two-stage culture in which SOD activity in S. lactis cells was enhanced by pressurizing the culture broth using hyperbaric O(2) after achievement of a high-concentration cultivation in the anerobic fermentation system with a microfiltration module.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal.

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

The effect of hydrogen peroxide on the activities of catalase and superoxide dismutase (SOD) in S. cerevisiae has been studied under different experimental conditions: various H₂O₂ concentrations, time exposures, yeast cell densities and media for stress induction. The yeast treatment with 0.25–0.50 mM H₂O₂ led to an increase in catalase activity by 2–3-fold. At the same time, hydrogen peroxide caused an elevation by 1.6-fold or no increase in SOD activity dependently on conditions used. This effect was cancelled by cycloheximide, an inhibitor of protein synthesis in eukaryotes. Weak elevation of catalase and SOD activities in cells treated with 0.25–0.50 mM H₂O₂ found in this study does not correspond to high level of synthesis of the respective enzyme molecules observed earlier by others. It is well known that exposure of microorganisms to low sublethal concentrations of hydrogen peroxide leads to the acquisition of cellular resistance to a subsequent lethal oxidative stress. Hence, it makes possible to suggest that S. cerevisiae cells treated with low sublethal doses of hydrogen peroxide accumulate non-active stress-protectant molecules of catalase and SOD to survive further lethal oxidant concentrations.     

Induction of catalase in Escherichia coli by ascorbic acid involves hydrogen peroxide

Ascorbic acid at concentrations between 0.57 and 5.7 mM in aerated medium caused an eight fold increase in catalase activity in Escherchia coli. The hydrogen peroxide concentrations resulting from ascorbate oxidation were between 20 and 120 μM and hydrogen peroxide by itself caused a similar increase in catalase levels in both aerobic and anaerobic media. Three catalase activity bands visualized on polyacrylamide gels were increased. Chloramphenicol which inhibits protein synthesis, anaerobic medium and EDTA, which prevent ascorbate oxidation, and exogenous catalase, which removes hydrogen peroxide from the medium, all prevented the increase in catalase in response to ascorbate. Superoxide dismutase activity was not affected by ascorbate.     

Scavenging ROS: superoxide dismutase/catalase mimetics by the use of an oxidation-sensitive nanocarrier/enzyme conjugate

Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then "mopped-up" by the polysulfides: this modus operandi is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides).     

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.     

Toxicity of Irradiated Media for Xenorhabdus spp

Bacterial isolates of the genus Xenorhabdus were shown to be extremely sensitive to photoproducts produced in a number of common media irradiated by fluorescent light. Two forms of toxic oxygen, hydrogen peroxide and superoxide radical, were produced in the media upon exposure to fluorescent light. The addition of pyruvate or catalase to the irradiated media eliminated the toxicity. The poor plating efficiencies previously reported for Xenorhabdus spp. are likely due to the uncontrolled exposure of media to ambient lighting.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

Mn porphyrin in combination with ascorbate acts as a pro-oxidant and mediates caspase-independent cancer cell death

Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy.     

Effects of Iso-osmotic Concentrations of NaCl and Mannitol on some Metabolic Activity in Calluses of Two Salicornia species

The comparative effects of salinity and drought on activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and polyphenol oxidase (PPO) and contents of protein, proline, hydrogen peroxide, malondialdehyde (MDA), and carotenoids were studied in calluses of Salicornia persica and Salicornia europaea. Calluses were cultured on Murashige and Skoog medium (MS medium) containing iso-osmotic concentrations of NaCl (100, 300, and 600 mM) and mannitol (200, 500, and 1000 mM). Proline content increased in calluses of both species under NaCl- and mannitol-induced stresses. Under both stresses, S. persica accumulated higher amounts of protein as compared to S. europaea. Under both stresses, protein content in S. persica decreased in comparison to control. Antioxidant enzyme activities showed different trends in the two Salicornia species under salt and drought stresses but were higher in S. europaea than in S. persica. Changes of hydrogen peroxide and MDA contents of calluses subjected to salt stress followed a pattern different from that observed under drought stress. MDA content showed similar changes to hydrogen peroxide content. Carotenoid content decreased in both species under both stresses. Overall, at the cellular level, S. europaea calluses were more tolerant to both NaCl- and mannitol-induced stresses than calluses of S. persica.     

Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism.

The consumption of molecular oxygen by Pseudomonas aeruginosa can lead to the production of reduced oxygen species, including superoxide, hydrogen peroxide, and the hydroxyl radical. As a first line of defense against potentially toxic levels of endogenous superoxide, P. aeruginosa possesses an iron- and manganese-cofactored superoxide dismutase (SOD) to limit the damage evoked by this radical. In this study, we have generated mutants which possess an interrupted sodA (encoding manganese SOD) or sodB (encoding iron SOD) gene and a sodA sodB double mutant. Mutagenesis of sodA did not significantly alter the aerobic growth rate in rich medium (Luria broth) or in glucose minimal medium in comparison with that of wild-type bacteria. In addition, total SOD activity in the sodA mutant was decreased only 15% relative to that of wild-type bacteria. In contrast, sodB mutants grew much more slowly than the sodA mutant or wild-type bacteria in both media, and sodB mutants possessed only 13% of the SOD activity of wild-type bacteria. There was also a progressive decrease in catalase activity in each of the mutants, with the sodA sodB double mutant possessing only 40% of the activity of wild-type bacteria. The sodA sodB double mutant grew very slowly in rich medium and required approximately 48 h to attain saturated growth in minimal medium. There was no difference in growth of either strain under anaerobic conditions. Accordingly, the sodB but not the sodA mutant demonstrated marked sensitivity to paraquat, a superoxide-generating agent. P. aeuroginosa synthesizes a blue, superoxide-generating antibiotic similar to paraquat in redox properties which is called pyocyanin, the synthesis of which is accompanied by increased iron SOD and catalase activities (D.J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). Pyocyanin production was completely abolished in the sodB and sodA sodB mutants and was decreased approximately 57% in sodA mutants relative to that of the wild-type organism. Furthermore, the addition of sublethal concentrations of paraquat to wild-type bacteria caused a concentration-dependent decrease in pyocyanin production, suggesting that part of the pyocyanin biosynthetic cascade is inhibited by superoxide. These results suggest that iron SOD is more important than manganese SOD for aerobic growth, resistance to paraquat, and optimal pyocyanin biosynthesis in P. aeruginosa.     

