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Department of Bacteriology and the Center for the Study of Nitrogen Fixation, University of Wisconsin—Madison, Madison, Wisconsin 53706
Received 15 May 2007/ Accepted 16 July 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AmtB proteins have been shown to interact with homologs of PII in several bacteria and in the archaeon Methanococcus jannaschii (8, 10, 17, 18, 44, 45, 47, 50, 53). The association of PII with AmtB can physically block the ammonia gas channels of AmtB under conditions of nitrogen sufficiency in the cell. In addition, the AmtB-PII complex is able to recruit at least one other protein to the membrane, dinitrogenase reductase-activating glycohydrolase (DRAG), in organisms capable of nitrogen fixation (22, 48). This membrane sequestration requires both an Amt protein and a PII protein and results in the inability of DRAG to activate dinitrogenase reductase in vivo. Finally, AmtB is able to remove equimolar amounts of PII from the cytoplasm, preventing PII from interacting with at least some other proteins. Although membrane sequestration of PII has been shown to be important in recovering from nitrogen starvation in Escherichia coli, this function of AmtB is still poorly understood (6).
PII homologs sense several small-molecule pools. The best-studied PII protein is GlnB from E. coli, and insights into PII protein function from this homolog apply to all PII proteins studied to date. Carbon status and perhaps energy status are sensed directly by the binding of 2-ketoglutarate (2-KG) and ATP to each subunit of the PII trimer (26). The binding of one 2-KG molecule or no molecules to PII signals carbon deficiency. At higher 2-KG concentrations, the binding of second and third 2-KG molecules to PII signals carbon sufficiency (26, 36). The binding of ATP lowers the Km of PII for 2-KG, which could act as a signal of energy sufficiency in the cell; however, previous studies have suggested that ATP may always present at saturating levels in E. coli (23). In contrast, cellular nitrogen status is indirectly sensed by the reversible modification of PII, rather than the direct binding of nitrogen-rich metabolites. In E. coli and other bacteria such as Rhodospirillum rubrum, the bifunctional, uridylyltransferase/uridylyl-removing GlnD protein carries out uridylylation of PII. In E. coli this function is regulated by glutamine levels: low levels of glutamine, signaling nitrogen deficiency, lead to uridylylation of PII (PII-UMP) by GlnD, while high levels of glutamine, signaling nitrogen sufficiency, lead to deuridylylation of PII-UMP by GlnD (3, 35). GlnD appears to sense 2-KG as well as glutamine in R. rubrum, where high levels of 2-KG stimulate uridylylation, and high levels of glutamine stimulate deuridylylation, of PII by GlnD (25).
The specificity and regulation of interaction between PII and AmtB proteins are not fully understood. There is evidence that both GlnB and GlnK of E. coli can interact with the sole AmtB species present in that organism in vivo, and it is suggested that GlnK may interact more strongly with AmtB than does GlnB (9). Likewise, both PII homologs of Azospirillum brasilense appear able to associate with AmtB at the membrane in vivo (21). Only unmodified PII has been found to interact with AmtB, and uridylylation of PII has been shown to prevent the formation of the PII-AmtB complex in E. coli, A. brasilense, and R. rubrum (11, 22, 53). Crystal structures of the E. coli complex reveal that the uridylyl group could sterically interfere with the surface of interaction between the two proteins (8, 17). However, not all PII proteins are regulated by uridylylation. In Streptomyces coelicolor the same tyrosine residue uridylylated in enteric bacteria is instead adenylylated (19), and in cyanobacteria a conserved serine in PII is phosphorylated in response to nitrogen limitation (16). In other organisms PII might not be modified at all (49). Regardless of the decoration's identity, it is likely to inhibit interaction with an AmtB protein because of the location of the modified residue, but this hypothesis has not been tested.
Finally, small molecules are known to have diverse regulatory effects on the PII-AmtB complex, depending on the organism studied. In E. coli purified AmtB-GlnK complexes disassociate only in the presence of both ATP and 2-KG, while in M. jannaschii purified GlnK1-Amt1 complexes disassociate in the presence of low levels of ATP (11, 50). These may reflect real physiological differences in regulation in different organisms or may be complicated by the buffers and detergents used to purify the protein complexes. To date, there has been no in vitro study of the regulation of this interaction using AmtB in its native state as an integral membrane protein.
