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J. Biol. Chem., Vol. 281, Issue 46, 34909-34917, November 17, 2006

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Interaction of the Membrane-bound GlnK-AmtB Complex with the Master Regulator of Nitrogen Metabolism TnrA in Bacillus subtilis^*

Annette Heinrich, Kathrin Woyda, Katja Brauburger, Gregor Meiss, Christian Detsch^¶, Jörg Stülke^||, and Karl Forchhammer¹

From the Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, Institut für Biochemie Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany, ^¶Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany, and ^||Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany

P_II proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the P_II-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other P_II proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other P_II-like proteins, Mg²⁺ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK- or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mM ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.

Received for publication, August 9, 2006 , and in revised form, September 20, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Fo 195/4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Construction of Plasmids and detailed Purification Protocols and Refs. 1–6 and 60 and 61.

¹ To whom correspondence should be addressed. Tel.: 49-641-9935545; Fax: 49-641-9935549; E-mail: Karl.Forchhammer{at}mikro.bio.uni-giessen.de.

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