fumarates and trimethyloxamine

The Escherichia coli K-12 ydhY-T operon, coding for a predicted oxidoreductase complex, is activated under anaerobic conditions and repressed in the presence of nitrate or nitrite. Anaerobic activation is mediated by the transcription factor FNR, and nitrate/nitrite repression is mediated by NarXL and NarQP. In vitro transcription reactions revealed that the DNA upstream of ydhY-T contains sufficient information for RNA polymerase alone to initiate transcription from five locations. FNR severely inhibited synthesis of two of these transcripts (located upstream of, and within, the FNR binding site) and activated the FNR-dependent promoter previously identified in vivo. Enhanced expression of ydhY-T in an hns mutant was consistent with the location of ydhY-T within a promoter island and the FNR-independent transcription observed in vitro. FNR-dependent transcription in vitro was decreased in the presence of NarL~P. DNaseI footprinting indicated that FNR and NarL~P simultaneously bound at the ydhY-T promoter region and that NarL~P-mediated repression was due to occupation of the 7-2-7 site located downstream of the FNR-dependent promoter. Expression of ydhY-T during the anaerobic growth cycle was repressed when nitrate was present but less so in the presence of nitrite. In vivo transcription measurements indicated that the alternative electron acceptors, DMSO and fumarate, could also lower ydhY-T expression, whereas trimethylamine-N-oxide (TMAO) permitted high expression. Therefore, expression of ydhY-T is subject to complex regulation in response to electron acceptor availability that involves at least three transcription factors, FNR (anaerobic activation), NarL~P (nitrate repression) and H-NS (repression in the absence of an antagonist; e.g. FNR).

Topics: Anaerobiosis; Bacterial Proteins; Dimethyl Sulfoxide; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Escherichia coli K12; Escherichia coli Proteins; Ferredoxins; Fumarates; Gene Expression Regulation, Bacterial; Iron-Sulfur Proteins; Methylamines; Oxidants; Oxidoreductases; Promoter Regions, Genetic

The human gastrointestinal pathogen Campylobacter jejuni is a microaerophilic bacterium with a respiratory metabolism. The genome sequence of C. jejuni strain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, and N- or S-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded by Cj0264c was shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth of C. jejuni under strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. The C. jejuni genome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth of C. jejuni. The results indicate that strict anaerobiosis is a stress condition for C. jejuni but that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.

Topics: Anaerobiosis; Campylobacter jejuni; Dimethyl Sulfoxide; Fumarates; Methylamines; Nitrates; Nitrites; Oxygen Consumption

The Escherichia coli SixA protein is the first discovered prokaryotic phospho-histidine phosphatase, which was implicated in a His-to-Asp phosphorelay. The sixA gene was originally identified as the one that interferes with, at its multi-copy state, the cross-phosphorelay between the histidine-containing phosphotransmitter (HPt) domain of the ArcB anaerobic sensor and its non-cognate OmpR response regulator. Nevertheless, no evidence has been provided that the SixA phosphatase is indeed involved in a signaling circuitry of the authentic ArcB-to-ArcA phosphorelay in a physiologically meaningful manner. In this study, a SixA-deficient mutant was characterized with special reference to the ArcB signaling, which allows E. coli cells to respond to not only external oxygen, but also certain anaerobic respiratory conditions. Here evidence is provided for the first time that the SixA phosphatase is a crucial regulatory factor that is involved in the ArcB signaling, particularly, under certain anaerobic respiratory growth conditions. We propose a novel mechanism, involving an HPt domain and a phospho-histidine phosphatase, by which a given multi-step His-to-Asp signaling can be modulated.

Topics: Anaerobiosis; Aspartic Acid; Bacterial Outer Membrane Proteins; Bacterial Proteins; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Escherichia coli Proteins; Fumarates; Genes, Bacterial; Genes, Reporter; Histidine; Kinetics; Membrane Proteins; Methylamines; Mutation; Nitrates; Oxygen; Phosphorylation; Protein Kinases; Protein Structure, Tertiary; Recombinant Fusion Proteins; Repressor Proteins; Signal Transduction

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.

Topics: Anaerobiosis; Animals; Cell Respiration; Decapodiformes; Dimethyl Sulfoxide; Ecosystem; Fishes; Fumarates; Methylamines; Nitrate Reductases; Nitrates; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxidoreductases, N-Demethylating; Photobacterium; Substrate Specificity; Symbiosis; Vibrio

Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. It participates as a member of the tricarboxylic acid cycle and the branched noncyclic pathways under aerobic and anaerobic cell growth conditions, respectively. To investigate how the mdh gene is expressed under these different conditions, an mdh-lacZ operon fusion was constructed and analyzed in vivo. The mdh-lacZ fusion was expressed about twofold higher under aerobic conditions than under anaerobic cell growth conditions on most media tested. This anaerobic response is modulated by the ArcA protein, which functions as a repressor of mdh gene expression under both aerobic and anaerobic conditions. In contrast, mutations in the fnr gene did not affect mdh gene expression. Interestingly, cells grown anaerobically with glycerol and trimethylamine N-oxide or fumarate showed higher levels of mdh expression than did cells that were grown aerobically. Depending on the type of carbon compound used for cell growth, mdh expression varied by 11-fold and 5-fold under aerobic and anaerobic conditions, respectively. While mdh transcription was shown to be inversely proportional to the cell growth rate, cellular heme limitation stimulated a fivefold increase in mdh gene expression. The mdh gene appears to be highly regulated to adapt to changing conditions of aerobic and anaerobic cell growth with various types of carbon substrates.