There are three PII homologs, GlnB, GlnJ, and GlnK, in R. rubrum that interact with up to five other proteins involved in nitrogen regulation (52, 53, 54). All three PII homologs can regulate the activity of GlnE (ATase), which reversibly adenylylates glutamine synthetase (52). Expressed from their native promoters, GlnB and GlnJ, but not GlnK, regulate the NtrBC two-component regulatory system, leading to expression of genes involved in nitrogen metabolism under conditions of nitrogen deficiency (52). GlnB and GlnJ also indirectly regulate nitrogen fixation by controlling the activities of two enzymes with opposing functions, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and DRAG, which reversibly modify dinitrogenase reductase (52). Finally, only uridylylated GlnB is able to interact with and activate NifA, which is a transcriptional activator of genes involved in nitrogen fixation (52). Thus, the three PII homologs in R. rubrum are not functionally interchangeable. The genes encoding the two GlnK-type PII proteins, glnJ and glnK, each have amtB homologs immediately downstream. However, only the amtB1 gene downstream of glnJ plays important roles in the regulation of PII in the cell (53). We therefore chose to examine whether specificity for a particular PII existed in the R. rubrum AmtB1 protein, as this could have substantial physiological consequences. Once such specificity was observed, we examined the role that small molecules have on the strength of interaction between a single PII, GlnJ, and AmtB1.
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MATERIALS AND METHODS |
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glnB3 glnK1::aacC1), and UR794 (amtB11::aacC1) (27, 40, 53). E. coli strains used in this study were UQ4037 (pUX754) and UQ4038 (pUX755). Similar to the construction of an R. rubrum GlnB overexpression vector (pUX753), a 0.4-kb fragment of R. rubrum glnK or glnJ was PCR amplified and cloned into pND706 at NdeI and EcoRI sites and then transformed into E. coli strain BK (glnB mdl-glnK::Kan), yielding UQ4037 (pUX754) and UQ4038 (pUX755), respectively (32, 44). R. rubrum strains were grown in rich medium (SMN) or minimal malate glutamate medium (MG), as described previously (14, 30). To prepare larger volumes of cells for membrane harvesting, MG-grown cells were prepared by inoculating 100:1 from fully grown SMN cultures into MG medium and grown anaerobically under light to an optical density at 600 nm (OD600) of about 0.9 (53). E. coli strains were grown in rich medium, Luria broth (LB) or LC (similar to LB medium but with NaCl reduced from 10 g/liter to 5 g/liter) (40). Protein purification. GlnJ and GlnK were purified using a protocol similar to that used previously to purify GlnB (54). UQ4037 (for GlnJ) and UQ4038 (for GlnK) were grown overnight in 10 ml of 2x LB and inoculated into 600 ml of 2x LB medium, both supplemented with 100 µg/ml ampicillin and 25 µg/ml kanamycin. Cultures were grown at 30°C to an OD600 of 0.8 to 1.1 and heat shocked at 44°C for an additional 4 to 5 h. Cells were concentrated by centrifugation at 10,800 x g for 10 min, resuspended in 50 mM Tris, pH 7.5, and broken by French press. Cell lysate was centrifuged as before, and then protein in the supernatant was precipitated with 45% (final) ammonium sulfate, centrifuged as before, and resuspended in 50 mM Tris, pH 7.5. GlnJ and GlnK were purified to greater than 75% purity by eluting the protein from a Q-Sepharose column with a gradient of 100 to 400 mM NaCl. GlnJ was further purified to over 95% purity on a G-100 size exclusion column. GlnK was further purified to over 90% purity on a hydroxyapatite column after elution with 10 mM KPO4 buffer, pH 7.5. Proteins were resuspended in 50 mM Tris, 100 mM NaCl, and 5% glycerol, pH 7.4.
