Evidence for 18O labeling of photorespiratory CO2 in photoautotrophic cell cultures of higher plants illuminated in the presence of 18O2

The ¹⁸O-enrichment of CO₂ produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular ¹⁸O₂. The only ¹⁸O-labeled species produced was C¹⁸O¹⁶O; no C¹⁸O¹⁸O could be detected. Production of C¹⁸O¹⁶O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO₂. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O₂ present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO₂ and H₂O, drastically diminished the ¹⁸O-enrichment of the produced CO₂. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the ¹⁸O labeling of photorespiratory CO₂ could be observed for the first time. These data allow the establishment of a direct link between O₂ consumption and CO₂ production in the light, and the conclusion that CO₂ produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C¹⁸O¹⁶O and CO₂ kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO₂ production in the light.     

O2 and H2O are each the source of one O in NO−2 produced from NH3 by Nitrosomonas: 15N-NMR evidence

The exchange of ¹⁸O between H₂¹⁸O and exogeneously added ¹⁵N¹⁶O^?₂ which occurs during oxidation of ammonia by Nitrosomonas is shown to occur one oxygen at a time. Conditions in which the exchange is diminished (notably the presence of ¹⁴NO^–₂ and CCCP) allowed demonstration that water and dioxygen are each the source of one oxygen in nitrite produced from ¹⁵NH₃. The nitrate produced in the presence of ¹⁸O₂ consisted of 67 and 0% ¹⁵N¹⁸O¹⁶O^? and ¹⁵N¹⁸O¹⁸O^?, respectively. Analysis was made using the ¹⁸O-isotope shift in ¹⁵N-NMR.     

Physiological Ecology of Clostridium glycolicum RD-1, an Aerotolerant Acetogen Isolated from Sea Grass Roots

An anaerobic, H₂-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H₂-CO₂, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O₂ in the headspace of static, horizontally incubated culture tubes; the concentration of O₂ decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O₂. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H₂ became significant end products when RD-1 was grown on glucose in the presence of O₂. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H₂ were produced. When the concentration of O₂ in the headspace exceeded 1% (vol/vol), supplemental H₂ was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288^T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288^T demonstrated that it had negligible H₂- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288^T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288^T confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O₂, (iii) oxic conditions favor the production of ethanol, lactate, and H₂ by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O₂ might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O₂.     

Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis

Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na⁺/K⁺ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H₂O₂) generated in the presence of heme in the Na⁺/K⁺ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H₂O₂ in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na⁺/K⁺ ATPase is through the generation of H₂O₂, we measured enzyme activity using increasing concentrations of H₂O₂ and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H₂O₂. To investigate the role of PKC in this signaling pathway, we observed the production of H₂O₂ in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H₂O₂. Furthermore, we found that PKC activity is increased in the presence of H₂O₂, and that in the presence of calphostin C, H₂O₂ is unable to activate the Na⁺/K⁺ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na⁺/K⁺ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na⁺/K⁺ ATPase by H₂O₂ opens new possibilities for understanding the signaling pathways of this parasite.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD⁺ or NADH at about the same operating redox potential as the NADH/NAD⁺ pool and comprise the NADH/NAD⁺ isopotential enzyme group. Complex I (specifically the flavin, site I_F) is often regarded as the major source of matrix superoxide/H₂O₂ production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H₂O₂ production. To differentiate the superoxide/H₂O₂-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H₂O₂ production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H₂O₂ production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)⁺ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H₂O₂ at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H₂O₂ was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site I_F of complex I. Depending on the substrates present, the dominant sites of superoxide/H₂O₂ production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.     

Stepwise oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase

Fungal peroxygenases have recently been shown to catalyze remarkable oxidation reactions. The present study addresses the mechanism of benzylic oxygenations catalyzed by the extracellular peroxygenase of the agaric basidiomycete Agrocybe aegerita. The peroxygenase oxidized toluene and 4-nitrotoluene via the corresponding alcohols and aldehydes to give benzoic acids. The reactions proceeded stepwise with total conversions of 93% for toluene and 12% for 4-nitrotoluene. Using H₂¹⁸O₂ as the co-substrate, we show here that H₂O₂ is the source of the oxygen introduced at each reaction step. A. aegerita peroxygenase resembles cytochromes P450 and heme chloroperoxidase in catalyzing benzylic hydroxylations.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase

