Interaction of the general transcription factor TnrA with the PII-like protein GlnK and glutamine synthetase in Bacillus subtilis Airat Kayumov1,2, Annette Heinrich3, Kseniya Fedorova2, Olga Ilinskaya2 and Karl Forchhammer3 Kazan State University of Architecture and Engineering, Russia Kazan Federal University, Department of Microbiology, Russia Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universitat Tubingen, Germany ă ă Keywords Bacillus subtilis; GlnK; glutamine synthetase; nitrogen regulation; PII protein; transcription factor TnrA Correspondence K Forchhammer, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universitat Tubingen, Auf der ă ă Morgenstelle 28, D-72076 Tubingen, ă Germany Fax: +49 7071295843 Tel: +49 70712972096 E-mail: karl.forchhammer@uni-tuebingen.de (Received 26 January 2011, revised 10 March 2011, accepted 14 March 2011) doi:10.1111/j.1742-4658.2011.08102.x TnrA is a master transcription factor regulating nitrogen metabolism in Bacillus subtilis under conditions of nitrogen limitation When the preferred nitrogen source is in excess, feedback-inhibited glutamine synthetase (GS) has been shown to bind TnrA and disable its activity In cells grown with an energetically unfavorable nitrogen source such as nitrate, TnrA is fully membrane-bound via a complex of AmtB and GlnK, which are the transmembrane ammonium transporter and its cognate regulator, respectively, originally termed NrgA and NrgB The complete removal of nitrate from the medium leads to rapid degradation of TnrA in wild-type cells In contrast, in AmtB-deficient or GlnK-deficient strains, TnrA is neither membrane-bound nor degraded in response to nitrate depletion Here, we show that TnrA forms either a stable soluble complex with GlnK in the absence of AmtB, or constitutively binds to GS in the absence of GlnK In vitro, the TnrA C-terminus is responsible for interactions with either GS or GlnK, and this region appears also to mediate proteolysis, suggesting that binding of GlnK or GS protects TnrA from degradation Surface plasmon resonance detection assays have demonstrated that GS binds to TnrA not only in its feedback-inhibited form, but also in its non-feedback-inhibited form, although less efficiently TnrA binding to GlnK or GS responds differentially to adenylate nucleotide levels, with ATP weakening interactions with both partners Structured digital abstract l tnrA binds to glnK by surface plasmon resonance (View interaction) l GS binds to tnrA by pull down (View interaction) l tnrA binds to glnK by pull down (View interaction) l tnrA binds to GS by pull down (View interaction) l GS physically interacts with tnrA by anti bait coimmunoprecipitation (View interaction) l glnK binds to tnrA by pull down (View interaction) l glnK physically interacts with tnrA by anti bait coimmunoprecipitation (View interaction) l tnrA physically interacts with GS by anti bait coimmunoprecipitation (View interaction) l tnrA physically interacts with glnK by anti bait coimmunoprecipitation (View interaction) l tnrA binds to tnrA by cross-linking study (View interaction) l tnrA binds to GS by surface plasmon resonance (View interaction) Abbreviations FC, flow cell; GlnK-ST, Strep-tag II-tagged variant of GlnK; GS, glutamine synthetase; GS-ST, Strep-tag II-tagged variant of glutamine synthetase; ITC, isothermal titration calorimetry; NAGK, N-acetyl-L-glutamate kinase; SPR, surface plasmon resonance FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1779 Interaction of TnrA with GlnK and GS A Kayumov et al Introduction Spore-forming bacteria of the genus Bacillus have a variety of regulatory responses to changes in the environment TnrA, a major transcription factor in Bacillus subtilis under nitrogen-limited conditions (conditions in which the nitrogen source becomes growth-limiting), controls gene expression in response to nitrogen availability During nitrogen-limited growth, TnrA serves either as an activator or a repressor of genes involved in nitrogen assimilation TnrA activates its own gene [1], the nitrate and nitrite utilization genes [2], the nrgAB (amtBglnK) operon (ammonium transport) [3], and some other target promoters [1,4,5] TnrA is a negative regulator of glnA and gltAB, encoding the ammonium assimilatory enzymes glutamine synthetase (GS) and glutamate synthase, respectively [6–8] TnrA belongs to the MerR family of transcription factors, and is present as a homodimer of two 12-kDa subunits [1] The signal for its activation remains unclear [1,6,8,9] Several lines of evidence indicate that GS acts as a sensor of nitrogen availability in B subtilis [1,9] The feedback-inhibited form of GS binds tightly to TnrA, preventing its binding to DNA, with the most effective feedback inhibitors of GS being glutamine and AMP [9] Mutations in TnrA that result in constitutive expression of the TnrA-activated amtB promoter all lie within the C-terminal region of TnrA, and impair the interaction between GS and TnrA [9,10] Another