Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobialeguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogencontaining compound excreted. Bacteroids incubated in the presence of 15 N 2 excreted alanine highly enriched in 15 N. The ammonium in these assays neither accumulated significantly nor was enriched in 15 N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.In 1956 (1), ammonium was shown to be the product of nitrogenase, the bacterial enzyme that reduces atmospheric dinitrogen. In free-living, nitrogen-fixing microorganisms, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase͞glutamate synthase pathway. Various transaminases utilize glutamate to generate all of the other amino acids, which are then used to synthesize proteins, nucleic acids and other nitrogen-containing molecules.In rhizobia-leguminous plant symbioses, the majority of the dinitrogen reduced by the microsymbionts, referred to as bacteroids, is transferred to the plant. Because the rhizobia are physically separated from the plant cytosol by a plant-derived symbiosome membrane, the ammonium must move across the bacteroid and symbiosome membranes. The ammonia diffusion hypothesis has been the prevalent model surmising the initial steps of leguminous nodule nitrogen transfer. In this model, the product of nitrogenase exists in two forms within the cell, ammonium [NH 4 ϩ ] and ammonia [NH 3 ], which are readily interconvertible. The ammonium ion cannot diffuse across membranes, but ammonia is believed to diffuse freely out of the bacteroid. Once outside the bacteroid but within the symbiosome space, the ammonia may either diffuse or be transported across the symbiosome membrane (2) where it is assimilated by the host plant into glutamate by the concerted action of glutamine synthetase and glutamate synthase. Glutamate serves as the central nitrogen metabolite in the plant nodule cells for the synthesis of the other amino acids, nucleic acids, and other nitrogen-containing compounds such as ureides that are the principal nitrogen compounds transported from the nodules to the plant shoots and leaves o...
Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-f3-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.
ABSTRACrThe activities of several enzymes of the citric acid and poly-4e-hydroxybutyrate cycles were measured in Rhizobiumjapeoicwm 3IIB-143 bacteroids which had been isolated from soybean nodules by sucrose gradient centrifupaton. Duing the period of developing nitrogenase activity, the specfic activity of fumuase, hydroxybutyrate dehydrogenase, ,-ketothiohlse, and pyruvate dehydrogenase complex incesed whereas acetoacetate-succinyl-CoA transferase and isocitrate dehydrogenase decreased (2,8,9,15,17,21,25,28,29).The carbon metabolism of developing and senescing nodules and the metabolism of bacteroids have been reviewed recently (5,19,27 PHB has been measured in many rhizobia species (7,12,14 The purpose of this investigation was, first, to establish that PDC, selected enzymes from the citric acid cycle, (IDH, MDH, and FMR) and of the PHB cycle (KT, HBD, and AST) were present during the period of nitrogen fixation and PHB accumulation in R. japonicum; second, to follow their activity as a function ofage; and third, to relate temporal changes in activities to those of nitrogenase and PHB accumulation through the use of dispersion diagrams.
Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular.
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