Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone.
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Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1. (+info)
Metabolite transport in isolated yeast mitochondria: fumarate/malate and succinate/malate antiports.
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In this study, we investigated the metabolite permeability of isolated coupled Saccharomyces cerevisiae mitochondria. The occurrence of a fumarate/malate antiporter activity was shown. The activity differs from that of the dicarboxylate carrier (which catalyses the succinate/malate antiport) in (a) kinetics (Km and Vmax values are about 27 microM and 22 nmol min(-1) mg protein(-1) and 70 microM and 4 nmol min(-1) mg protein(-1), respectively), (b) sensitivity to inhibitors, (c) Ki for the competitive inhibitor phenylsuccinate and (d) pH profiles. (+info)
Voltammetry of a flavocytochrome c(3): the lowest potential heme modulates fumarate reduction rates.
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Iron-induced flavocytochrome c(3), Ifc(3), from Shewanella frigidimarina NCIMB400, derivatized with a 2-pyridyl disulfide label, self-assembles on gold electrodes as a functional array whose fumarate reductase activity as viewed by direct electrochemistry is indistinguishable from that of Ifc(3) adsorbed on gold or graphite electrodes. The enhanced stability of the labeled protein's array permits analysis at a rotating electrode and limiting catalytic currents fit well to a Michaelis-Menten description of enzyme kinetics with K(M) = 56 +/- 20 microM, pH 7.5, comparable to that obtained in solution assays. At fumarate concentrations above 145 microM cyclic voltammetry shows the catalytic response to contain two features. The position and width of the lower potential component centered on -290 mV and corresponding to a one-electron wave implicates the oxidation state of the lowest potential heme of Ifc(3) as a defining feature in the mechanism of fumarate reduction at high turnover rates. We propose the operation of dual pathways for electron transfer to the active site of Ifc(3) with the lowest potential heme acting as an electron relay on one of these pathways. (+info)
Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation.
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The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral uptake and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, gamma-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia. (+info)
The SixA phospho-histidine phosphatase modulates the ArcB phosphorelay signal transduction in Escherichia coli.
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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. (+info)
Denitrovibrio acetiphilus, a novel genus and species of dissimilatory nitrate-reducing bacterium isolated from an oil reservoir model column.
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A novel dissimilatory, nitrate-reducing bacterium, designated strain N2460T, was isolated from an oil reservoir model column. Strain N2460T is a mesophilic, obligately anaerobic, marine, gram-negative bacterium. The cells are vibrio-shaped and motile by a bipolar flagellum. Strain N2460T reduces nitrate to ammonia in a mineral medium supplied by acetate. The presence of a 2-oxoglutarate dehydrogenase activity indicates that acetate is oxidized via the citric acid cycle. No growth is obtained on formate, higher fatty acids, malate, fumarate, benzoate, alcohols, sugar, yeast extract, crude oil, alkanes, proline, hydrogen, sulfur or thiosulfate with nitrate as electron acceptor. Oxygen, sulfate, thiosulfate and sulfur are not utilized as alternative electron acceptors. Strain N2460T grows fermentatively on fumarate, but not on pyruvate. The G+C content of the DNA is 42.6 mol%. 16S rRNA gene analysis shows that strain N2460T belongs to the Bacteria and that the closest relative is 'Geovibrio ferrireducens' (sequence similarity 86.9%). On the basis of phylogenetic as well as phenotypic data, it is proposed that strain N2460T represents the type strain of a new genus and species, Denitrovibrio acetiphilus gen. nov., sp. nov. (+info)
Anaerobic transport in Escherichia coli membrane vesicles.
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Anaerobic lactose and/or amino acid transport by membrane vesicles prepared from Escherichia coli ML 308-225 can be coupled to at least four electron transfer systems: alpha-glycerol-P-dehydrogenase:nitrate reductase, formate dehydrogenase:nitrate reductase, alpha-glycerol-P dehydrogenase:fumarate reductase, and formate dehydrogenase:fumarate reductase. Vesicles contain one or more of these electron transfer systems depending on the growth conditions of the parent cells. alpha-Glycerol-P dehydrogenase and fumarate reductase are present only in vesicles prepared from cells grown in the presence of glycerol or fumarate, respectively. Formate dehydrogenase and nitrate reductase activities, on the other hand, are present in vesicles from cells grown on a variety of media. alpha-Glycerol-P and formate are able to drive aerobic transport in vesicles prepared from anaerobically grown cells, indicating coupling between aerobic and anaerobic electron transfer systems. (+info)
Aspartate transcarbamylase of Escherichia coli. Mechanisms of inhibition and activation by dicarboxylic acids and other anions.
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The interactions of several dicarboxylic acids and monoanions with Escherichia coli aspartate transcarbamylase and with its catalytic subunit have been studied by ultraviolet difference spectroscopy and steady state kinetics, with the following major findings. 1. A variety of dicarboxylic acids compete with carbamyl-P for the active sites of unliganded catalytic subunit, with steric requirements very different from those important for competition with L-aspartate for the subunit/carbamyl-P complex. Competition with carbamyl-P is much reduced if the dicarboxylic acid has a positively charged amino group. Acetate and chloride also compete. 2. At pH 7, equal concentrations of lysine acetate and L-aspartate are equally effective in displacing the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) from the active sites of the concentrations of L-aspartate and lysine acetate is constant, increasing the concentration of L-aspartate does not relieve inhibition of the enzyme by PALA (Collins, K.C., and Stark, G. R. (1971) J. Biol. Chem. 246, 6599-6605). Therefore, the L-aspartate/subunit complex, like the acetate/subunit complex, must be incapable of participating in the catalytic reaction. We conclude that the kinetic mechanism is ordered, in agreement with the recent findings of Wedler and Gasser (Wedler, F.C., and Gasser, F.J. (1974), Arch. Biochem. Biophys. 163, 57-68) and in disagreement with the interpretation of Heyde et al. (Heyde, E., Nagabhushanam, A., And Morrison, J.F. (1973) Biochemistry 12, 4718-4726)... (+info)