mechanism for controlling TnrA activity was recently found When B subtilis cells were grown with a poor nitrogen source such as nitrate, TnrA was found to be almost completely associated with the cell membrane via the ammonium uptake proteins AmtB and GlnK, originally termed NrgA and NrgB, respectively [11,12] AmtB is a homotrimeric transmembrane ammonium transporter that is active under nitrogenlimited conditions [13] GlnK consists of three 12-kDa monomers, and is a small regulatory protein that belongs to the PII protein family GlnK homologs bind to AmtB and regulate its activity, depending on the cellular nitrogen status [14] Like other GlnK proteins, B subtilis GlnK was shown to bind to the membrane in an AmtB-dependent manner [11,12] Furthermore, B subtilis GlnK exhibits the unique feature of lacking a response to 2-oxoglutarate, but seeming to primarily respond to ATP Depending on the ATP levels, B subtilis GlnK was shown in vitro to be soluble or membrane-bound: mm ATP caused almost full solubilization of GlnK [12] In wild-type B subtilis, TnrA was shown to bind specifically to the membranebound AmtB–GlnK complex, but not to soluble, ATP- 1780 saturated GlnK TnrA-dependent expression of the nrgAB (amtBglnK) promoter was shown to be reduced in a GlnK-deficient strain under conditions of ammonium-limited growth [11], indicating that GlnK could be involved in fine-tuning TnrA-dependent gene expression Furthermore, the cellular levels of TnrA are modulated by proteolysis [15] After shifting of nitrate-grown cells to a medium containing no usable nitrogen source, TnrA is released from the membrane and, concomitantly, it is degraded within 15 by proteolysis By contrast, no degradation of TnrA was observed during this kind of shift experiment in B subtilis AmtB-deficient and GlnK-deficient strains, despite TnrA being soluble in these cells [12,15] To gain deeper insights in the involvement of proteolysis in modulating TnrA-dependent gene expression, we aimed to elucidate why TnrA is resistant to proteolysis in the GlnK-deficient and AmtB-deficient strains Results Immunoprecipitation of TnrA with GlnK or GS in B subtilis In contrast to what is seen in wild-type cells, in amtB or glnK knockout mutants TnrA was only detectable in the soluble fraction of cell-free extracts, but was never membrane-bound and no proteolytic degradation occurred after nitrate depletion [12,15] To explain the mechanism of TnrA protection from proteolysis, we investigated which proteins TnrA is bound to in these mutants, considering GlnK and GS, in particular, as potential partner proteins of TnrA To this end, immunoprecipitation assays were performed with cellfree extracts from both mutant and wild-type nitrategrown cells, and from cells shifted to nitrogen-depleted medium Cell-free extracts were incubated with TnrAspecific, GlnK-specific or GS-specific antibodies coupled to Protein A Sepharose, in the presence of nonionic detergent These antigen–antibody complexes were collected, and after rigorous washing in nonionic detergent-containing buffer and elution of antibodybound protein, the samples were separated by SDS ⁄ PAGE and analyzed by immunoblotting In agreement with earlier data [12], GlnK was coprecipitated with TnrA from crude extracts of nitrategrown wild-type cells, when antibodies against TnrA were used for immunoprecipitation (Fig 1A) When the cells were shifted to nitrate-deprived medium prior to extraction of the proteins, much less TnrA was immunoprecipitated, and, in consequence, less GlnK FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS A Kayumov et al Interaction of TnrA with GlnK and GS whether the cells were nitrate-grown or shifted to nitrate-deprived medium By contrast, no GS was copurified with TnrA in the wild-type or AmtB-deficient mutant under either condition (Fig 1A) These observations were confirmed by reversing the immunoprecipitation experiments, using antibodies against GlnK or GS TnrA was copurified with immunoprecipitated GlnK from extracts of both wild-type and AmtB-deficient cells (Fig 1B), and was recovered by GS immunoprecipitation only in the GlnK-deficient mutant (Fig 1C) Taken together, these data demonstrate, that TnrA binds constitutively to GlnK in AmtB-deficient mutants, and to GS in GlnK-deficient mutants This constitutive binding in the mutant strains probably protects TnrA from proteolytic degradation Surface plasmon resonance analysis (SPR) of the GlnK–TnrA interaction Fig Coimmunoprecipitation of TnrA, GlnK and GS Immunoprecipitation experiments were performed with either TnrA-specific (A), GlnK-specific (B) or GS-specific (C) antibodies Cells were grown under nitrogen-limited conditions in SMM supplemented with 20 mM NaNO3 (I) At late exponential growth phase, cells were washed and shifted to combined nitrogen-free medium, incubated at 37 °C with shaking for 10 min, and then harvested (II) The crude cell extracts were used for immunoprecipitation as described