Membrane preparation. Cell harvesting, breakage by sonication, and separation of whole-cell clarified extract into cytoplasmic and membrane fractions using ultracentrifugation were performed as described previously (53). Separation of the PII-AmtB1-containing membrane into soluble and membrane-bound protein fractions was done in the following manner. Samples were centrifuged at 200,000 x g for 30 min at 4°C, and 80% of the total volume was removed as the supernatant fraction. The remaining supernatant was discarded, and the membrane pellet was resuspended in an equal volume of 50 mM NaPO4 buffer, pH 6.8. The resuspended membrane was frozen at –80°C until use.
Membrane extract from UR757 (glnB glnK) without bound PII was prepared by incubating membranes for 1 h at 30°C in the presence of 10 mM 2-KG and 3 mM ATP, followed by two rounds of ultracentrifugation at 200,000 x g for 30 min at 4°C. All supernatant was removed, and the pellet was resuspended in an equal volume of 50 mM NaPO4 buffer, pH 6.8, after each centrifugation to remove 2-KG and ATP from the final membrane preparation. No GlnJ was present in the membrane extract following such treatment, as determined by Western blotting using anti-GlnJ antibodies.
PII disassociation and reassociation assays.
Both disassociation and reassociation reactions took place in reaction buffer with a final concentration of 50 mM NaPO4 buffer, pH 6.8, 100 mM NaCl, 25 mM MgCl2, 1 mM MnCl2, and 1 mM dithiothreitol, adapted from previous reports (9, 24, 54). ATP, ADP, and AMP-PNP [adenylyl 5'-(ß,
-imido)triphosphate tetralithium salt], at 95% or greater purity (Sigma-Aldrich, St. Louis, MO), and 2-KG were added to reactions. Membrane extract (250 µl), normalized to an OD581 of 0.14, was added to 250 µl of 2x reaction buffer, and the mixture was mixed and incubated for 1 h at 30°C. Afterwards, the reaction mixtures were immediately placed on ice and then transferred to ultracentrifugation tubes for separation of soluble proteins from membranes and insoluble proteins by ultracentrifugation as described above. Incubation of reaction mixtures on ice prevented further changes in association between GlnJ and AmtB1 (data not shown).
Immunoblotting of R. rubrum GlnB, GlnJ, and GlnK. Trichloroacetic acid precipitation was used to extract and concentrate protein from all protein samples (51). Proteins samples were loaded on low-cross-linker tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels as described previously (41, 53). Proteins were electrophoretically transferred to a nitrocellulose membrane and probed with polyclonal antibody against R. rubrum GlnJ or GlnB. Visualization was performed with either enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ) and Kodak film for protein detection or with SuperSignal West Dura extended-duration substrate (Pierce, Rockford, IL) for quantitation using a Storm 860 phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Software. Storm scanner control, version 5.03 (Amersham Biosciences), was used to operate the Storm 860 phosphorimager, and ImageQuant, version 5.0, build 050 (Molecular Dynamics), was used to quantitate bands from Western blots. Data were then exported to Excel 2000 (Microsoft Corporation, Redmond, WA) and graphed and analyzed in KaleidaGraph, version 4.02 (Synergy Software, Reading, PA). Global adjustments to contrast and brightness of Western blots were performed using Photoshop CS2 (Adobe Systems Incorporated, San Jose, CA).
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RESULTS |
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As expected, in Fig. 1 both an upper uridylylated GlnJ band and a lower nonuridylylated GlnJ band are visible in whole-cell extracts of the glnB glnK mutant UR757. Only nonuridylylated GlnJ is present in the membrane extract, while uridylylated GlnJ predominates in the cytoplasmic extract. This is in agreement with our earlier observation that nonuridylylated PII in wild-type extracts preferentially interacts with an AmtB1-containing membrane compared to uridylylated PII, as well as prior observations for E. coli by others (9, 53). The proportion of total PII in membrane extract versus cytoplasmic extract was also similar to the proportions we found in wild-type extracts. It is also consistent with recent structural model predictions that indicate that an uridylyl group on Tyr51 would sterically hinder interaction between PII and AmtB in E. coli (8, 17).