Molecular hydrogen (H₂) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H₂ production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H₂ syntheses rely mainly on light, water, and CO₂ and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H₂-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O₂) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O₂-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O₂-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O₂ sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O₂ tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH⁺ to utilize H₂ as sole electron source to fix CO₂. Syn_ReSH⁺ also was able to produce H₂ under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H₂ production.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

Effect of 1-Aminocyclopropane-1-Carboxylic Acid on the Production of Ethylene in Senescing Flowers of Ipomoea tricolor Cav

Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to rib segments excised from flowers of Ipomoea tricolor Cav. resulted in the formation of C₂H₄ in greater quantities than produced under natural conditions. The ability of ACC to enhance C₂H₄ production was independent of the physiological age of the tissue and its capacity to synthesize C₂H₄ without applied ACC. When ACC was fed to rib segments that had been treated with [¹⁴C]methionine, incorporation of radioactivity into C₂H₄ was reduced by 80%. Aminoethoxyvinylglycine and aminooxyacetic acid inhibited C₂H₄ production in rib segments of I. tricolor but had no effect on ACC-enhanced C₂H₄ production. Protoplasts obtained from flower tissue of I. tricolor did not form C₂H₄, even when incubated with methionine or selenomethionine. They produced C₂H₄ upon incubation with ACC, however. ACC-dependent C₂H₄ production in protoplasts was inhibited by n-propyl gallate, AgCl, CoCl₂, KCN, Na₂S, and NaN₃. ACC-dependent C₂H₄ synthesis in rib segments and protoplasts was dependent on O₂, the K_m for O₂ being 1.0 to 1.4% (v/v). These results confirm the following pathway for C₂H₄ biosynthesis in I. tricolor. methionine [selenomethionine] → S-adenosylmethionine [selenoadenosylmethionine] → ACC → C₂H₄.     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria

Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H₂O₂/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca²⁺ also markedly stimulated H₂O₂ production by rat heart mitochondria. The enhancement of H₂O₂ generation in antimycin-supplemented mitochondria and the increased O₂ uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca²⁺ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H₂O₂ production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O₂⁻, the precursor of mitochondrial H₂O₂. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c₁³⁺ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O₂⁻. The effect of protophores, ionophores and Ca²⁺ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c₁ [Wikstrom & Berden (1972) Biochim. Biophys. Acta 283, 403–420; Mitchell (1976) J. Theor. Biol. 62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O₂⁻ production, is proposed as the molecular mechanism for the enhancement of H₂O₂ formation rates observed on addition of protophores, ionophores and Ca²⁺.     

Potential role of Noxes in the protection of mucosae: H2O2 as abacterial repellent

Duox proteins are members of the NADPH oxidase (Nox) family and are responsible for hydrogen peroxide (H₂O₂) production by various tissue types including bronchial and intestinal mucosae. The antimicrobial killing role of H₂O₂ in leukocytes and macrophages is generally considered as the paradigm of its function. We investigated here the positive role of H₂O₂ in the prevention of cellular invasion by Salmonella. We show that H₂O₂, under conditions that preserved bacterial growth, has a repellent effect on Salmonella motility on agar plates. In addition, H₂O₂ produced by PCCl3, a rat thyroid cell line, reduces bacterial invasion of the cells by around 40%. To test whether the observed phenotype is attributable to H₂O₂ production, we constructed a CHO stable cell line expressing Duox2 protein at the cell surface (CHO-D2). The transfected cells produce a high amount of H₂O₂. Upon infection with Salmonella, the invasion of CHO-D2 cells was reduced by up to 60%. In both PCCl3 and CHO expressing Duox2 cells, normal invasion was restored upon incubation with catalase. Our data suggest that H₂O₂ at reduced concentrations acts as a repellent for bacteria, keeping them away from cells, a situation that could naturally prevent mucosal cells infection in vivo.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Production and stabilization of cells of Bacillus popilliae and Bacillus lentimorbus