in Experimental procedures The washed immunoprecipitates were analyzed by immunoblotting with antibodies against TnrA, GlnK, or GS, as indicated on the right was detected, as TnrA is degraded by proteolysis following the shift to nitrate-deprived medium [15] By contrast, in the AmtB-deficient mutant strain, the same amount of TnrA was immunoprecipitated and the same amount of GlnK was copurified with TnrA in both nitrogen regimes (Fig 1A, lanes I and II) It should be noted that GlnK is present only as soluble protein in the AmtB-deficient mutant, whereas, in wild-type cells, it is predominantly bound to the transmembrane AmtB channel, and only AmtB-bound GlnK was able to interact with TnrA [11,12,15] In the TnrA immunoprecipitate of GlnK-deficient cells, again no effect was observed on the recovery of TnrA following nitrate deprivation, in agreement with the lack of TnrA degradation in this strain GS was copurified with TnrA and the recovery of GS was independent of As a next step, the interaction of TnrA with GlnK was investigated in vitro by BIAcore SPR detection For this analysis, a Strep-tag II-tagged variant of GlnK (GlnK-ST) and a His-tagged recombinant TnrA were overproduced in Escherichia coli BL21 and purified to apparent electrophoretic homogeneity [12] His6-tagged TnrA was immobilized on flow cell (FC) of a chelating nitrilotriacetic acid sensor chip, and GlnK-ST was used as an analyte His6-N-acetyl-l-glutamate kinase (NAGK) from Synechococcus elongatus [16] was bound to the reference cell (FC 1) as a control for nonspecific interactions Figure 2A shows a response difference sensorgram (FC2 – FC1) of interactions of GlnK with immobilized TnrA For this analysis, an analyte concentration of 40 nm GlnK (trimer) was used Binding of GlnK was not observed when another His-tagged protein (His6-NtcA from S elongatus) was immobilized on the sensor chip (not shown), revealing that the observed binding was specific for TnrA The GlnK–TnrA complex appeared to be quite stable, as revealed by the very slow dissociation of the complex following the injection phase (Fig 2A) In the course of the measurements, we found that mm ATP (in the absence of Mg2+) led to rapid dissociation of the GlnK–TnrA complex (see below), which was subsequently used to regenerate the TnrA-coated chip surface The dissociating effect of mm ATP on the GlnK–TnrA complex is shown in Fig 2A Immediately after application of mm ATP to the preformed GlnK–TnrA complex, rapid dissociation was observed, reaching the basal levels of resonance units (GlnK free surface) within seconds To test the effects of various molecules on the interaction of TnrA with GlnK, 40 nm GlnK (trimer) FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1781 Interaction of TnrA with GlnK and GS A Kayumov et al Fig BIAcore analysis of GlnK–TnrA complex formation and ATP effect on dissociation of the GlnK–TnrA complex The analyte GlnK was injected in a volume of 30 lL at a flow rate of 15 lLỈmin)1 The graph shows the response difference between FC (His6TnrA) and FC (His6-NAGK) (A) ATP effect on dissociation of the GlnK–TnrA complex First, GlnK (40 nM trimers) was injected onto the His6-TnrA surface After 50 s of washing with HBS buffer, 25 lL of mK ATP was injected (indicated by the arrow), which removed the GlnK bound to the His6-TnrA surface within a few seconds (B) Binding of GlnK to TnrA in the presence of different Mg2+ or Mn2+ concentrations with or without mM ATP and mM 2-oxoglutarate (2-OG) present, as indicated GlnK in pure HBS buffer served as a control (set as 100% binding) was incubated with various effector molecules, and the mixture was used as an analyte in SPR analysis ATP and 2-oxoglutarate are known to be the primary effectors involved in PII signaling, and they strongly affect interactions of many GlnK proteins with their receptors [17] The divalent cations Mg2+ or Mn2+ were previously shown to negatively affect the binding of ATP to GlnK [12] Therefore, we investigated the binding of TnrA to GlnK in the presence of different mixtures of Mg2+ or Mn2+ with the effector molecules ATP and 2-oxoglutarate As shown in Fig 2B, MgCl2 or MnCl2 alone did not affect TnrA binding to GlnK However, Mg2+ and Mn2+ gradually relieved the inhibitory effect of ATP on the GlnK–TnrA interaction, so that, in the presence of mm Mg2+ or Mn2+, ATP at mm was not fully inhibitory, and mm Mg2+ or Mn2+ restored more than 50% of the 1782 Fig Influence of various effector molecules on the interaction of GlnK with the His6-TnrA surface GlnK was preincubated with effector molecules at the concentrations indicated, and injected onto the His6-TnrA surface GlnK incubated in pure HBS buffer served as a control (set as 100% binding) (A) Effect of increasing ATP concentrations (as indicated), with or without mM 2-oxoglutarate (2-OG) present (B) Effects of various nucleotides (ATP, ADP, AMP, and GTP) and 2-OG at different concentrations on GlnK binding to TnrA GlnK–TnrA interaction