IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Monday 28 November 2011 Concurrent Session 2 – Function & Control of Nitrogenase 1530 - 1650 Authors: Svetlana N. Yurgel 1 , Jennifer Rice 1 , and Michael L. Kahn 1,2 1 Institute of Biological Chemistry,Washington State University, Pullman, WA. 99164. 2 School of Molecular Biosciences,Washington State University, Pullman, WA. 99164. Presentation Title: GlnD and symbiosis: Control beyond the nitrogen stress response? Presentation Time: 1610 - 1630 To establish an effective symbiosis with alfalfa, S. meliloti Rm1021 needs normal operation of the GlnD protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a nitrogen stress response (NSR) (Yurgel & Kahn 2008). Both of the two known targets of GlnD modification, the PII proteins GlnB and GlnK, are not needed for effectiveness (Yurgel et al., 2010). However, the glnD-sm2 mutation that leads to a Fix+Eff¯ phenotype is defective in uridylylation and cells are thought to only have unmodified PII proteins. To generate GlnB in the state expected in a glnD-sm2 mutant, we introduced a Tyr�Phe variant of GlnB that cannot be uridylylated into a glnBglnK background and found that this strain was Fix+Eff+. We also generated a glnBglnKglnD triple mutant and used this and other mutants to dissect the role of these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting that S. meliloti GlnD can communicate information to the cell by alternate mechanisms. Yurgel SN & Kahn ML (2008). A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective Sinorhizobium meliloti symbiosis with alfalfa. Proc Natl Acad Sci U S A 105(48):18958-18963. Yurgel SN, Rice J, Mulder M & Kahn ML (2010). GlnB/GlnK PII proteins and regulation of the Sinorhizobium meliloti Rm1021 nitrogen stress response and symbiotic function. J Bacteriol 192:2473-2481. 28 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Monday 28 November 2011 Concurrent Session 2 – Function & Control of Nitrogenase 1530 - 1650 Authors: Emilio Jiménez-Vicente, César Poza-Carrión & Luis M Rubio Centre for Plant Biotechnology and Genomics U.P.M. – I.N.I.A. Parque Científico y Tecnológico de la U.P.M. Campus de Montegancedo 28223 Pozuelo de Alarcón (Madrid) Presentation Title: The FdxN protein is required for the biosynthesis and activity of the NifDK and NifH nitrogenase components in Azotobacter vinelandii Presentation Time: 1630 - 1650 The molybdenum nitrogenase enzyme is composed of two [Fe-S] cluster containing proteins designated as dinitrogenase (NifDK) and dinitrogenase reductase (NifH). The biosynthesis of nitrogenase [Fe-S] clusters starts by the concerted activity of the NifS cysteine desulfurase and the NifU scaffolding protein. It is expected that the processes of [Fe4-S4] cluster formation on NifU and/or their transfer to apo-NifH and apo-NifDK require an electron donor. The FdxN ferredoxin is a likely candidate to perform such a role in A. vinelandii. This study shows the effects of deleting fdxN on NifH and NifDK. The fdxN mutant exhibited much lower diazotrophic growth rate and in vivo nitrogenase activity than the wild type. In vitro activ fdxN strain was defective in the activity of both component proteins, fdxN NifDK protein exhibited 20% acetylene reduction activity and had lower Fe content than wild- fdxN NifDK could not be reconstituted in vitro by FeMo-co suggesting that it is defective in [Fe4-S4] cluster incorporation or P-cluster synthesis. Time-course analysis of nifH, nifD and nifK fdxN strain had a profile of delayed nifHDK expression and lower nifD and nifK expression rates than wild type. Consistently, NifDK protein levels were lower in the strain. On the other hand, the strain accumulated higher levels of the FeMo-co biosynthetic proteins NifEN and NifB, a phenotype previously observed in mutants with defects in FeMo-co synthesis. In summary, fdxN deletion had a profound negative effect on NifDK expression, maturation and enzymatic activity, and a noticeable effect on cellular NifH activity level. The fact that fdxN deletion affects both NifH and NifDK suggests that FdxN could be involved in the synthesis or insertion of [Fe-S] clusters for both nitrogenase component proteins. 29 2011
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