Fumarate Respiration Research Articles

Requirement for the Proton‐Pumping NADH Dehydrogenase I of Escherichia Coli in Respiration of NADH to Fumarate and Its Bioenergetic Implications

In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration. The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around -309, -277, and downstream of -231 bp, respectively, relative to the transcriptional-start site. The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL. For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II. The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I. The mutant used acetyl-CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol. Therefore, NADH dehydrogenase I is essential for NADH-->fumarate respiration, and is able to use menaquinone as an electron acceptor. NADH-->dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I. The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed.

Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange.

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.

Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud).

A delta sud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The delta psr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity.

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and13C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 μM selenate and 100 μM selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.

Anaerobic respiration of Bacillus macerans with fumarate, TMAO, nitrate and nitrite and regulation of the pathways by oxygen and nitrate

In Bacillus macerans, anaerobic respiratory pathways and the regulation of facultatively anaerobic catabolism by electron acceptors were analysed. In addition to fermentative growth, B. macerans was able to grow anaerobically by fumarate, trimethylamine N-oxide, nitrate, and nitrite respiration with glycerol as donor. During growth by fumarate respiration, a membrane-bound fumarate reductase was present that was different from succinate dehydrogenase. The end product of nitrate and nitrite respiration was ammonia. No N2 or NO and only traces of N2O could be detected. O2 repressed the activity of nitrate and fumarate reductases and the fermentation of glucose, presumably at the transcriptional level. Nitrate repressed fumarate reductase activity and partially glucose fermentation. Thus energy metabolism and the regulatory hierarchy with respect to the use of electron acceptors were very similar to that known from E. coli; B. macerans can be regarded as a truly facultative anaerobic bacterium. In addition, the anaerobic growth capabilities of some other Bacillus strains are described.

Bacterial fumarate respiration

Bacterial fumarate respiration

Anaerobic fumarate transport in Escherichia coli by an fnr-dependent dicarboxylate uptake system which is different from the aerobic dicarboxylate uptake system.

Escherichia coli grown anaerobically with fumarate as electron acceptor is able to take up C4-dicarboxylates by a specific transport system. The system differs in all tested parameters from the known aerobic C4-dicarboxylate transporter. The anaerobic transport system shows higher transport rates (95 mumol/g [dry weight] per min versus 30 mumol/g/min) and higher Kms (400 versus 30 microM) for fumarate than for the aerobic system. Mutants lacking the aerobic dicarboxylate uptake system are able to grow anaerobically at the expense of fumarate respiration and transport dicarboxylates with wild-type rates after anaerobic but not after aerobic growth. Transport by the anaerobic system is stimulated by preloading the bacteria with dicarboxylates. The anaerobic transport system catalyzes homologous and heterologous antiport of dicarboxylates, whereas the aerobic system operates only in the unidirectional mode. The anaerobic antiport is measurable only in anaerobically grown bacteria with fnr+ backgrounds. Additionally, the system is inhibited by incubation of resting bacteria with physiological electron acceptors such as O2, nitrate, dimethyl sulfoxide, and fumarate. The inhibition is reversed by the presence of reducing agents. It is suggested that the physiological role of the system is a fumarate/succinate antiport under conditions of fumarate respiration.

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418

No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.

Anaerobic respiration of fumarate as a differential test betweenCampylobacter fetus andCampylobacter jejuni

The effect of formate and of various electron acceptors—fumarate, aspartate, or nitrate—on the growth of 36 catalase-producingCampylobacter strains was quantitatively investigated in a semisynthetic medium, under aerobic (5% oxygen, 10% carbon dioxide, 85% nitrogen) or anaerobic (10% carbon dioxide, 90% nitrogen) conditions. Under anaerobic conditions, 24 strains ofC. jejuni failed to grow, or grew poorly, in the presence of fumarate, whereas ten strains ofC. fetus subsp.fetus and two strains ofC. fetus subsp.venerealis grew abundantly, rather better than under aerobic conditions. The quantitative differences of growth yields were very significant between the two species with fumarate, but less pronounced with aspartate or nitrate. The activity of fumarate-reductase inC. fetus was substantiated by identification of relevant metabolites by gas liquid chromatography and by mass spectrometry. The anaerobic fumarate respiration in the genusCampylobacter has taxonomic implications.

The dependence on quinone specificity of terminal electron transport of bacteria

Bacterial quinones were extracted with pentane, and homologues or other quinones were reincorporated. In spite of the redox potential difference of 110 mV, menaquinone and demethylmenaquinone could replace each other in aerobic electron transport and fumarate respiration ofHaemophilus influenzae RAMC 18 Bensted andProteus mirabilis Harding & Nicholson. The enzymes involved may recognize the naphthoquinone structure and are not specific for menaquinone or demethylmenaquinone. Ubiquinone was not replaced in aerobic electron transport by naphthoquinones withPseudomonas fluorescens 28/5 Rhodes orAcinetobacter sp. 661/60 Mannheim, probably owing to the specificity for benzoquinones of the enzymes involved, since the redox potential difference between demethylmenaquinone and ubiquinone is only 76 mV.Haemophilus parainfluenzae 429 Pittman, which resembles aerobic bacteria with respect to the terminal electron transport system, could incorporate demethylmenaquinone or menaquinone. This organism seems to be defective in the synthesis of naphthoquinones but possesses the enzyme system for fumarate respiration.Haemophilus influenzae RAMC 18 Bensted, which produces only demethylmenaquinone, seems to be defective in synthesizing ubiquinone, but it also possesses the enzymes for a ubiquinonemediated aerobic respiration.