in the presence of mm ATP On the other hand, 2-oxoglutarate did not influence the GlnK–TnrA interaction, either alone, in the absence of divalent metals, or in combination with ATP and Mg2+ or Mn2+ To resolve the inhibitory effect of ATP on the GlnK–TnrA interaction in the absence of divalent cations more clearly, ATP was titrated to the binding assays in the absence or presence of 2-oxolguatarate As shown in Fig 3A, 0.2 mm ATP was sufficient to inhibit 50% of the GlnK–TnrA interaction The inhibitory effect of ATP was not further enhanced by 2-oxoglutarate, in agreement with earlier studies showing that B subtilits GlnK does not respond to 2-oxoglutarate [12] Other nucleotides, such as ADP, AMP, and GTP, at concentrations of FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS A Kayumov et al 1–3 mm had only a small effect on the GlnK–TnrA interaction, except for ADP, which was moderately inhibitory, although less so than ATP (Fig 3B) Taken together, these measurements, although performed under rather artificial conditions, indicate that the GlnK–TnrA complex could be stable in vivo in the presence of divalent cations and that the complex is negatively affected most efficiently by ATP Interaction of TnrA with GlnK and GS (Kd3 = ± 0.05 mm) No binding of other nucleotides (ADP, AMP, and GTP) was detectable (Fig S1), confirming their weak effect on the GlnK–TnrA interaction (Fig 3) These data support the idea that, in B subtilis, GlnK senses the intracellular ATP level at site 3, as the binding affinity of this site is in the millimolar range, which is considered to be physiologically relevant, and that this signal is then transmitted to TnrA Isothermal titration calorimetry (ITC) The affinity of binding of GlnK to nucleotides, which affected the GlnK–TnrA interaction as revealed by SPR analysis, was quantified by ITC Previously, binding of different combinations of ATP and 2-oxoglutarate to GlnK was measured by this method [12] Under optimal binding conditions, strong binding of ATP was observed (Fig 4), which could be perfectly fitted with a three sequential binding sites model Data analysis resolved two high-affinity binding sites (dissociation constant for the first two sites: Kd1 = 12 ± lm and Kd2 = 77 ± 15 lm) and one low-affinity site (site 3) Fig ITC of ATP binding to GlnK The raw data were fitted with a three-site binding model for a PII trimer The upper panel shows the raw data in the form of the heat effect during the titration of 25 lM GlnK solution (trimer concentration) with ATP (titration from 2.1 to 73.5 lM) The lower panels show the binding isotherm and the best-fit curve according to the three sequential binding sites model BIAcore analysis of the GS–TnrA interaction The results of the immunoprecipitation experiments revealed a constitutively present GS–TnrA complex in the GlnK-deficient cells transferred to nitrate-deprived conditions, as well as in nitrate-grown cells Under these conditions, GS is supposed to be in an active state, whereas only feedback-inhibited GS was previously reported to bind TnrA [9,18] To test whether, indeed, non-feedback-inhibited GS can also bind TnrA, a Strep II-tagged variant of GS (GS-ST) was overproduced in E coli BL21, purified to apparent electrophoretic homogeneity [12], and used in BIAcore analysis on immobilized His6-tagged TnrA immobilized on a chelating nitrilotriacetic acid sensor chip NAGK from S elongatus was bound to the reference cell as a control for nonspecific interactions The response difference sensogram (FC2 – FC1) in Fig 5A shows the binding of non-feedback-inhibited GS to immobilized TnrA The GS–TnrA complex was quite stable under the conditions used: almost no complex dissociation appeared after the injection phase In contrast to what was found for GlnK, no efficient effector molecule was found to remove GS from the His6-TnrA surface ATp at 10 mm caused only partial release of GS from TnrA (Fig 5A) The effects of various metabolites on the GS–TnrA interaction were also investigated GS at 40 nm was incubated with effector molecules for in ice, and the mixture was used as an analyte in SPR analysis AMP and glutamine are known to be the most effective inhibitors of GS [9] Figure 5B shows the effect of the feedback inhibitors AMP and glutamine on GS binding to a TnrA-coated sensor chip The presence of either AMP or glutamine led to an approximately twofold signal increase in comparison with non-feedbackinhibited GS ATP, at a physiological concentration, negatively affected GS binding to the TnrA sensor surface At a concentration of 2.5 mm, it decreased complex formation by approximately 60%, and at a concentration of mm, by 80% (Fig 5C) ATP at 10 mm was required to completely abolish complex formation; however, once the complex was formed, FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1783 Interaction of TnrA with GlnK and GS A Kayumov et al Fig BIAcore analysis of GS–TnrA complex formation (A) GS– TnrA interaction