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ATP and 2-KG affect the stability of GlnJ interaction with the AmtB1-containing membrane. Proposed models of PII interaction with AmtB suggest that all three sites of uridylylation, the Tyr51 residue of each monomer in the PII trimer, would be inaccessible for modification by GlnD when GlnJ is bound to AmtB1, and recent crystal structures of PII-AmtB complexes support this hypothesis (2, 8, 17). Thus, although PII uridylylation could prevent association between PII and AmtB, the uridylylation process should not be able to cause complex disassociation. It seemed reasonable that small-molecule binding, not uridylylation, would be the primary regulator of interaction between these two proteins. This idea was supported by results obtained using purified E. coli GlnK-AmtB complexes, where 3.5 mM ATP and 1 mM 2-KG led to complex disassociation (11). However, these results were obtained with AmtB dissolved in detergent rather than in its native membrane environment. We therefore studied this issue in the physiologically relevant in vivo environment of purified membrane extracts containing AmtB1 and bound GlnJ; membrane extracts were prepared by sonication and therefore consisted primarily of inside-out vesicles exposing the GlnJ-AmtB1 complex to solvent (42).
As seen in Fig. 2, when purified membrane extract from an R. rubrum glnB glnK mutant was incubated in buffer lacking exogenous ATP or 2-KG, almost all GlnJ remained associated with the membrane. In the presence of 3 mM ATP and 10 mM 2-KG, all GlnJ was released from the membrane and was found solely in the supernatant. To clarify whether ATP or 2-KG affected the stability of the GlnJ-AmtB1 complex, either 3 mM ATP or 10 mM 2-KG was added separately. While 3 mM ATP alone had no effect, 10 mM 2-KG alone was able to efficiently remove GlnJ from the membrane. Increasing ATP levels up to 15 mM ATP had very little effect on the stability of GlnJ binding (data not shown). In contrast, in the absence of ATP lower levels of 2-KG (1 mM) caused only a small amount of GlnJ to disassociate (Fig. 2).
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The ratio of ATP to ADP affects the stability of the GlnJ-AmtB1 complex. Recent crystal structures show ADP bound to PII in the E. coli PII-AmtB protein complex (17). Although the absolute level of ATP is thought to be the most important indicator of cellular energy status sensed by PII, this result suggested that ADP levels could also be an important factor in controlling the interaction between GlnJ and AmtB1 in R. rubrum. Specifically, we hypothesized that ADP might be able to compete with ATP for binding sites on PII, thus lowering the effective concentration of available ATP. This would lead to an increase in the proportion of GlnJ bound to AmtB1 in our in vitro assay using an isolated R. rubrum membrane. If this were true, it would suggest that PII homologs may be able to sense the ratio of ATP to ADP as well as the absolute level of ATP in the cell as a measure of energy status.
We tested the stability of GlnJ binding to the AmtB1-containing membrane as in the previous section (Fig. 3A). In the presence of 500 µM 2-KG alone, there was little disassociation of GlnJ from the membrane compared to the 0 µM 2-KG control sample. As expected, a large fraction of GlnJ disassociated when 500 µM ATP was added to the reaction mixture in the presence of 500 µM 2-KG. We then increased ADP from 0 mM to 2.5 mM while keeping 2-KG and ATP levels constant and assayed the amount of GlnJ found at the membrane. There was no difference in levels of GlnJ association with the membrane when 250 µM ADP was added, but when 500 µM, 1 mM, or 2.5 mM ADP was added, an increasing amount of GlnJ was bound to the AmtB1-containing membrane. Finally, the presence of 2.5 mM ADP had no effect on GlnJ membrane association in the absence of ATP. This demonstrates that ADP is able to interfere with the ability of ATP to disrupt GlnJ-AmtB1 interaction in the presence of 2-KG, presumably by direct competition for the three primary nucleotide binding sites on the PII trimer.
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GlnB and GlnK bind the AmtB1-containing membrane less efficiently than does GlnJ in vitro. Although we had shown that GlnJ is capable of interacting with AmtB1 both in vivo and in vitro, it was unclear whether other PII homologs in R. rubrum could bind AmtB1 as strongly as GlnJ could. If the GlnJ-AmtB1 complex could be reconstituted in vitro, we would be able to test the ability of GlnB or GlnK to also complex with AmtB1 in vitro. We took advantage of our observation that addition of high levels of ATP and 2-KG caused complete release of GlnJ from isolated membranes to prepare AmtB1-containing membranes stripped of GlnJ. Both GlnJ and small molecules were subsequently washed away with repeated ultracentrifugation steps, producing AmtB1-containing membrane extract that we expected would be able to rebind GlnJ.
