title: Pyruvate is synthesized by two pathways in pea bacteroids with different efficiencies for nitrogen fixation.
abstract: Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD+ malic enzyme (Dme) but not NADP+ malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N2 fixation, but the PckA/PykA pathway supports N2 fixation at only 60% of that for Dme. Double mutants of dme and pckA/pykA did not fix N2. Furthermore, dme pykA double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N2 fixation. When PckA was expressed from a constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Mulley
authors.forename: Miguel
authors.initials: M
authors.lastname: Lopez-Gomez
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jason
authors.initials: J
authors.lastname: Terpolilli
authors.forename: Jurgen
authors.initials: J
authors.lastname: Prell
authors.forename: Turlough
authors.initials: T
authors.lastname: Finan
authors.forename: Philip
authors.initials: P
authors.lastname: Poole
mesh_terms: D000114:Acetylene / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003998:Dicarboxylic Acids / Q000378:metabolism; D000455:Medicago sativa / Q000382:microbiology; D008954:Models, Biological; D009586:Nitrogen Fixation / Q000235:genetics / Q000502:physiology*; D018532:Pisum sativum / Q000382:microbiology*; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016961:Rhizobium leguminosarum / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003998:Dicarboxylic Acids; D019289:Pyruvic Acid; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase; D000114:Acetylene