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.     

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.     

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.     

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.     

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.     

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.     

Dimethyl sulfoxide reductase of Escherichia coli: an investigation of function and assembly by use of in vivo complementation.

Dimethyl sulfoxide (DMSO) reductase of Escherichia coli is a membrane-bound, terminal anaerobic electron transfer enzyme composed of three nonidentical subunits. The DmsAB subunits are hydrophilic and are localized on the cytoplasmic side of the plasma membrane. DmsC is the membrane-intrinsic polypeptide, proposed to anchor the extrinsic subunits. We have constructed a number of strains lacking portions of the chromosomal dmsABC operon. These mutant strains failed to grow anaerobically on glycerol minimal medium with DMSO as the sole terminal oxidant but exhibited normal growth with nitrate, fumarate, and trimethylamine N-oxide, indicating that DMSO reductase is solely responsible for growth on DMSO. In vivo complementation of the mutant with plasmids carrying various dms genes, singly or in combination, revealed that the expression of all three subunits is essential to restore anaerobic growth. Expression of the DmsAB subunits without DmsC results in accumulation of the catalytically active dimer in the cytoplasm. The dimer is thermolabile and catalyzes the reduction of various substrates in the presence of artificial electron donors. Dimethylnaphthoquinol (an analog of the physiological electron donor menaquinone) was oxidized only by the holoenzyme. These results suggest that the membrane-intrinsic subunit is necessary for anchoring, stability, and electron transport. The C-terminal region of DmsB appears to interact with the anchor peptide and facilitates the membrane assembly of the catalytic dimer.     

Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.     

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.     

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

Localization of cytochromes to the outer membrane of anaerobically grown Shewanella putrefaciens MR-1.

In gram-negative bacteria, numerous cell functions, including respiration-linked electron transport, have been ascribed to the cytoplasmic membrane. Gram-negative bacteria which use solid substrates (e.g., oxidized manganese or iron) as terminal electron acceptors for anaerobic respiration are presented with a unique problem: they must somehow establish an electron transport link across the outer membrane between large particulate metal oxides and the electron transport chain in the cytoplasmic membrane. When the metal-reducing bacterium Shewanella putrefaciens MR-1 is grown under anaerobic conditions and membrane fractions are purified from cells lysed by an EDTA-lysozyme-polyoxyethylene cetyl ether (Brij 58) protocol, approximately 80% of its membrane-bound cytochromes are localized in its outer membrane. These outer membrane cytochromes could not be dislodged by treatment with chaotropic agents or by increased concentrations of the nonionic detergent Brij 58, suggesting that they are integral membrane proteins. Cytochrome distribution in cells lysed by a French press protocol confirm the localization of cytochromes to the outer membrane of anaerobically grown cells. This novel cytochrome distribution could play a key role in the anaerobic respiratory capabilities of this bacterium, especially in its ability to mediate manganese and iron reduction.     

Oxygen and nitrate-dependent regulation of <Emphasis Type="Italic">dmsABC</Emphasis> operon expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>: sites for Fnr and NarL protein interactions

Background  

Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.     

Nucleic acid spot hybridization for detection of Chlamydia trachomatis

Abstract The TMAO reductase activity of Escherichia coli grown anaerobically in the presence or absence of TMAO was analysed on linear sucrose gradients and on non-denaturing polyacrylamide gels. The results, together with those obtained by analysis of some other properties of TMAO reductase, showed that there are significant differences between the enzyme synthesized in the absence of TMAO ("constitutive" enzyme) and that synthesized in its presence ("inducible" enzyme).
A similar study of a tor mutant specifically altered in the structural gene for TMAO reductase, showed that the enzymes synthesized under the 2 growth conditions are probably 2 distinct enzymes encoded by different genes.     

Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC 33456.

Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III). The reduced chromium was predominantly present in the external medium. Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E. coli ATCC 33456 was a largely soluble reductase activity. In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E. coli ATCC 33456. Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone. The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI). Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force.     

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     

A soluble fumarate reductase in Trypanosoma brucei procyclic trypomastigotes

The enzyme NADH-fumarate reductase associated with the membrane fraction of Trypanosoma brucei procyclic trypomastigotes, can be solubilized by more than 50% when increasing the ionic strength to the equivalent of 150 mM KCl. The apparent KMs for NADH (125 microM) and fumarate (50 microM) remain close to those previously reported for the membrane-bound form of this enzyme. Other electron acceptors (i.e. oxygen or cytochrome c) appear to accept electrons in the absence of fumarate (KM for cytochrome c = 50 microM). The drug L-092,201 (Merck, Sharp and Dohme Research Laboratories, Rahway, NJ), an inhibitor of the membrane-bound fumarate reductase, also blocked the solubilized enzyme. Given the relatively high ionic strength of the intracellular environment we propose that, in vivo, the enzyme fumarate reductase is in the mitochondrial matrix or in the soluble fraction of another intracellular compartment.     

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.