Topics: 2,2'-Dipyridyl; Aerobiosis; Anaerobiosis; Bacterial Outer Membrane Proteins; Bacterial Proteins; Carbon; Culture Media; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Glycerol; Heme; Iron Chelating Agents; Iron-Sulfur Proteins; Malate Dehydrogenase; Methylamines; Oxidants; Oxygen; Recombinant Fusion Proteins; Repressor Proteins

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.

Topics: Carbon Tetrachloride; Electron Transport; Fumarates; Gram-Negative Facultatively Anaerobic Rods; Halogens; Methylamines; Models, Biological; Oxidation-Reduction; Oxygen

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

Topics: Bacteria, Anaerobic; Carbon; Chemotaxis; Dimethyl Sulfoxide; Electron Transport; Environmental Microbiology; Fumarates; Methylamines; Nitrates; Nitrites; Oxidants; Oxidation-Reduction; Thiosulfates

The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al. 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents. In contrast to the wild-type, strain AN70 contained only DMK, but no MK. The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor. The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates. DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (less than or equal to 8% of wild-type), although the respective enzymes were present in wild-type rates. The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al. 1990). It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration. In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis. Synthesis of MK from DMK appears to depend on the same gene (ubiE).

Topics: Anaerobiosis; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Fumarates; Methylamines; Mutation; Nitrates; Oxidants; Oxidation-Reduction; Vitamin K; Vitamin K 2

The respiratory activities of E. coli with H2 as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to less than or equal to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K1 [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K1 or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was less than or equal to 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K1 or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.

Topics: Anaerobiosis; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Fumarates; Methylamines; Nitrates; Oxidation-Reduction; Oxidoreductases; Vitamin K; Vitamin K 2

The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molybdoenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from an E. coli gene bank. Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO and trimethylamine N-oxide (TMAO) reductase activities 13- to 15-fold compared with wild-type cells. Amplification was highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reduced levels of enzyme amplification. Growth on nitrate or aerobic growth repressed expression of the enzyme. A 6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vector pBR322, yielding a recombinant DMSO reductase plasmid, pDMS159. Two polypeptides were amplified and identified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E. coli HB101 harboring pDMS159: a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight 23,600. Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 were detected by in vivo transcription/translation studies. The smallest subunit was poorly defined and not detectable by Coomassie blue staining. The DMSO reductase operon was localized to the 20.0-min position on the E. coli linkage map.

Topics: Anaerobiosis; Bacterial Proteins; Chromosome Mapping; Cloning, Molecular; Dimethyl Sulfoxide; DNA Restriction Enzymes; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fumarates; Gene Amplification; Gene Expression Regulation; Genes, Bacterial; Iron-Sulfur Proteins; Methylamines; Mutation; NADH, NADPH Oxidoreductases; Nitrates; Operon; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-NH Group Donors; Plasmids; Substrate Specificity

The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E. coli chromosome at the lambda attachment site. Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic cell growth, increased an additional 1.5-fold by the presence of fumarate, the substrate, and decreased 23-fold by nitrate, a preferred electron acceptor. The addition of trimethylamine-N-oxide as an electron acceptor did not significantly alter frdA'-'lacZ expression. Control of frd operon expression is therefore exerted at the transcriptional level in response to the availability of the electron acceptors oxygen, fumarate, and nitrate. Anaerobic induction of frdA'-'lacZ expression was impaired in an fnr mutant and was restored when the fnr+ gene was provided in trans, thus establishing that the fnr gene product, Fnr, is responsible for the anaerobic activation of frd operon expression. Nitrate repression of frdA'-'lacZ expression was observed under either aerobic or anaerobic cell growth conditions in both wild-type and fnr mutant strains, demonstrating that the mechanism for nitrate repression is independent of nitrate respiration and oxygen control imparted by Fnr. Studies performed with a fnr'-'lacZ protein fusion confirmed that the fnr gene is expressed both aerobically and anaerobically. A model is proposed for the regulation of frdABCD operon expression in response to the availability of the alternate terminal electron acceptors oxygen, nitrate, and fumarate.

Topics: Aerobiosis; Anaerobiosis; Chromosomes, Bacterial; Electron Transport; Escherichia coli; Fumarates; Gene Expression Regulation; Genes, Bacterial; Methylamines; Nitrates; Operon; Oxygen; Recombinant Fusion Proteins; Repressor Proteins; Succinate Dehydrogenase

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.

Topics: Aerobiosis; Anaerobiosis; Escherichia coli; Formates; Fumarates; Methylamines; Nitrates; Oxidative Phosphorylation