First, non-feedback-inhibited GS was injected onto the His6-TnrA surface After 180 s of washing with HBS buffer, 25 lL of 10 mM ATP was injected (indicated by the arrow), which partially removed the GS bound to the His6-TnrA surface (B, C) Effects of AMP, glutamine and ATP on GS binding to TnrA GS was preincubated with effector molecules at the concentrations indicated, and injected onto the His6-TnrA surface GS incubated in pure HBS buffer served as a control this concentration could not efficiently dissociate the complex (see above) The C-terminus of TnrA is required for interaction with both GlnK and GS, as well as for intracellular proteolysis Previously, it had been reported that the DNA-binding domain of TnrA is located on its N-terminus, whereas 1784 the C-terminus is responsible for GS binding [10] Six amino acids required for this interaction on the C-terminus were identified (Met96, Leu97, Gln100, Leu101, Ala103, and Phe105) (Fig S2) A previous study showed [19] that the TnrA-dependent nrg and nasB promoters were constitutively expressed when seven or 20 amino acids were deleted from the C-terminus of TnrA, whereas deletion of 34 amino acids from the C-terminus resulted in a TnrA null mutation phenotype This implied that the TnrA signal transduction domain is most likely located at the C-terminus In nitrate-grown cells, TnrA is almost completely membrane-bound via GlnK [12] We have speculated that GlnK may also interact with the C-terminus of TnrA, and may play a role in the regulation of TnrA activity and its proteolysis [15] To test this assumption, various truncations of TnrA (lacking six, 20 and 35 amino acids from the C-terminus) were constructed and overproduced in E coli (Fig S2) Glutaraldehyde crosslinking assays revealed that all proteins were in a dimeric state, confirming that the C-terminus is not required for dimerization (Fig S3) [10,19] Interactions of the truncated TnrA proteins with GlnK and with GS were determined by pulldown and SPR analysis, as described above (Fig 6) As expected, the C-terminus of TnrA was absolutely required for GS binding: deletion of even six amino acids abolished this interaction (Fig 6A,B) Truncated forms of TnrA with the C-terminus lacking six or 20 amino acids still bound to GlnK; however, removal of 35 amino acids completely abolished binding of GlnK (Fig 6A,C) This result implies that a region in TnrA located between 20 and 35 amino acids from the C-terminus is required for GlnK interaction, whereas the ultimate C-terminal amino acids of TnrA are apparently needed for GS binding In addition, the in vitro proteolysis of truncated TnrA variants was investigated, as described previously for full-length TnrA [15] B subtilis 168 (wild-type) cells were grown in SMM medium supplemented with sodium nitrate until the late exponential growth phase, and the cells were then washed and resuspended in SMM without nitrate, and finally incubated for a further 20 Samples were taken before and after the shift, and soluble cell-free extract was prepared by ultracentrifugation The soluble extract (containing 20 lg of total protein) was supplemented with 50 ng of TnrA, and the mixture was incubated at 37 °C for 60 min; TnrA incubated in buffer served as a control The fate of TnrA in the samples was then analyzed by immunoblotting with TnrA-specific antibodies (Fig 7) During incubation in the soluble cytoplasmic extract, TnrA6, TnrA20 and wild-type TnrA were almost completely degraded, but not TnrA35 This indicates that a FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS A Kayumov et al Interaction of TnrA with GlnK and GS Fig In vitro proteolysis of truncated TnrA proteins Soluble cellfree extracts were prepared from (I) cells that had been shifted into nitrogen-free medium and incubated for 20 min, and (II) nonshifted cells Purified TnrA protein variants, full-length or different C-terminal truncations (each 50 ng of protein), were incubated with these extracts for 30 (as described in [15]) TnrA incubated in assay buffer served as a control (C) Subsequently, proteolytic removal of TnrA was analyzed by western blotting recognition site (see above) This finding agrees with the assumption that binding of GS or GlnK protects TnrA from proteolytic degradation [15], as these proteins would shed the recognition site for proteolytic degradation As soon as GlnK or GS dissociate from TnrA, the C-terminus becomes accessible to proteolysis Discussion Fig The interaction of truncated TnrA proteins with GlnK and GS (A) BIAcore analysis of GlnK and GS binding to wild-type TnrA (TnrAwt), TnrA6, TnrA20, and TnrA35 The analyte (40 nM GlnK or GS oligomers) was injected in a volume of 30 lL onto the TnrA surface at a flow rate of 15 lLỈmin)1 His6-NAGK served as a control in FC (B) Pulldown analysis of GS binding to TnrAwt, TnrA6, TnrA20, and TnrA35 (see Experimental procedures for details) (C) Pulldown analysis of GlnK binding to TnrAwt, TnrA6, TnrA20, and TnrA35 TnrA (dimer) at 10 nM was premixed with 10 nM GS (12mer) or 10 nM GlnK (trimer), and incubated in buffer B at 20 °C for 30 The protein mix was loaded onto an Ni2+–nitrilotriacetic acid Sepharose column to affinity-purify TnrA (I) or Strep-Tactin Sepharose to affinity-purify GS or GlnK (II), after the columns had been washed with buffer B Proteins were eluted with 250 mM imidazole (I) or with 2.5 mM destiobiotin (II), and the eluates were analyzed by western blot with TnrA-specific, GlnK-specific and GSspecific antibodies, as indicated on the left region located between 20 and 35 amino acids from the C-terminus of TnrA is required for protease recognition and, at the same time, overlaps with the GlnK TnrA, a major transcription factor in B subtilis for the control of nitrogen assimilation, is active under nitrogen-limited conditions and is membrane-bound via the AmtB–GlnK complex [6,12] Its activity was shown to be regulated by complex formation with feedbackinhibited GS, and in the absence of a nitrogen source TnrA is eliminated from the cells by proteolysis [9,10,15] The findings in the present study strongly imply that, in vivo, TnrA is stable only in a complex with a partner protein In nitrate-grown wild-type cells, TnrA is active and bound to the GlnK–AmtB complex [12] (Fig 1A,B) After shifting of the cells to nitrate-free medium, this complex dissociates and TnrA becomes degraded, whereas no degradation occurs in AmtB-deficient or GlnK-deficient cells [12] Our data show that, in these mutant strains, TnrA interacts constitutively with either soluble GlnK or GS, respectively, and in consequence is protected from proteolysis (Fig 1) The reason for this protection, according to the present study, is that binding of GS or GlnK to the C-terminus FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1785 Interaction of TnrA with GlnK and GS A Kayumov et al of TnrA shields the recognition site for proteolytic degradation of TnrA (Figs and 7) In the AmtB-deficient strain, GlnK is located in the cytoplasm and constitutively binds TnrA Previously, the AmtB-deficient strain (with constitutive GlnK– TnrA binding) was shown to display high levels of transcription from the TnrA-dependent nrgAB promoter under ammonia-limited conditions (ammonium at low pH) [11] This suggests that TnrA bound to GlnK is still able to activate gene expression The assumption that GlnK binding does not impair TnrA activity is consistent with the observation that, in nitrate-grown wildtype cells, TnrA is bound to the AmtB–GlnK complex despite being transcriptionally active Nitrate depletion leads to dissociation of the AmtB– GlnK–TnrA complex and subsequent TnrA degradation, whereas in AmtB mutants TnrA remains bound to GlnK and is therefore protected from proteolysis This suggests a role of AmtB in dissociation of the GlnK–TnrA complex A possible regulatory role of AmtB proteins has been suggested previously [20]; however, the mechanism leading to AmtB-dependent GlnK–TnrA dissociation has remained elusive so far In B subtilis wild-type cells growing on a poor nitrogen source (nitrate), GS is active and does not bind TnrA (Fig 1A,C), and the latter is sequestered by the AmtB–GlnK complex However, in the GlnK mutant, TnrA is constitutively bound to GS; a shift to a nitrate-deprived medium does not lead to dissociation of the complex, and TnrA remains protected from proteolysis The constitutive binding of TnrA to GS seems to contradict previous reports that only feedback-inhibited GS is able to bind TnrA [9] However, the sensitive SPR analysis has demonstrated that nonfeedback-inhibited GS is, in fact, able to bind TnrA, although with reduced affinity as compared with feedback-inhibited GS (Fig 5) The reduced affinity could account for the fact that this interaction is not detected by examining it indirectly through TnrA–DNA binding assays [9,10,18] Constitutive binding of GS to TnrA in the GlnK-deficient strain provides an explanation for the so-far elusive observation that TnrA-dependent transcription from the nrgAB promoter is impaired in a GlnK-deficeint strain growing under ammonialimited conditions (ammonium at low pH) [11], as GS binding was shown to depress the transcriptional activity of TnrA [9,10,18] Taken together, the results from this investigation provide indications of the physiological role of the GlnK–TnrA interaction, which has previously been unclear In the GlnK-bound state, TnrA is protected from proteolysis without affecting its ability to induce gene expression When TnrA dissociates from the 1786 AmtB–GlnK complex (after a shift to nitrate-deprived conditions), it becomes rapidly degraded Under these conditions, GS should be in a highly active, nonfeedback-inhibited state, which has reduced affinity for TnrA, Therefore, TnrA could be preferentially recognized by a protease as an idle protein and degraded, as has been proposed for many proteins in B subtilis [21] When, however, TnrA is complexed by GS before nitrate