The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans

Dimethylsulphoxide (DMSO) and trimethylamine oxide (TMAO) sustained anaerobic growth of Proteus vulgaris with the non-fermentable substrate lactate. Cytoplasmic membrane vesicles energized by electron transfer from formate to DMSO displayed anaerobic uptake of serine, which was hindered by metabolic inhibitors known to destroy the proton motive force. This showed that DMSO reduction was coupled with a chemiosmotic mechanism of energy conversion; similar data for TMAO respiration have been presented previously. All biochemical tests applied indicated that the oxides were reduced by the same reductase system. The DMSO and TMAO reductase activities showed the same mobility on ion-exchange chromatography, and polyacrylamide disc gel electrophoresis (pH 8.9), gradient gel electrophoresis, and gel isoelectric focusing; mol. wt. and pI determined were 95,000 and 4.6, respectively. DMSO inhibited reduction of [¹⁴C]TMAO in vesicles. The reductase was inducible to a certain extent; both oxides being equally efficient as inducers. TMAO was reduced at a higher rate than DMSO, explaining faster growth of cells and increased uptake of serine in vesicles with TMAO as electron acceptor. Comparative studies with Escherichia coli also gave evidence for common TMAO and DMSO reductase systems.Abbreviations TMAO trimethylamine oxide - DMSO dimethylsulphoxide     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735.

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Trimethylamine oxide respiration in Proteus sp. strain NTHC153: electron transfer-dependent phosphorylation and L-serine transport

Cells of Proteus sp. strains NTHC153 grown anaerobically with glucose and trimethylamine oxide (TMAO) were converted to spheroplasts by the penicillin method. The spheroplasts were lysed by osmotic shock, and the membrane vesicles were purified by sucrose gradient centrifugation. Vesicles energized electron transfer from formate to TMAO displayed active anaerobic transport of serine. An anaerobic cell-free extract of Proteus sp. disrupted in a French pressure cell reduced TMAO with formate and NADH with the concomitant formation of organic phosphate. The net P/2e- ratios determined were 0.1 and 0.3, respectively. The NADH- and TMAO-dependent phosphorylation was sensitive to uncouplers of oxidative phosphorylation (protonophores), and the formate- and TMAO-dependent serine transport was sensitive to ionophores and protonophores. We conclude that TMAO reduction in Proteus sp. fulfills the essential features of anaerobic respiration.     

A conductance method for the assay and study of bacterial trimethylamine oxide reduction

Bacterial growth and trimethylamine oxide (TMAO) reduction were measured by following the change in conductance of the growth medium. The method was used as a reliable taxonomic test for the ability of bacteria to reduce TMAO. Conductance measurements were also applied to assaying the enzyme TMAO reductase in resting cells of the marine alteromonad NCMB 400: the enzyme was only active under anaerobic conditions with pyruvate, lactate and formate being good donors; the K_mTMAO was 93 ± 16 μmol/1; TMAO reductase activity was inhibited by several N -oxides including nitrite and nitrate, and was relatively resistant to cyanide. The relevance of conductance measurements and the significance of TMAO reduction are discussed.     

Trimethylamine oxide transport across plasma membranes of elasmobranch erythrocytes.

The aim of this study was to characterize the erythrocyte cell membrane transport of trimethylamine oxide (TMAO) in the little skate, Raja erincea. Uptake of TMAO occurs by two processes, Na(+)-dependent and Na(+)-independent. 2,4 dinitrophenol (2,4 DNP), a known ATP synthesis inhibitor, inhibited TMAO uptake, suggesting the involvement of the Na(+)/K(+)-ATP pump in Na(+)-dependent TMAO transport. Na(+)-independent TMAO uptake was stimulated by cell swelling when erythrocytes were incubated in hypotonic elasmobranch incubation medium. Swelling-activated, Na(+)-independent TMAO uptake was inhibited by the anion transport inhibitors quinine and 4, 4'-diisthiocyanostilbene-2,2'-disulfonic acid (DIDS), two blockers of the swelling-activated osmolyte channel in skate erythrocytes. TMAO efflux was stimulated by hypotonic stress in the erythrocytes of the spiny dogfish, Squalus acanthias. DIDS also inhibited this efflux, indicating that TMAO is transported by the organic osmolyte channel in the erythrocytes of this elasmobranch as well. J. Exp. Zool. 284:605-609, 1999.     

Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c -type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N -oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.     

Effects of electron donor and acceptor conditions on reductive dehalogenation of tetrachloromethane by Shewanella putrefaciens 200.

Shewanella putrefaciens 200 is a nonfermentative bacterium that is capable of dehalogenating tetrachloromethane to chloroform and other, unidentified products under anaerobic conditions. Since S. putrefaciens 200 can respire anaerobically by using a variety of terminal electron acceptors, including NO3-, NO2-, and Fe(III), it provides a unique opportunity to study the competitive effects of different electron acceptors on dehalogenation in a single organism. The results of batch studies showed that dehalogenation of CT by S. putrefaciens 200 was inhibited by O2, 10 mM NO3-, and 3 mM NO2-, but not by 15 mM Fe(III), 15 mM fumarate, or 15 mM trimethylamine oxide. Using measured O2, Fe(III), NO2-, and NO3- reduction rates, we developed a speculative model of electron transport to explain inhibition patterns on the basis of (i) the kinetics of electron transfer at branch points in the electron transport chain, and (ii) possible direct inhibition by nitrogen oxides. In additional experiments in which we used 20 mM lactate, 20 mM glucose, 20 mM glycerol, 20 mM pyruvate, or 20 mM formate as the electron donor, dehalogenation rates were independent of the electron donor used. The results of other experiments suggested that sufficient quantities of endogenous substrates were present to support transformation of tetrachloromethane even in the absence of an exogenous electron donor. Our results should be significant for evaluating (i) the bioremediation potential at sites contaminated with both halogenated organic compounds and nitrogen oxides, and (ii) the bioremediation potential of iron-reducing bacteria at contaminated locations containing significant amounts of iron-bearing minerals.     

