Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Location of anaerobic respiratory enzyme trimethylamine N-oxide reductase in the cytoplasmic membrane ofEscherichia coli strain K10

Trimethylamine N-oxide (TMAO) reductase, which is anaerobically induced by TMAO, is a terminal enzyme in anaerobic electron transport inEscherichia coli. When the organism was anaerobically grown with TMAO, a marked increase in the specific activity of TMAO reductase was observed mainly in a cell membrane fraction and stopped after exhausting TMAO. On the other hand, activity was moderately increased in a soluble fraction of the cell even after exhaustion of TMAO. Immunoblot analysis with an antiserum against the TMAO reductase purified from the soluble fractions showed that the cells growing with TMAO contained only a membrane-bound enzyme, which has a molecular mass of 94 kDa, while a soluble enzyme with 92 kDa appeared in the stationary growth phase lacking TMAO. Experiments with right-side-out and inside-out vesicles of cytoplasmic membrane indicated that the membrane-bound enzyme faces the cytoplasm. The soluble enzyme was mainly found in the cytoplasm of the cell, but also at a negligible amount in the periplasm. The membrane-bound form of TMAO reductase functioning in anaerobic electron transport seems to be cleaved and released into the cytoplasm as soluble enzyme after exhaustion of TMAO.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

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.     

Effects of ISSo2 insertions in structural and regulatory genes of the trimethylamine oxide reductase of Shewanella oneidensis

We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration. The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS. Although TorA is not the sole enzyme reducing TMAO in S. oneidensis, growth analysis showed that it is the main respiratory TMAO reductase. Use of a plasmid-borne torE'-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.     

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.     

TorT, a member of a new periplasmic binding protein family, triggers induction of the Tor respiratory system upon trimethylamine N-oxide electron-acceptor binding in Escherichia coli

In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 mum. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.     

Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.     

Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri.

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.     

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.     

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.     

Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k _cat /K _m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t _1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T _m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.     

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea <Emphasis Type="Italic">Photobacterium phosphoreum</Emphasis> ANT-2200

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.     

Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, M_r=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.Abbreviations TMAO trimethylamine-N-oxide - DMSO dimethylsulphoxide - PMS phenazine methosulphate - cytoplasmic membrane potential     

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.     

Time-dependent effects of trimethylamine-N-oxide/urea on lactate dehydrogenase activity: an unexplored dimension of the adaptation paradigm.

Given that enzymes in urea-rich cells are believed to be just as sensitive to urea effects as enzymes in non-urea-rich cells, it is argued that time-dependent inactivation of enzymes by urea could become a factor of overriding importance in the biology of urea-rich cells. Time-independent parameters (e.g. Tm, k(cat), and Km) involving protein stability and enzyme function have generally been the focus of inquiries into the efficacy of naturally occurring osmolytes like trimethylamine-N-oxide (TMAO), to offset the deleterious effects of urea on the intracellular proteins in the urea-rich cells of elasmobranchs. However, using urea concentrations found in urea-rich cells of elasmobranches, we have found time-dependent effects on lactate dehydrogenase activity which indicate that TMAO plays the important biological role of slowing urea-induced dissociation of multimeric intracellular proteins. TMAO greatly diminishes the rate of lactate dehydrogenase dissociation and affords significant protection of the enzyme against urea-induced time-dependent inactivation. The effects of TMAO on enzyme inactivation by urea adds a temporal dimension that is an important part of the biology of the adaptation paradigm.     

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membranebound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membranebound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.Abbreviations PMS phenazine methosulfate - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - CHAPSO [3-(3-cholamidopropyldimethylammonia)-1-(2-hydroxy-1-propanesulfonate)] National Research Council Research Fellow     

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