Bacillus popilliae and B. lentimorbus grew most rapidly and to the greatest extent in aerated cultures at 30 to 32 C with oxygen absorption rates of 1 mmole of O₂ per min per liter, or above. The control of pH also increased the maximal populations attained. Media were developed which consistently produced cell populations of about 10⁹ within 24 to 48 hr in aerated cultures of these two species. The acetic acid produced in highly aerated cultures was shown not to be responsible for the rapid loss of cell viability in stationary phase cultures. However, H₂O₂ was very lethal to cells of B. popilliae, and this species is known to have the capacity to produce it. Stationary-phase cells were partially stabilized by reducing the availability of oxygen after 24 hr of incubation on a shaker, and the addition of low levels of glucose further stabilized the cells. The most stable cells were those produced in a medium in which 4% Trypticase (BBL) and 0.1% barbituric acid were incorporated. A high percentage of these cells contained refractile bodies visible under a phase microscope. Although these bodies were not heat-resistant and lacked other characteristics of endospores, cells in cultures containing them had reasonably high viability for extended periods, as compared with those in control cultures.     

Mechanism of aromatic ring cleavage of β-O-4 lignin substructure models by lignin peroxidase

This investigation examined the aromatic ring cleavage of β-O-4 lignin substructure model compounds by lignin peroxidase of Phanerochaete chrysosporium. Based on tracer experiments using H₂¹⁸O and ¹⁸O₂, mechanisms of the aromatic ring cleavage of the β-O-4 lignin models were proposed. The mechanisms involve one-electron oxidation of the β-O-4 lignin models by the enzyme followed by attack of nucleophiles and radical coupling with O₂.     

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Agarwood is highly valuable resinous and fragrant heartwood, produced principally from tropical tree species in the genus Aquilaria, which is used widely in countries of the Middle East, Southeast Asia and Japan. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. In this study, the pruning of actively growing saplings of Aquilaria sinensis resulted in hydrogen peroxide (H₂O₂) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Treatment of the pruned stems with scavenger of H₂O₂ (ascorbate, AsA) greatly reduced the amount of H₂O₂ released, the number of vessel occlusions, and the amount of sesquiterpenes produced. In addition, exogenous H₂O₂ also induced A. sinensis plants to form vessel occlusions and produce sesquiterpenes as pruning treatment. The results indicated that H₂O₂ may be an important post-wounding signal in A. sinensis that leads to the induction of vessel occlusions formation and sesquiterpene biosynthesis, and thus H₂O₂ might play a vital role in agarwood formation in pruned stems of A. sinensis.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Growth Pattern and Yield of a Chemoautotrophic Beggiatoa sp. in Oxygen-Sulfide Microgradients

Recently developed techniques involving opposed, gel-stabilized gradients of O₂ and H₂S permit cultivation of a marine Beggiatoa strain as a chemolithoautotroph which uses gliding motility to precisely track the interface between H₂S and O₂. In the current study with microelectrodes, vertical profiles of H₂, O₂, and pH were measured in replicate cultures grown for various intervals. After an initial period of exponential biomass increase (doubling time, 11 h), linear growth prevailed throughout much of the time course. This H₂S-limited growth was followed by a transition to stationary phase when the declining H₂S flux was sufficient only to supply maintenance energy. During late-exponential and linear growth phases, the Beggiatoa sp. consumed a constant 0.6 mol of H₂S for each 1.0 mol of O₂, the ratio anticipated for balanced lithoautotrophic growth at the expense of complete oxidation of H₂S to SO₄²⁻. Over the entire range of conditions studied, this consumption ratio varied by approximately twofold. By measuring the extent to which the presence of the bacterial plate diminished the overlap of O₂ and H₂S, we demonstrated that oxidation of H₂S by Beggiatoa sp. is approximately 3 orders of magnitude faster than spontaneous chemical oxidation. By integrating sulfide profiles and comparing sulfide consumed with biomass produced, a growth yield of 8.4 g (dry weight) mol⁻¹ of H₂S was computed. This is higher than that found for sulfide-grown thiobacilli, indicating very efficient growth of Beggiatoa sp. as a chemoautotroph. The methods used here offer a unique opportunity to determine the yield of H₂S-oxidizing chemolithoautotrophs while avoiding several problems inherent in the use of homogeneous liquid culture. Finally, by monitoring time-dependent formation of H₂S profiles under anoxic conditions, we demonstrate a method for calculating the molecular diffusion coefficient of soluble substrates in gel-stabilized media.