Trimethylamine oxide: a terminal electron acceptor in anaerobic respiration of bacteria.

Trimethylamine oxide (TMAO) stimulated both the anaerobic growth rate and the growth yield of Proteus NTHC 153. The molar growth yield from glucose and pyruvate in tryptone/yeast extract medium doubled in the presence of TMAO, and the organism grew anaerobically on the non-fermentable substrates L-lactate and formate when TMAO was added to the medium. We conclude that TMAO stimulated growth by serving as a terminal electron acceptor in an oxidative phosphorylation process.     

The formation of hydroxyproline in collagen by cells grown in the absence of serum.

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggests that reductants other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

The formation of hydroxyproline in collagen by cells grown in the absence of serum

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggest that reductant other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium.

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

Maleimides stimulate oxygen reduction in illuminated thylakoids

N-ethylmaleimide (NEM) and N,N'-(1,4-phenylene)dimaleimide (PDM) were discovered to stimulate light-induced oxygen uptake in isolated thylakoids, and PDM provided the same stimulation at one order less concentrations. Oxygen uptake rate increased promptly after NEM or PDM addition to thylakoids. The inhibitors of photosynthetic electron transport as well as catalase decreased this rate close to zero, whereas ascorbate increased it almost three-fold. Dithiothreitol suppressed oxygen uptake stimulated by NEM. NEM stimulated light-induced reduction of cytochrome c, and this stimulation was suppressed by superoxide dismutase. It was concluded that NEM and PDM being reduced can effectively reduce molecules O(2) producing superoxide radicals.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

The influence of pH on the inhibition of oxidative phosphorylation and electron transport by triethyltin

The ability of triethyltin to inhibit oxidative phosphorylation and electron transport in tightly coupled rat liver mitochondria is very dependent on the pH and the ionic constitution of the assay medium.

1. 1. In an assay medium containing Cl⁻ at an alkaline pH, above 7.1, triethyltin inhibited both the ADP stimulated rate of oxygen uptake and the dinitrophenol-induced ATPase (EC 3.6.1.3) but had no effect on the dinitrophenol-stimulated rate of oxygen uptake. If the pH was reduced to below 6.9 the pattern of inhibition changed and both the ADP and dinitrophenol-stimulated rates of oxygen uptake were inhibited by triethyltin.

2. 2. In the absence of Cl⁻ in the medium triethyltin inhibited both the ADP-stimulated rate of oxygen uptake and dinitrophenol-induced ATPase and had no effect on the dinitrophenol-stimulated rate of oxygen uptake at either pH 7.4 or 6.6.

3. 3. In either the presence or absence of Cl⁻ the ability of triethyltin to inhibit ATP synthesis appears to markedly decrease as the pH is lowered from 7.4 to 6.6.

4. 4. The significance of these observations is discussed in relation to the operation of a Cl⁻/OH⁻ antiport in the coupling membrane.

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.     

Photosynthetic activity of plant cells solubilized in water-in-oil microemulsions

With the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water-in-oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2-ethyl-hexyl-sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N', N'-tetramethyl-p-phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.     

Effects of inhibitors on respiratory oscillations in synchronised Bacillus subtilis

Respiratory activity increased in a stepwise fashion during the cell cycle of Bacillus subtilis. This was true either for cells in growth medium or for those removed by filtration and supplied with an exogenous electron donor. KCN was used to probe terminal oxidase activity and cell cycle-dependent oscillations in the degree of inhibition were measured. The extent of stimulation by an uncoupling agent (CCCP) also varied during the cycle rising to two maxima at 0.14 and 0.69, intermediate to the step rises in oxygen uptake rates (0.4 and 0.8 of the cycle). However the net effect of the uncoupler was to convert the discontinuous pattern of oxygen uptake to a continuous one. These data are consistent with control of respiratory chain activity during the cell cycle of B. subtilis, and not of the amount of its components.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Submicromolar Ag+ increases passive Na+ permeability and inhibits the respiration-supported formation of Na+ gradient in Bacillus FTU vesicles

The effect of Ag+ on Na+ pumping by Na(+)-motive NADH-quinone reductase and terminal oxidase has been studied in Bacillus FTU inside-out vesicles. Very low concentrations of Ag+ (C1/2 = 1 x 10(-8) M or 2 x 10(-12) g ion.mg protein-1) are shown to inhibit the uphill Na+ uptake coupled to the oxidation of NADH by fumarate or of ascorbate + TMPD by oxygen but exert no effect on the H+ uptake by the H(+)-motive respiratory chain. Low Ag+ also induces a specific increase in the Na+ permeability of the vesicles. HQNO, added before and not after Ag+, prevents the Ag(+)-induced permeability increase, with effective HQNO concentrations being similar to those inhibiting the uphill Na(+)-uptake coupled to the NADH-fumarate oxidoreduction. Reduction of terminal oxidase by ascorbate + TMPD in the presence of cyanide sensitizes the Na+ permeability to Ag+. It is suggested that low [Ag+], known as a specific inhibitor of electron transport by the Na(+)-motive NADH-quinone reductase, uncouples the electron and Na+ transports so that the Ag(+)-modified NADH-quinone reductase operates as an Na+ channel rather than an Na+ pump. This effect is discussed in connection with the antibacterial action of Ag+.     

