Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O₂ ^-·) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H₂O₂ while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H₂O₂, because it was abolished by the addition of catalase and lengthened by H₂O₂ added in addition to H₂O₂ formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.Abbreviations O₂ ^-· superoxide anion radical - NBT p-nitro blue tetrazolium chloride - ABTS 2,2-azino-di-[3-ethylbenzthiazoline sulfonate (6)] - TL-medium tryptose-lactate medium     

Growth yield and energy generation in anaerobically-grown Campylobacter spec.

An anaerobic continuous culture study was made with Campylobacter spec. to determine growth yields under various growth conditions. The growth media contained 0.1% (w/v) yeast extract as carbon source. When grown in an aspartate-limited culture Y _asp ^max was 4.6. Inclusion of formate in the culture medium hardly affected the true growth yield. The number of ATP equivalents generated in the fumaratereductase system was 0.66 and the Y _ATP ^max was 7.0. In the nitrate reduction with formate 1.7 ATP equivalents were generated, and a YNO _3- ^max of 12.2 was observed. The true growth yield obtained with a mixture of lactate and aspartate was lower than that found with aspartate alone.     

Oxygen consumption by Campylobacter sputorum subspecies Bubulus with formate as substrate

The kinetics of oxygen utilization by the microaerophile Campylobacter sputorum subspecies bubulus was studied. With formate as substrate two enzyme systems were found to be responsible for electron transfer between formate and oxygen. In the case of lactate oxidation one enzyme system could account for the activity measured. One of the formateoxidizing systems possessed a high affinity for oxygen [K _m(O₂)=approx. 4M O₂]. From inhibitor studies it was concluded that a respiratory chain was involved in its activity. Respiration by this system must be responsible for proton translocation and electron transport-linked phosphorylation at formate oxidation. The other enzyme system had an extremely low affinity for oxygen [K _m (O₂)=approx. 1 mM O₂]. It was tentatively identified as the H₂O₂-producing formate oxidase previously found in C. sputorum. The H₂O₂ production by this enzyme is implicated in an explanation of the microaerophilic nature of C. sputorum. Sensitivity of formate dehydrogenase to H₂O₂ was demonstrated. The influence of the formate concentration on aerobic formate oxidation was determined. The pH- and temperature dependencies of oxygen uptake with formate as substrate were examined at airsaturation and at a low dissolved oxygen tension.Abbreviations TL medium tryptose-lactate medium - TF medium tryptose-formate medium - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - SHAM salicylhydroxamic acid - DCPIP 2,6-dichlorophenolindophenol     

Reconsideration of the efficiency of energy transduction in Paracoccus denitrificans during growth under a variety of culture conditions

1. Theoretical overall P/2e^- ratios were calculated for growth of Paracoccus denitrificans under a variety of culture conditions on the basis of a growth model and recently published schemes of electron transport and associated proton translocation. 2. Experimental overall P/2e^- ratios were calculated, using the specific rate of ATP synthesis determined in cultures grown under aerobic carbon source-limited conditions. 3. The experimental P/2e^- was equal to the theoretical P/2e^- for aerobic sulphate-limited growth with gluconate or succinate as carbon source, on the assumption that site 1 phosphorylation was completely absent. 4. The experimental and the theoretical P/2e^- were similar for growth on nitrate as terminal electron acceptor and gluconate, mannitol or succinate as carbon source, on the assumption that nitrate enters the cell via the electroneutral nitrate-nitrite antiport system. 5. The experimental and theoretical P/2e^- were similar for growth on nitrite as terminal electron acceptor and mannitol or succinate as carbon source. 6. The experimental P/2e^- was substantially lower than the theoretical P/2e^- for aerobic growth with nitrate as nitrogen source and gluconate or mannitol as carbon source. The amount of energy needed for assimilative reduction of nitrate to ammonia was calculated from this difference.Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Growth yields and growth rates of Desulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources

