Posttranslational mechanisms of nitrogenase regulation in Rhodopseudomonas palustris CGA009

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Posttranslational mechanisms of nitrogenase regulation in Rhodopseudomonas palustris CGA009

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Title: Posttranslational mechanisms of nitrogenase regulation in Rhodopseudomonas palustris CGA009
Author: Heiniger, Erin Kron
Abstract: Nitrogenase catalyzes the conversion of dinitrogen gas (N2) and protons to ammonia and hydrogen gas (H2). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris express nitrogenase genes constitutively and produce H2 when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of nitrogenase. In this study I used mutation analysis and gene expression studies to show that posttranslational regulation of nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated P(II) protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 nitrogenase modification enzyme. In addition, the NifA* strain had elevated nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H2 production by a nifA* mutant strain grown with ammonium. Inactivation of the nitrogenase posttranslational modification system by a draT2 mutation resulted in increased H2 production by ammonium-grown NifA* cells. I also showed that the two alternative nitrogenases of R. palustris are subject to DraT regulation. The vnf cluster has two different phylogenies, the vnfDGK genes were horizontally acquired while the vnfH gene is the product of a gene duplication event. Transcriptional regulation of V-nitrogenase in R. palustris is poorly understood and I have isolated and described a mutant which expresses V-nitrogenase constitutively.
Description: Thesis (Ph.D.)--University of Washington, 2012
URI: http://hdl.handle.net/1773/20572
Author requested restriction: Restrict to UW for 6 months -- then make Open Access

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