Anaerobic metabolism of cyclohexanol by denitrifying bacteria

Three strains of denitrifying bacteria were anaerobically enriched and isolated from oxic or anoxic habitats with cyclohexanol or cyclohexanone as sole electron donor and carbon source and with nitrate as electron acceptor. The bacteria were facultatively anaerobic, Gramnegative and metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as terminal electron acceptor. Cyclohexanol and cyclohexanone were degraded both anaerobically and aerobically. Aromatic compounds were oxidized in the presence of molecular oxygen only. One of the bacterial strains was further characterized. During anaerobic cyclohexanol degradation approximately 40% of the substrate was oxidized to phenol, which accumulated as dead-endproduct in the growth medium; 60% of cyclohexanol was completely oxidized to CO₂ and assimilated, respectively. In addition to phenol formation, transient accumulation of cyclohexanone, 2-cyclohexenone and 1,3-cyclohexanedione was observed. Based on these findings we propose a pathway for anaerobic cyclohexanol degradation involving these intermediates.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Bioenergetic aspects of aerobic glucose metabolism of Escherichia coli K-12 under varying specific growth rates and glucose concentrations.

An attempt was made to find a bioenergetical explanation for the differential effect of specific growth rate and glucose concentration on glucose metabolism of Escherichia coli K-12 with the help of 2,4-dinitrophenol (DNP). The effect of DNP on biomass occurred only at high glucose concentrations. The presence of this uncoupler strongly stimulated glucose uptake rates and oxygen uptake rates, but repressed severly Yg values. Increase in glucose concentration, however, sharply decreased QO2. The amount of oxygen required for maintenance was not affected by DNP, but Yomax values were much lower in the presence of DNP. The results are discussed and it is suggested that aerobic fermentation is caused by a severe reduction of site 1 of the respiratory chain region, whereas biomass formation is affected by repression of the terminal cytochrome a2. In comparing the effect of glucose on biomass formation at similar Qglucose levels aerobic and anaerobic fermentation, repression occurred in both cases at glucose concentrations of 0.3% and above. Although the analyses of 15 enzymes established the metabolic differences, the repression of growth was common to both fermentation types.     

Glucagon stimulation of mitochondrial respiration.

Acute glucagon treatment of intact rats has been found to cause a stimulation of hepatic mitochondrial respiration as measured by monitoring oxygen uptake polarographically. Rates of State 3 respiration with several NAD-linked substrates and succinate were increased significantly after hormonal treatment and isolation of mitochondria. This stimulation cannot be ascribed to a partial uncoupling effect since State 4 respiration as measured by monitoring oxygen uptake polarographically. Rates of State 3 respiration with either slightly increased or unchanged. Furthermore, rates of uncoupled respiration with these substrates were also stimulated after hormonal treatment. On the other hand, respiratory rates (State 3, 4, and uncoupled) with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine as substrate were unaffected by glucagon treatment. The hormonally stimulated rates of respiration produced a corresponding increase in the rate of generation of high energy state as indicated in measurements of Ca2+ uptake by isolated mitochondria. Rates of Ca2+ uptake were monitored by two methods: measurement of initial rates of proton ejection following CaCl2 additions and measurement of disappearance of Ca2+ from the suspension medium using murexide as indicator in a dual wavelength spectrophotometer. A significant stimulation in the initial rate of succinate-dependent Ca2+ uptake was noted after glucagon treatment of animals and isolation of hepatic mitochondria. No effect of the hormonal treatment was seen on the extent of Ca2+ uptake or the stoichiometry of H+ ejected per Ca2+ taken up. That the hormonal effect on Ca2+ transport is at the level of the substrate-induced generation of high energy state is indicated by the observation that no effect of glucagon treatment is seen on ATP-dependent Ca2+ uptake. Glucagon-induced changes in the activities of substrate-metabolizing enzymes are considered unlikely for the following reasons: (a) previously published data showed a lack of a hormonal effect on pyruvate-metabolizing enzymes and (b) data in this study showing no effect of glucagon treatment on the activity of NAD-malate dehydrogenase as measured in mitochondrial lysates. All of these observations are consistent with either an activation of mitochondrial substrate transport and/or a stimulation of mitochondrial electron transport by glucagon treatment. Regardless of the exact mechanism involved, the effect of the hormonal treatment is to produce an increase in ATP synthetic and ion-pumping capability during a period of increased energy demand, i.e. increased gluconeogenesis.     

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.