downshift, as is the case in the GlnK-deficient mutant, it remains bound and is protected from proteolysis Experimental procedures Bacterial strains and growth conditions The B subtilis strains used in this study – strain 168 (wild type), the AmtB-deficient strain GP 254, and the GlnKdeficient mutant GP 253 – have been described previously [11] B subtilis cells were grown in Spizizen minimal medium (SMM) [22] containing glucose [0.5% (w ⁄ v)] as a carbon source Sodium nitrate (20 mm) served as a nitrogen source l-Tryptophan was added to a final concentration of 50 mg L)1 Protein preparation TnrA from B subtilis 168 cells and NAGK from S elongatus, carrying His6-tags on their N-terminal, were overproduced in E coli BL21 with the pET15b expression vector (Novagene, San Diego, CA, USA), and purified on Ni2+– nitrilotriacetic acid columns to apparent electrophoretic homogeneity, as described previously [12,16] GlnK-ST and GS-ST were overproduced in E coli BL21 with the pDG148 expression vector and purified with a Strep-Tactin column (IBA, Gottingen, Germany), as described in detail in Doc S1 ă Immunoblot analysis For immunoblot analysis, the samples were separated on 15% SDS ⁄ PAGE gels After electrophoresis, the proteins were transferred to a nitrocellulose membrane by semi-dry electroblotting Antibodies were visualized with secondary antibodies (anti-rabbit IgG–POD) (Sigma-Aldrich, Taufkirchen, Germany) and the LumiLight detection system (Roche Diagnostics, Mannheim, Germany) Coupling antibodies to Protein A Sepharose One hundred milligrams of Protein A Sepharose beads (GE Healthcare, Munich, Germany) were incubated for h at 24 °C in 0.5 mL of NaCl ⁄ Pi (4.3 mm Na2HPO4, 1.8 mm KH2PO4, 137 mm NaCl, 2.7 mm KCl, pH 8.0) The beads were harvested by short centrifugation (11 500 g, 30 s, FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS A Kayumov et al °C), and incubated with 0.5 mL of antiserum for h at 24 °C with gentle shaking After being washed times with mL of 0.2 m Na3BO3 (pH 9.0), the Sepharose beads were resuspended in mL of 0.2 m Na3BO3 (pH 9.0), and dimethyl pimelimidate dihydrochloride was added to a final concentration of 20 mm The incubation was continued for 30 at 24 °C with gentle shaking The Protein A Sepharose beads were washed twice with mL of 0.2 m ethanolamine (pH 8.0), resuspended in mL of this, and incubated for h at 24 °C To remove the unbound antibodies, the beads were washed twice with mL of NaCl ⁄ Pi, twice with mL of 100 mm glycine (pH 3.0), and twice with NaCl ⁄ Pi, and resuspended in 0.5 mL of NaCl ⁄ Pi Immunoprecipitation The immunoprecipitation experiments were performed as described in [23] Cultures of B subtilis were grown in SMM with 20 mm NaNO3 to a D600 nm of 0.8, harvested by centrifugation (8500 g, 10 min, °C), resuspended in buffer I (50 mm Hepes ⁄ NaOH, pH 7.0, 50 mm KCl, 100 mm EDTA, mm MgCl2, mm benzamidine), and broken with a FastPrep-24 (M.P Biomedical, Irvine, CA, USA) After centrifugation (15 000 g, 10 min, °C) to remove debris and unbroken cells, the samples, containing mg of total protein, were diluted with detergent-containing buffer [NET buffer I: 50 mm Tris ⁄ HCl, pH 7.0, 150 mm NaCl, 0.1% (v ⁄ v) nonionic detergent Nonidet P-40, mm EDTA] to a total volume of 1.5 mL, and following a 15-min incubation at 24 °C, the sample was briefly centrifuged (16 000 g, 30 s) to remove debris To this extract, 100 lL of a suspension of Protein A Sepharose beads with coupled antibodies was added After a 3-h incubation at °C, Sepharose beads were harvested by centrifugation (16 000 g, 30 s, °C), and the sediment was washed twice with NET buffer I, once with NET buffer II (NET buffer I with 500 mm NaCl), and once with buffer IP [10 mm Tris ⁄ HCl, pH 7.5, 0.1% (v ⁄ v) Nonidet P-40] The bound proteins were eluted from Protein A Sepharose by 10 consecutive additions of 50 lL each of buffer IE (100 mm glycine, pH 2.4), and the elutions were pooled and analyzed by immunoblot analysis with TnrAspecific, GlnK-specific and GS-specific antibodies BIAcore SPR detection SPR experiments were performed with a BIAcore X biosensor system (Biacore AB, Uppsala, Sweden) To immobilize His6-TnrA on the nitrilotriacetic acid biosensor surface, Ni2+ was first bound to the nitrilotriacetic acid surfaces of both flow chambers through injection of 10 lL of a mm NiSO4 solution Then, His6-TnrA was injected into FC in a volume of 50 lL at a concentration of nmol ⁄ mL in HBS buffer (10 mm Hepes, 200 mm NaCl, 0.005% Nonidet P-40, pH 7.5) His6-NAGK from S elongatus was injected into FC in a volume of 50 lL at a concentration of nmol ⁄ mL Interaction of TnrA with GlnK and GS in HBS buffer This resulted in increases in resonance units of 500 in FC and 800 in FC Experiments were performed at 25 °C in HBS buffer at a flow rate of 15 lLỈmin)1, with GlnK-ST or GS-ST as analyte at the concentrations indicated To analyze the effect of small molecules on GlnK or GS binding to the His6-TnrA surface, the