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.     

Mitigation of sodium chloride toxicity in Solanum lycopersicum L. by supplementation of jasmonic acid and nitric oxide

We investigated the effects of exogenous application of jasmonic acid (JA) and nitric oxide (NO) on growth, antioxidant metabolism, physio-biochemical attributes and metabolite accumulation, in tomato (Solanum lycopersicum L.) plants exposed to salt stress. Treating the plants with NaCl (200 mM) resulted in considerable growth inhibition in terms of biomass, relative water content, and chlorophyll content, all of which were significantly improved upon application of JA and NO under both normal and NaCl-stress treatments. Salt treatment particularly 200 mM NaCl caused an apparent increase in electrolyte leakage, lipid peroxidation, and hydrogen peroxide production, which were reduced by exogenous application of JA and NO. Salt treatment triggered the induction of antioxidant system by enhancing the activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Application of JA and NO separately as well as in combination caused a significant improvement in activities of SOD, CAT, APX, and GR activities. JA and NO either applied individually or in combination boosted the flavonoid, proline and glycine betaine synthesis under NaCl treatments. In conclusion, the exogenous application of JA and NO protected tomato plants from NaCl-induced damage by up-regulating the antioxidant metabolism, osmolyte synthesis, and metabolite accumulation.     

The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild-type E. coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.     

Role of catalase and superoxide dismutase in the yeast Saccharomyces cerevisiae response to hydrogen peroxide in exponential phase of growth

The role of catalase and superoxide dismutase (SOD) in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide in the middle-exponential phase has been investigated. It was shown that cell survival is significantly decreased after yeast exposure to hydrogen peroxide in the strains defective in cytosolic or peroxisomal catalases. Treatment of the wild-type cells with 0.5 mM H2O2 for 30 min causes an increase in the activity of catalase and superoxide dismutase, but the effect was not observed in all strains investigated. It was also shown that hydrogen peroxide leads to an increase in the activities of both catalases and Cu,Zn-containing SOD. The effect was cancelled by cycloheximide, an inhibitor of protein synthesis.     

Heat stress results in loss of chloroplast Cu/Zn superoxide dismutase and increased damage to Photosystem II in combined drought‐heat stressed Lotus japonicus

Drought and heat stress have been studied extensively in plants, but most reports involve analysis of response to only one of these stresses. Studies in which both stresses were studied in combination have less commonly been reported. We report the combined effect of drought and heat stress on Photosystem II (PSII) of Lotus japonicus cv. Gifu plants. Photochemistry of PSII was not affected by drought or heat stress alone, but the two stresses together decreased PSII activity as determined by fluorescence emission. Heat stress alone resulted in degradation of D1 and CP47 proteins, and D2 protein was also degraded by combined drought–heat stress. None of these proteins were degraded by drought stress alone. Drought alone induced accumulation of hydrogen peroxide but the drought–heat combination led to an increase in superoxide levels and a decrease in hydrogen peroxide levels. Furthermore, combined drought–heat stress was correlated with an increase in oxidative damage as determined by increased levels of thiobarbituric acid reactive substances. Heat also induced degradation of chloroplast Cu/Zn superoxide dismutase (SOD: EC 1.15.1.1) as shown by reduced protein levels and isozyme‐specific SOD activity. Loss of Cu/Zn SOD and induction of catalase (CAT: EC 1.11.1.6) activity would explain the altered balance between hydrogen peroxide and superoxide in response to drought vs combined drought–heat stress. Degradation of PSII could thus be caused by the loss of components of chloroplast antioxidant defence systems and subsequent decreased function of PSII. A possible explanation for energy dissipation by L. japonicus under stress conditions is discussed.     

Modulation of catalase peroxidatic and catalatic activity by nitric oxide

Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch. Biochem. Biophys. 316:327-334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide. Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide.     

Effect of hydrogen peroxide on antioxidant enzyme activities in Saccharomyces cerevisiae is strain-specific

The effect of hydrogen peroxide on the survival and activity of antioxidant and associated enzymes in Saccharomyces cerevisiae has been studied. A difference found in the response of wild-type yeast strains treated with hydrogen peroxide was probably related to the different protective effects of antioxidant enzymes in these strains. Exposure of wild-type YPH250 cells to 0.25 mM H₂O₂ for 30 min increased activities of catalase and superoxide dismutase (SOD) by 3.4-and 2-fold, respectively. However, no activation of catalase in the EG103 strain, as well as of SOD in the YPH98 and EG103 wild strains was detected, which was in parallel to lower survival of these strains under oxidative stress. There is a strong positive correlation (R ² = 0.95) between activities of catalase and SOD in YPH250 cells treated with different concentrations of hydrogen peroxide. It is conceivable that catalase would protect SOD against inactivation caused by oxidative stress and vice versa. Finally, yeast cell treatment with hydrogen peroxide can lead to either a H₂O₂-induced increase in activities of antioxidant and associated enzymes or their decrease depending on the H₂O₂ concentration used or the yeast strain specificity. Published in Russion in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1243–1252.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.