Desulfovibrio vulgaris (Marburg) was grown on H₂ plus sulfate and H₂ plus thiosulfate as the sole energy sources and acetate plus CO₂ as the sole carbon sources. Conditions are described under which the bacteria grew exponentially. Specific growth rates () and molar growth yields (Y) at different pH were determined. and Y were found to be strongly dependent on the pH. Highest growth rates and molar growth yields were observed for growth on H₂ plus sulfate at pH 6.5 (=0.15h^-1; Y _{SO 4} ^2- =8.3g·mol^-1) and for growth on H₂ plus thiosulfate at pH 6.8 (=0.21h^-1; Y _{S 2}O ₃ ² =16.9g·mol^-1).The growth yields were found to increase with increasing growth rates: plots of 1/Y versus 1/ were linear. Via extrapolation to infinite growth rates a Y _SO4 ^2- /max of 12.2g·mol^-1 and a YS₂O ₃ ^2- /max of 33.5g·mol^-1 was obtained.The growth yield data are interpred to indicate that dissimilatory sulfate reduction to sulfide is associated with a net synthesis of 1 mol of ATP and that near to 3 mol of ATP are formed during dissimilatory sulfite reduction to sulfide.     

Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

An assessment of growth yields and energy coupling inDesulfovibrio

The yield coefficients forDesulfovibrio vulgaris andD. gigas varied with the electron donoracceptor combinations and with the bacterial strain. The only evidence for electron transport coupled formation of adenosine triphosphate (ATP) was with sulfate as the electron acceptor. WithD. vulgaris the ATP formation coupling to electron flow with pyruvate oxidation was 1:4 electrons and with lactate oxidation was 1:8 electrons. WithD. gigas these ratios were 1:8 electrons and 1:16 electrons for the oxidation of pyruvate and lactate. The clearest resolution of energy coupling was withD. vulgaris growing on formatesulfate medium where 2 ATP appear to be formed with the transfer of electrons from formate to adenosine phosphosulfate and one ATP with the transfer of electrons from formate to sulfite.     

Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.     

Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture

Desulfovibrio vulgaris (strain Marburg) was grown on H₂ and sulfate as sole energy source in a chemostat limited by the sulfate supply. The biomass concentration and the sulfate concentration in the culture were determined as a function of the dilution rate. From the data a K _S (saturation constant) for sulfate of 10 M, a _max of 0.23 h^–1, and a of 13 g/mol were calculated. The organism was also grown in chemostat culture on H₂ and sulfite, H₂ and thiosulfate, and pyruvate (without sulfate). was found to be 35 g/mol, 36 g/mol, and Y _pyr ^max 10 g/mol. The growth yields are discussed with respect to ATP gains in dissimilatory sulfate reduction.     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

Utilization of hydrogen and formate by Campylobacter spec. under aerobic and anaerobic conditions

A free-living aspartate-fermenting Campylobacter spec. was shown to utilize hydrogen produced in mixed culture by Clostridium cochlearium from glutamate. Resting cells of Campylobacter were shown to reduce aspartate, fumarate and malate as well as nitrate, nitrite, hydroxylamine, sulphite, thiosulphate and elemental sulphur with molecular hydrogen. Growth of Campylobacter spec. was demonstrated with formate as electron donor and nitrate, thiosulphate, elemental sulphur or oxygen as electron acceptor in the presence of acetate as carbon source.     

Mixotrophic and heterotrophic growth of Beggiatoa alba in continuous culture

Beggiatoa alba strain B18LD was grown in continuous culture under heterotrophic conditions on acetate or acetate and asparagine and under mixotrophic conditions on acetate plus either 1 mM sodium sulfide or 1 mM sodium thiosulfate. Considerable differences were observed between the yields and the cell compositions of heterotrophic and mixotrophic cultures at all dilution rates tested. The dry weight yield per gram acetate utilized was approximately three times higher in the acetate-sulfide mixotrophic culture than in the acetate heterotrophic culture, whereas the poly--hydroxybutyric acid and carbohydrate contents were much higher in the heterotrophic cultures. The high yields (0.52–0.75, corrected for the weight of the sulfur) obtained with the mixotrophic cultures imply that the acetate was utilized mainly for biosynthesis. Thus, the oxidation of sulfide supplied energy. The addition of catalase to the chemostat cultures increased yields slightly, but it was insufficient to explain the differences between the heterotrophic and the mixotrophic cultures.     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

A continuous culture study of an ATPase-negative mutant of Escherichia coli

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for Y _ATP ^max . For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli Y _ATP ^max values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51–58% of the total ATP production is used for energetization of the membrane. Using the Y _ATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.     