We first tested the ability of washed membranes to rebind GlnJ. Washed membranes treated with a twofold excess of purified GlnJ protein, compared to the original membrane extract, were able to bind approximately 60% of the GlnJ found in extract before treatment with ATP and 2-KG (data not shown). The decrease in GlnJ binding capacity of washed membranes was probably due to membrane loss during the washing and resuspension steps or imperfect resuspension of the membrane after ultracentrifugation, which might mask some AmtB1 inside membrane particles. However, the important observation remained that a membrane stripped of GlnJ could rebind GlnJ in our buffer conditions. This allowed us to test the ability of the AmtB1-containing membrane to individually bind each of the three PII homologs in R. rubrum.
Data shown in Fig. 4 are from representative experiments, all using the same preparation of membrane extract from wild-type cells; different preparations differed slightly in GlnJ binding capacity but showed the same trends. Different amounts (0.21 µg to 10 µg) of R. rubrum GlnJ were added to washed membranes, corresponding to a final PII concentration of about 11 to 540 nM. The maximal amount of added GlnJ, 10 µg, was 2.5-fold more than the amount of GlnJ found in the isolated membrane and about 4-fold more than the maximal amount of rebound GlnJ. The amounts of GlnJ recovered from soluble and membrane-bound fractions were quantitated by Western blotting. The amount of GlnJ associated with the membrane increased as more GlnJ was added to the membrane extract but approached saturation of the membrane's binding capacity at about 2.5 µg total GlnJ, a concentration of about 135 nM GlnJ (Fig. 4A).
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ATP and 2-KG act synergistically to promote release of GlnJ from the AmtB1-containing membrane. The strong, relatively stable interaction between GlnJ and AmtB1 in membrane extracts allowed us to further explore the effects of small molecules on the stability of this complex. Specifically, we examined a broad range of both ATP and 2-KG concentrations to estimate the disassociation constants for each small molecule in the presence or absence of the other. After treatment with small molecules, Western blotting quantitated the proportions of GlnJ found associated with the membrane and free in solution. These values were then normalized to the maximal proportion of GlnJ found at the membrane under any condition, as a small amount of GlnJ, about 5%, disassociated from the membrane during treatment under any condition.
We first examined the effects of 2-KG on the stability of the GlnJ-AmtB1 complex (Fig. 5A). In the absence of ATP, levels of 2-KG under 1 mM had no significant effect on complex stability. Increasing levels of 2-KG resulted in more release of GlnJ; however, even 15 mM 2-KG failed to cause complete disassociation of GlnJ. The Kd of GlnJ-AmtB1 for 2-KG in the absence of ATP was calculated to be 5,000 ± 430 µM by curve fitting a standard disassociation equation to the data with the software program KaleidaGraph. Next, we examined the effects of 2-KG in the presence of 3 mM ATP. This concentration of ATP was chosen as a physiologically relevant level that may be present under conditions of energy sufficiency (34). ATP dramatically lowered the levels of 2-KG required for destabilization of the GlnJ-AmtB1 complex. Furthermore, complete disassociation of GlnJ was seen only in the presence of ATP. The Kd of the GlnJ-AmtB1 complex for 2-KG in the presence of 3 mM ATP was approximately 340 ± 30 µM. This synergistic effect of 2-KG and ATP is also observed in the model of cooperative binding found for E. coli and Synechococcus sp. PII proteins (15, 26, 35). R. rubrum PII also demonstrates cooperative binding of ATP and 2-KG in in vitro modification experiments using R. rubrum GlnD and GlnB proteins (data not shown).
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Finally, we tested the effect of small molecules on the ability of the GlnJ-AmtB1 complex to reform in vitro. The results, shown in Fig. 5C, are almost identical to those in Fig. 5B. This was expected, as both reaction mixtures were incubated for an extended period of time and should have reached equilibrium between bound and unbound GlnJ in the samples. This is also a confirmation that the reconstituted PII-AmtB1 complexes behave similarly to those isolated from R. rubrum.