Electron transport phosphorylation coupled to fumarate reduction by H2- and Mg2+-dependent adenosine triphosphatase activity in extracts of the rumen anaerobe Vibrio succinogenes.

Vibrio succinogenes, an anaerobic bacterium, obtains its energy for growth from H2 or formate oxidation coupled to the reduction of fumarate to succinate. Membrane preparations have been obtained from this organism that catalyze the synthesis of ATP during H2 oxidation coupled to fumarate reduction. Esterification of orthophosphate is dependent on electron transfer, as evidenced by the requirement for both H2 and fumarate. Phosphorylation is also dependent on ADP and is destroyed by boiling the membrane preparations. H2 utilized for fumarate reduction and succinate formed are stoichiometric. The phosphorylation is markedly uncoupled by pentachlorophenol and gramicidin, but to a lesser extent by dinitrophenol and methyl viologen. 2-n-Heptyl-4-hydroxyquinoline-N-oxide causes severe inhibition of H2 oxidation as well as phosphorylation, but oligomycin or antimycin A has no demonstrable effect. Among several electron acceptors tested, significant phosphorylation is observed only with fumarate. A Mg2+-dependent adenosine triphosphatase activity is present in both the membrane and soluble protein fractions. Highest activity is obtained with ATP as the substrate, and considerably less activity is obtained with other nucleoside triphosphates. The possibility that phosphorylation during "fumarate respiration" may play an important physiological role in the growth of many anaerobic and facultatively anaerobic bacteria is discussed.

Classification of the Flavobacterium-Cytophaga Complex on the Basis of Respiratory Quinones and Fumarate Respiration

Respiratory quinones and the ability to use fumarate as a terminal electron acceptor in anaerobic respiration were investigated in 49 bacterial strains representing a variety of conventional Flavobacterium or Cytophaga species. The organisms examined were subdivided into two categories according to their quinones. (i) Ubiquinones are used by the neotype strain of Flavobacterium aquatile and by cultures representing F. acidificum, F. capsulatum, F. devorans, F. halmephilum, and some unnamed Flavobacterium species. (ii) Menaquinones are produced by both typical Cytophaga strains and many so-called Flavobacterium or “Flavobacterium/Cytophaga” cultures. Several members of category ii exhibited low to medium reduced nicotinamide adenine dinucleotide-fumarate reductase activities when grown in unaerated complex media supplemented with fumarate. In addition, with F. meningosepticum, “group IIb” organisms, and a strain of F. odoratum, the yields of oxygen-limited growth were markedly increased by fumarate, indicating an energetic use of fumarate respiration. On the basis of these findings, restriction of the genus Flavobacterium to “low-guanine-plus-cytosine” organisms containing ubiquinones and resembling F. aquatile is proposed. The incorporation of some former “flavobacteria” into a natural group of organisms containing menaquinones and placement in the vicinity of the C. hutchinsonii guanine-plus-cytosine ratio are discussed.

Taxonomic Significance of Respiratory Quinones and Fumarate Respiration in Actinobacillus and Pasteurella

The lipoquinones and associated respiratory functions of 34 cultures representing 17 bacterial species hitherto assigned to the genus Actinobacillus or Pasteurella were investigated. Strains representing recognized Actinobacillus or Pasteurella species regularly contained desmethylmenaquinones and were capable of anaerobic electron transport with fumarate as a terminal acceptor when grown in oxygen-limited cultures. On aeration, the majority of the strains produced ubiquinones in addition to desmethylmenaquinones, with inter- and intraspecific variations. The use of lipoquinone patterns and features indicating fumarate respiration, such as stimulation of oxygen-limited growth by fumarate, in the classification and identification of Actinobacillus, Pasteurella, and phenotypically similar organisms is discussed.

Osservazioni prellminari sul meccanismo di azione della cocliobolina

Abstract MECHANISM OF COCHLIOBOLIN ACTIVITY: A PREVENTIVE NOTE. — Coch-liobolin caused leakage of phosphate and organic compounds from corn rootlets and potato discs, at room temperature. The leakage does not occur at 1–5°C, but when potato discs are incubated at this temperature and then thoroughly washed and brought at 25°C, a sharp increase of phosphate may be noticed in the incubation solutions. Cochliobolin inhibited partially the aerobic respiration of glucose and endogenous carbon in Micrococcus pyogenes var. aureus resting cells. Aerobic respiration of pyruvate, succinate, fumarate and malate was completely inhibited, while the inhibition of lactate and Lketoglutarate respiration required a short lag period. The hydrogen-ion concentration of the media seemed to be an important factor controlling the rapidity of action of the inhibitor, because at pH 4 and 5 at least 120 minutes were required prior any effect could be observed, while only 30 minutes were required at pH 6. The effects of cochliobolin on Micrococcus resting cells were irreversible In contrast, respiratory activities of acetonic powders were refractory to the substance under aerobic conditions, and oxidation of pyruvate, malate, fumarate, succinate and α-ketoglutarate were not affected by saturated solutions of cochliobolin. It is suggested that the first site of attack is the cell wall-cell membrane unit, altering cell permeability, so that inorganic ions and other cofactors essential to respiration are lost in consequence of the leakage through the cell membrane.