Influence of respiratory substrate on the cytochrome content of Shewanella putrefaciens

Shewanella putrefaciens can use trimethylamine oxide (TMAO) as electron acceptor under anoxic conditions. The associated cytochromes induced during growth under various respiratory conditions have been separated by liquid chromatography (DEAE Sepharose CL6b) and SDS-PAGE and characterized spectrophotometrically and by redox potentiometry. Two major low potential cytochromes and at least three minor low potential cytochromes, likely to be involved in TMAO reduction, were found. No cytochrome specific for TMAO reductase was found.     

Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of +130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto-TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto-substituted TMAO reductase (W-TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo-TMAO reductase (Mo-TorA). W-TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo-TorA. Most importantly, the W-TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.     

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.     

Redox mechanisms in “oxidant-dependent” hexose fermentation by Rhodopseudomonas capsulata

Trimethylamine N-oxide (TMAO) can function as an electron acceptor in the anaerobic metabolism of both Rhodopseudomonas capsulata and Escherichia coli. In both bacteria, anaerobic growth in the presence of TMAO induces a system that can reduce TMAO to trimethylamine (TMA). Comparative studies, however, show that TMAO reduction serves different purposes in the organisms noted. In E. coli, anaerobic growth on sugars does not require the presence of TMAO, but in cells induced for TMAO reductase, TMAO can act as the terminal electron acceptor for membrane-associated oxidative phosphorylation. Anaerobic dark growth of R. capsulata is dependent on the presence of TMAO (or an analog) and in this organism a soluble system catalyzes anaerobic oxidation of NADH with TMAO. The mechanism, in R. capsulata, appears to involve a flavoprotein of the flavodoxin type and presumably represents a system for maintenance of redox balance during anaerobic dark fermentation of hexoses and related compounds.     

Anaerobic growth of Escherichia coli on formate by reduction of nitrate, fumarate, and trimethylamine N-oxide.

Anaerobic growth of E. coli, strain K-10, depending on formate oxidation by nitrate, fumarate, and trimethylamine N-oxide was followed in a medium containing peptone. The presence of formate and peptone was indispensable for growth with fumarate and trimethylamine N-oxide reduction. While there was no growth in the absence of acceptor, growth was observed in the absence of formate by nitrate reduction though not as much as under aerobic conditions. Per mole consumed formate equimolar succinate or trimethylamine was formed, but 1.2 mole of nitrate was produced, probably depending partly on peptone oxidation. The molar growth yield on formate was found to be 6.5, 7.6, and 7.0 g cells/mole depending on the reduction of nitrate, fumarate, and trimethylamine N-oxide, respectively, suggesting the formation of one mole ATP coupled to the anaerobic electron transfers from formate.     

Anaerobic Fish Spoilage by Bacteria II. Kinetics of Bacterial Growth and Substrate Conversions in Herring Extracts

The time course of the conversions of chemical components in herring extracts during anaerobic growth of Proteus sp., str. NTHC 153, Aeromonas sp., str. NTHC 154, and Enterobacter sp., str. NTHC 151 (Strøm & Larsen 1979) has been studied. When the Proteus sp. or the Aeromonas sp. were inoculated into the herring extracts and incubated at 15°C under anaerobic conditions, the sugar components (i.e. mainly ribose, free and bound) were the first substrates utilized. These compounds were converted to acetate and CO₂ by the use of trimethylamine oxide (TMAO) as an external hydrogen acceptor. Growth of bacteria ceased when all TMAO was reduced to trimethylamine (TMA). By adding an extra amount of TMAO to the herring extracts an increased growth of the Proteus sp. and the Aeromonas sp. ensued. The increased growth occurred concomitantly with a further conversion of TMAO to TMA and of lactate to acetate and CO₂. The Enterobacter sp., which did not utilize lactate, did not give an increased growth in herring extracts enriched with TMAO.     

Trimethylamine N-oxide respiration by aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Erythrobacter sp. OCh 114, an aerobic photosynthetic bacterium, had trimethylamine N-oxide (TMAO) reductase activity, which increased when the culture medium contained TMAO. The reductase was located in the periplasm. The bacteria grew anaerobically in the presence of TMAO. These results suggested that Erythrobacter OCh 114 has the ability to reduce TMAO through the respiratory chain. The TMAO respiration system of this organism was different from those of facultative purple photosynthetic bacteria in two respects: (a) TMAO reductase did not have activity to reduce dimethyl sulfoxide and (b) soluble c-type cytochrome, cytochrome c551, and cytochrome b-c1 complex appeared to be involved. The photochemical activity, which is usually inoperative in the anaerobic cell suspension, was restored by TMAO, suggesting that the photosynthesis and the TMAO respiration share a common electron transfer chain.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.