analyte was preincubated for on ice with the various effector molecules as indicated, and was then injected into the sensor chip For novel reloading of the nitrilotriacetic acid sensor chip with fresh His6-TnrA, 50 lL of 0.5 m EDTA was injected to completely remove His6-TnrA and Ni2+ Subsequently, the chip was loaded again with Ni2+ and His6-TnrA or His6NAGK as described above This procedure was performed when the performance of analyte binding to the His6-TnrA surface started to decrease ITC ITC experiments were performed on a VP-ITC microcalorimeter (MicroCal, LCC, New York, USA) in 10 mm Hepes ⁄ NaOH, 50 mm KCl and 100 mm NaCl (pH 7.4) at 20 °C [24] For determination of ATP, ADP, AMP and GTP binding isotherms for wild-type GlnK, 25 lm protein (trimer concentration) was titrated with mm ATP, mm ADP, mm AMP, or mm GTP, respectively The ligand (5 lL) was injected 35 times into the 1.4285-mL cell with stirring at 350 r.p.m The binding isotherms were calculated from received data, and fitted to a three-site binding model with MicroCal origin software (Northampton, MA, USA) Construction of mutant tnrA genes All DNA manipulations were performed by standard methods as described in [23] Mutant tnrA genes were amplified with pfu polymerase from chromosomal DNA of B subtilis 168 Briefly, the tnrA gene coding for the protein with deletion of six amino acids from C-terminus was obtained with primers TnrAN (5¢-GCT CGA GGA TCC GAT GAC CAC AGA AGA TCA TTC TT-3¢) and TnrA6 (5¢-TTA ACG GGA TCC GTA CCG TTA GTG AGC ATT AAG3¢) The PCR products were purified, digested with BamHI, and ligated into the BamHI-digested pET-15b vector (Novagen) This vector provides N-terminally His6-tagged protein overexpression in E coli BL21 cells To obtain TnrA proteins lacking 20 and 35 amino acids from the C-terminus, TnrA20 (5¢-TCC AGC GGA TCC TTC CGC ACT TAC GGA TC-3¢) and TnrA35 (5¢-TTC TTT GGA TCC CAT ATC CTT TTA AAT CTC TGC-3¢) oligonucleotides were used, respectively, instead of TnrA6 The sequences of all cloned genes were confirmed by DNA sequencing Pulldown For these assays, the purified His6-tagged TnrA proteins (wild-type and truncated versions), GlnK-ST and GS-ST FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1787 Interaction of TnrA with GlnK and GS A Kayumov et al were used Initially, the proteins (10 nm each protein) were diluted in 300 lL of buffer B (100 mm Tris ⁄ HCl, pH 8.0, 200 mm NaCl, mm MgCl2, mm EDTA) and incubated at 20 °C for 30 Afterwards, the protein mixture was loaded onto Ni2+–nitrilotriacetic acid Sepharose (Qiagen, Hilden, Germany) or Strep-Tactin Sepharose (IBA, Gottină gen, Germany) equilibrated with 10 column volumes (10 · 0.2 mL) of buffer B, with subsequent washing four times with five volumes of the same buffer Proteins were eluted with buffer E (buffer B supplemented with 250 mm imidazole from Ni2+–nitrilotriacetic acid Sepharose or 2.5 mm destiobiotin from the Strep-Tactin column) The samples were collected and analyzed by western blot with TnrA-specific, GlnK-specific and GS-specific antibodies 10 11 Acknowledgements J Stulke (Gottingen) is gratefully acknowledged for ¨ ¨ providing B subtilis strains This work was supported by DFG grant Fo195, the Russian–German program ‘Michail Lomonosov’ A ⁄ 08 ⁄ 75091, and the Ministry of Education and Science of the Russian Federation (government contract No P2573 from 25 November 2009) References 13 14 Fisher SH (1999) Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! 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resistant to purine analogs J Bacteriol 169, 2977–2983 23 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York, NY 24 Fokina O, Chellamuthu VR, Zeth K & Forchhammer K (2010) A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK–P(II) complex formation J Mol Biol 399, 410–421 Interaction of TnrA with GlnK and GS Fig S2 TnrA C-terminal truncations Fig S3 Crosslinking analysis of truncated TnrA proteins Doc S1 Purification of His6-tagged TnrA proteins, purification of GlnK-ST and GS-ST, and glutaraldehyde crosslinking assays This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors Supporting information The following supplementary material is available: Fig S1 Isothermal titration calorimetry of (A) ADP, (B) AMP and (C) GTP binding to GlnK FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1789 ... concentrations on GlnK binding to TnrA GlnK? ? ?TnrA interaction in the presence of mm ATP On the other hand, 2-oxoglutarate did not in? ??uence the GlnK? ? ?TnrA interaction, either alone, in the absence of divalent... interaction of truncated TnrA proteins with GlnK and GS (A) BIAcore analysis of GlnK and GS binding to wild-type TnrA (TnrAwt), TnrA6 , TnrA2 0, and TnrA3 5 The analyte (40 nM GlnK or GS oligomers) was injected... with the lack of TnrA degradation in this strain GS was copurified with TnrA and the recovery of GS was independent of As a next step, the interaction of TnrA with GlnK was investigated in vitro by