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DISCUSSION |
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Why would there be specificity in interaction between PII and AmtB proteins? A known difference between PII homologs in R. rubrum is that only GlnB is able to activate NifA, which activates transcription of genes involved in nitrogen fixation; GlnJ and GlnK are unable to activate NifA. In addition, while the glnJ amtB1 operon is expressed only under nitrogen-limiting conditions, glnB is expressed constitutively, although expression is also upregulated under nitrogen-limiting conditions (3, 54). We have found that, when R. rubrum experiences an increase in nitrogen levels in the cell, GlnJ may be the preferred PII homolog sequestered to the membrane. This would leave a relatively large proportion of GlnB free in the cytoplasm, which might then reactivate NifA upon subsequent decrease in nitrogen levels. Therefore, one possible reason for specificity between PII and AmtB is to regulate which functions the remaining free PII is capable of performing. Additionally, if the level of 2-KG required for uridylylation of GlnB by R. rubrum GlnD is less than that required for disassociation of GlnJ from AmtB1, then GlnJ would be modified after GlnB in vivo; this would again allow a specific uridylylated PII to function despite the presence of multiple PII proteins in the cell. However, the minimal level of 2-KG required for efficient modification of R. rubrum PII in vivo is not yet known (25).
In organisms such as E. coli, the two PII homologs GlnB and GlnK appear to be functionally interchangeable when expressed at equal levels (4). In such a case, PII binding to AmtB still has the effective consequence of transiently and reversibly sequestering the majority of cytoplasmic PII to the membrane. This would enable the cell to more rapidly recover from a transient increase in nitrogen status by quickly increasing the concentration of active PII in the cell (6). While GlnK is preferentially bound to AmtB at the membrane over GlnB, there is no clear requirement for this specificity of interaction between GlnK and AmtB in E. coli (9).
The small-molecule requirements we observed for release of GlnJ from AmtB1 in R. rubrum are similar to those observed for E. coli: both ATP and 2-KG are required for GlnJ release when only 1 mM of 2-KG is added to high levels of ATP (11). However, 2-KG alone led to complex disassociation at millimolar concentrations, and high ATP levels neither stabilized nor destabilized the complex, whereas ATP was required for complex stability in E. coli. Higher levels of 2-KG were not tested in E. coli, so it is unclear if 2-KG alone would have effects on complex stability in this organism (11). In contrast, ATP alone was sufficient to disrupt the PII-AmtB complex in Bacillus subtilis and the presence of 2-KG had almost no effect on complex stability (18). Low levels of ATP (100 µM) in the absence of 2-KG were also sufficient to disrupt a purified GlnK1-Amt1 complex from M. jannaschii (50).
Both allosteric effectors of PII, ATP and 2-KG, affected the stability of the GlnJ-AmtB1 complex in R. rubrum. Overall, the Kd values obtained for both ATP and 2-KG were significantly higher than values others have observed for binding of one ATP or one 2-KG molecule in E. coli GlnB (24). In vitro experiments with purified R. rubrum PII homologs and purified E. coli GlnD suggest that the Kd of R. rubrum PII for 2-KG is similar to that of E. coli (our unpublished results). The large increase in measured binding constants for 2-KG and ATP could be attributed to two possibilities: either multiple molecules of 2-KG and ATP must bind the GlnJ-AmtB1 complex for it to disassociate or the Kd of GlnJ for 2-KG is dramatically altered when GlnJ is bound to AmtB1. Our results favor the former model, as the calculated binding constants were similar whether GlnJ was already bound to the AmtB1-containing membrane or whether it was added to a membrane stripped of PII. However, while we predict that the binding of one 2-KG molecule has little effect on GlnJ-AmtB1 complex stability, it is still unclear whether only two or all three binding sites for 2-KG on GlnJ must be filled to allow release of GlnJ from AmtB1. It has been suggested that an intermediate state of 2-KG binding has physiological relevance in E. coli, where PII with three ATP molecules and one 2-KG molecule bound is likely to interact with NtrB (23, 26, 35). In addition, the ability of high concentrations of 2-KG and ATP to prevent interaction between PII and AmtB proteins is the simplest explanation for the recent observation for Rhodobacter capsulatus that fully unmodified GlnK fails to be sequestered in the membrane in a glnB mutant despite the presence of AmtB protein (47).