A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis

Summary Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a -value of 0.22. Above this growth rate protease production was repressed. Dependent on 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products was 4.1.Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y _glu ^max and YO ₂ ^max were 83.8 and 53.3, respectively. When Y _glu ^max was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y _ATP ^max . Anaerobic batch experiments showed a Y _ATP of 14.6.Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel ^-) and non-producing cells stringent (rel ⁺).Glossary specific growth rate (h^-1) - Y _sub growth yield permol substrate (g biomass/mol) - Y ^max maximum molar growth yield, corrected for maintenance requirements (g biomass/mol) - Y ^max(corr) Y ^max corrected for product formation (g biomass/mol) - m _sub maintenance requirements (mol/g biomass·h) - m _sub(corr) maintenance requirements corrected for product formation (mol/g biomass·h) - Y _c fraction of organic substrate converted in biomass - z fraction of organic substrate converted in exocellular products - d fraction of organic substrate converted in CO₂ (g mol/g atom C) - Crec% carbon recovery % - average degree of reduction of exocellular products - P/O amount of ATP produced during electron-transport of 2 electrons to oxygen     

Energy conservation during aerobic growth in Paracoccus denitrificans

Paracoccus denitrificans was aerobically grown in chemostat culture with succinate or gluconate as carbon source. Due to the presence of two phosphorylation sites in the respiratory chain and the absence of branching, theoretical P/O ratios of 1.71 and 1.82 were calculated for cells growing respectively with succinate and gluconate as carbon source. Using these data, 95% confidence intervals for the P/O ratio were determined, via a mathematical model, at 0.91–1.15 and 1.00–1.37 for sulphate-limited cultures, with respectively succinate and gluconate as carbon source.These results and measurements of P/O ratios in membrane particles and of proton translocation in whole cells have led to the conclusion that site I phosphorylation is affected under sulphate-limited conditions.Under conditions of carbon source-limitation the endogenous H⁺/O ratio is about 7–8. Average values of 3.40 and 4.78 were respectively found for sulphate-limited succinate- and gluconate grown cells. For starved cells, oxidizing succinate as exogenous substrate, the H⁺/O ratios were determined at about 3–4, independent of the growth limiting factor. It is concluded that the number of protons ejected per pair of electrons per energy-conserving site (H⁺/site ratio) is about 3–4, instead of 2 as postulated by the chemiosmotic hypothesis.     

Growth yields and glucose metabolism of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> at different culture pH values

Gluconacetobacter diazotrophicus was grown in chemostat under N₂-fixing conditions at different culture pH values (from 2.5 to 7.5) with glucose as the C-source. Maximum glucose and oxygen utilization yields were observed at pH values between 5.0 and 6.5. Yields, although lower, were not severely affected at acidic (2.5–4.5) and moderate alkaline (7.5) pH values. But, at pH values just over 7.5, cultures became unstable and washed out. Maximum biomass yields coincided with optimal activity (and minimal synthesis) of pyrroloquinoline quinone (PQQ)-linked glucose dehydrogenase (PQQ-GDH). At external pH values of 7.0 and above, whereas PQQ-GDH was actively synthesized, a very low in situ activity could be detected. The lack of PQQ-GDH activity at moderate alkaline pH values seems to be the cause of lack of growth of this organism under these conditions.     

Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors

In Wolinella succinogenes ATP synthesis and consequently bacterial growth can be driven by the reduction of either nitrate (E₀=+0.42 V), nitrite (E₀=+0.36 V), fumarate (E₀=+0.03 V) or sulphur (E₀=-0.27 V) with formate as the electron donor. Bacteria growing in the presence of nitrate and fumarate were found to reduce both acceptors simultaneously, while the reduction of both nitrate and fumarate is blocked during growth with sulphur. These observations were paralleled by the presence and absence of the corresponding bacterial reductase activities. Using a specific antiserum, fumarate reductase was shown to be present in bacteria grown with fumarate and nitrate, and to be nearly absent from bacteria grown in the presence of sulphur. The contents of polysulphide reductase, too, corresponded to the enzyme activities found in the bacteria. This suggests that the activities of anaerobic respiration are regulated at the biosynthetic level in W. succinogenes. Thus nitrate and fumarate reduction are repressed by the most electronegative acceptor of anacrobic respiration, sulphur. By contrast, in Escherichia coli a similar effect is exerted by the most electropositive acceptor, O₂. W. succinogenes also differs from E. coli in that fumarate reductase is not repressed by nitrate.Abbreviations BV benzyl viologen - DMN 2,3-dimethyl-1,4-naphthoquinone - DMSO dimethylsulfoxide - TMAO trimethylamine-N-oxide     

Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

Abstract The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h⁻¹. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h⁻¹. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.