rubrum     S1 Wild type   E. coli     BL21 (DE3) pLysS Host for expression of PII proteins, Cmr Invitrogen BL21 Star (DE3) Host for expression

of GlnE Invitrogen RB9040 ΔglnD; host for expression of GlnD, Tcr [19] Plasmids     pETGlnE pET101 derivative containing glnE, Apr [5] pGEXGlnD pGEX6P-3 derivative containing glnD, Apr [11] pMJET pET15b derivative containing glnB, Apr [20] pETGlnJ pET15b derivative containing glnJ, Apr [5] pETGlnJR17K pETGlnJ derivative encoding GlnJR17K, Apr This study pETGlnJQ42H pETGlnJ derivative encoding GlnJQ42H, Apr This study pETGlnJN54D pETGlnJ derivative encoding GlnJN54D, Apr This study pETGlnJK85R pETGlnJ derivative encoding GlnJK85R, Apr This study pETGlnJV100I pETGlnJ derivative encoding GlnJV100I, Apr This Selleck MCC 950 study pETGlnJE109G pETGlnJ derivative encoding GlnJE109G, Apr This study pETGlnJQ42HK85R pETGlnJ derivative encoding GlnJQ42HK85R, Apr This study pETGlnBH42Q pMJET derivative encoding GlnBH42Q, Apr This study pETGlnBR85K pMJET derivative encoding GlnBR85K, Apr This study pETGlnBH42QR85K pMJET derivative encoding see more GlnBH42QR85K, Apr This study Ap ampicillin; Tc tetracycline; Cm chloramphenicol. Site-directed

mutagenesis All GlnJ and GlnB variants were generated by standard PCR-mediated site-directed mutagenesis using the QuikChange kit (Stratagene) and according to the manufacturer’s instruction. The templates used were pETGlnJ [5] and pMJET [20]. Purification of R. rubrum PII proteins All constructs used to express PII proteins were pET15b derivatives, generating proteins with an N-terminal poly-histidine tag. All PII proteins were purified using HiTrap 1 ml columns (GE Healthcare)

according to [5]. Purification of R. rubrum glutamine synthetase, GlnE and GlnD proteins GlnD was purified as a GST fusion-protein according to [11]. Glutamine KPT-8602 chemical structure synthetase was purified from wild type R. rubrum and GlnE was purified with a C-terminal poly-histidine tag as previously described [5]. Uridylylation assays Each reaction (final volume 50 μl) contained 50 mM Tris–HCl pH 7.6, 3.5 μM PII protein (GlnJ, GlnB check or a variant), 0.2 μM GlnD, 100 mM KCl, 1 mM ATP, 1 mM dithiothreitol, 0.5 mM UTP and either 3 mM MnCl2 and 60 μM 2-OG or 25 mM MgCl2 and 250 μM 2-OG (in the control reactions the divalent cations were omitted and 2-OG was at 250 μM). After 30 min (or as indicated) the reaction was stopped by the addition of 5X native loading buffer (125 mM Tris–HCl pH 6.8, 50 mM EDTA, 50% glycerol, 5% sorbitol) and a 20 μl sample was loaded onto a 12.5% native PAGE prepared according to [21]. After electrophoresis the gels were stained with Coomassie brilliant blue R250. Adenylylation assays Adenylylation reactions were performed as previously described [13] and GS activity measured using the γ-glutamyl transferase reaction [5, 22].