Our results indicate that competition between ADP and ATP for binding sites on PII can be a relevant factor in regulating interaction between PII and AmtB. Other proteins have also been shown to be regulated by the ratio of ATP to ADP such as SpoIIAB in Bacillus subtilis, where one of two potential protein targets is bound according to which nucleotide is in the majority (1). In our system, high levels of ADP are able to stabilize the GlnJ-AmtB1 complex in the presence of ATP and 2-KG, which would otherwise destabilize the complex. Sensing the ATP to ADP ratio allows yet another means to exert subtle control over the degree of response to energy depletion and nitrogen deficiency. ADP has also recently been found to influence the ability of E. coli PII to interact with receptors GlnE and NtrB and the signal transduction protein GlnD; in each case, ADP appears to signal energy limitation and limits the degree of response by PII to nitrogen limitation (P. Jiang and A. J. Ninfa, personal communication).
Although effects on the stability of the AmtB1-GlnJ complex of R. rubrum were seen only when the ratio of ADP to ATP was greater than 1 in our experiments, these data still support the idea of energy charge being an important regulatory input of PII protein function. Adenylate energy charge is determined by formula ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]) and has commonly been estimated to be near 0.85 during logarithmic growth in bacteria, implying there is much less ADP than ATP in these cells (7). However, many reports of energy charge give a ratio of ADP to ATP that is significantly higher. Specifically, in R. rubrum under conditions identical to those used during collection of cells for determining the localization of PII to the membrane, the estimated energy charge is only about 0.66 and the ratio of ADP to ATP is approximately 1 within the limits of experimental error (38). Also, adenylate energy charge has been measured at significantly lower levels in both log and stationary-phase growth conditions in multiple bacterial species, where the ratio of ADP to ATP is as much as 3 (5, 12, 31, 37). Given that nonlogarithmic growth conditions are more likely to be relevant in the wild, we predict that energy sensing by PII is an important function of this protein.
Uridylylation of GlnJ in R. rubrum provides an additional level of regulation of interaction between GlnJ and AmtB1, above and beyond small-molecule concentrations. Only nonuridylylated PII interacts with AmtB, and recent crystal structures show that modification of PII would sterically block interaction. Therefore, there are two effective concentrations of PII in the cell: total PII and that fraction of PII that is unmodified and therefore available for interaction with AmtB1. Even if the concentrations of small molecules in the cell are permissive of interaction between PII and AmtB, there necessarily is a delay while PII modifications are removed before interaction can take place.
In summary, we have demonstrated that AmtB1 of R. rubrum shows some specificity for binding the PII homolog GlnJ over the other two PII homologs present, GlnB and GlnK, and that only nonuridylylated GlnJ interacts strongly with AmtB1. The binding of ATP and 2-KG to GlnJ specifically disrupts the interaction between GlnJ and AmtB1, but ATP hydrolysis is not required for this process. The level of 2-KG required suggests that multiple 2-KG molecules may need to bind GlnJ for complex disassociation in R. rubrum. Finally, ADP stabilizes the GlnJ-AmtB1 complex in the presence of ATP and 2-KG, suggesting that the ratio of ADP to ATP may be sensed by PII as a measure of cellular energy status for at least some PII functions.
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ACKNOWLEDGMENTS |
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We thank Edward Pohlmann and Jonathan Jacobs for technical assistance and Jose Serate for purifying GlnJ and GlnK proteins. We also thank Alex Ninfa for helpful comments on the manuscript and permission to cite unpublished work.
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FOOTNOTES |
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Published ahead of print on 20 July 2007.
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-ketoglutarate and divalent cations, but not by glutamine. J. Bacteriol. 189:3471-3478.-ketoglutarate that regulate nitrogen metabolism. Curr. Opin. Microbiol. 8:168-173.[CrossRef][Medline]This article has been cited by other articles:
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