Involvement of GlnK, a P II protein, in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1
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ORIGINAL PAPER
Involvement of GlnK, a PII protein, in control of nitrogen Wxation and ammonia assimilation in Pseudomonas stutzeri A1501 Sheng He · Ming Chen · Zhihong Xie · Yongliang Yan · Hongquan Li · Ying Fan · Shuzhen Ping · Min Lin · Claudine Elmerich
Received: 2 October 2007 / Revised: 14 January 2008 / Accepted: 22 January 2008 / Published online: 15 February 2008 © Springer-Verlag 2008
Abstract The nitrogen-Wxing, root-associated strain Pseudomonas stutzeri A1501 carries a single gene encoding a protein from the PII family, designated glnK. The glnK gene is co-transcribed with two distantly related copies of amtB genes encoding putative ammonium channels. Transcription of glnK was decreased in the presence of ammonia and was partly dependent on NtrC and RpoN under nitrogen-limiting conditions. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity, auxotrophic for glutamine and unable to deadenylylate glutamine synthetase, while inactivation of amtB1 led to a prototro-
Communicated by Jack Meeks. S. He and M. Chen contributed equally to the work. S. He · M. Chen · Z. Xie · Y. Yan · H. Li · Y. Fan · S. Ping · M. Lin (&) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081 Beijing, People’s Republic of China e-mail: [email protected] S. He Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, Guangxi University, 530005 Nanning, Guangxi, People’s Republic of China e-mail: [email protected] C. Elmerich Département de Microbiologie, Institut Pasteur, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, 75724 Paris Cedex 15, France Present Address: Z. Xie Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA
phic and Nif+ mutant strain. RT-PCR analysis showed that nifA transcription was abolished in the glnK mutant, while glnA remained transcribed. Using the yeast two-hybrid system, an interaction between GlnK and the C-terminal domain of NifL was observed, suggesting GlnK-dependent control of NifA activity by NifL. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen Wxation to the glnK mutant, and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in control of ammonia assimilation, of nifA expression and in modulation of NifA activity by NifL. Keywords Pseudomonas stutzeri · Nitrogen Wxation · Ammonia assimilation · Glutamine synthetase · GlnK · PII · nif gene regulation · Yeast-two hybrid assay
Introduction Proteins that belong to the PII family are intracellular, small trimeric signal proteins that play a major role in overall regulation of nitrogen metabolism in Bacteria and Archaea (Arcondéguy et al. 2001; Merrick 2004; Pedrosa and Elmerich 2007). Several genes encoding PII paralogues have been identiWed in Proteobacteria (e.g. glnB, glnK, glnJ, glnY, glnZ) (Merrick 2004; Pedrosa and Elmerich 2007 and references therein). In alpha-Proteobacteria
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