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Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Department of Bacteriology and the Center for the Study of Nitrogen Fixation, University of Wisconsin-Madison, Madison, WI 53706, USA

Correspondence
Gary P. Roberts
groberts{at}bact.wisc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The AmtB protein transports uncharged NH₃ into the cell, but it also interacts with the nitrogen regulatory protein P_II, which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P_II homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB₁ and amtB₂, have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB₁ are defective in their response to both addition and darkness, while mutants lacking AmtB₂ show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB₁ is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB₁-P_II regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P_II is proposed. Other results demonstrate a degree of specificity in interaction of AmtB₁ with the different P_II homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P_II proteins regulate processes involved in nitrogen acquisition and utilization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Nitrogen is a major nutrient for the living cell and its availability is often the growth-limiting factor. Therefore, all organisms have evolved highly effective but complicated regulatory systems for nitrogen acquisition and utilization. The first step in the acquisition of ammonium, the transport of ammonium through the membrane, is facilitated by the ammonium/methylammonium transport (Amt) proteins, which serve as transmembrane channels (Khademi et al., 2004; Soupene et al., 1998). The Amt protein family [also called methylammonium permease (Mep)] has been found in all three domains of life (Howitt & Udvardi, 2000; Marini et al., 1997; Saier et al., 1999). AmtB in enteric bacteria has been studied in the most detail (Blakey et al., 2002; Soupene et al., 2002a, 1998; Thomas et al., 2000b) and X-ray crystal structures of AmtB from Escherichia coli and Archaeoglobus fulgidus have been solved at a high resolution (Andrade et al., 2005; Khademi et al., 2004; Zheng et al., 2004). It appears that AmtB is a passive gas channel, rather than an active transporter (Javelle et al., 2005; Khademi et al., 2004; Soupene et al., 2001, 2002a, 1998, 2002b).

After NH₃ is transported into the cell, it is assimilated into glutamine by either the glutamine synthetase/glutamate synthetase (GS/GOGAT) pathway or the glutamate dehydrogenase (GDH) pathway. Glutamine synthetase (GS) activity is particularly critical and it is closely regulated transcriptionally and post-translationally, based on the nitrogen status of the cell (Arcondéguy et al., 2001; Stadtman, 2001). There are two sensors of nitrogen status in the enterics, reflecting the pools of two small molecules: nitrogen-rich glutamine and carbon-rich 2-oxoglutarate (-ketoglutarate, -KG). The primary nitrogen sensor is GlnD, a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR, the product of glnD), which uridylylates or deuridylylates the other central protein in nitrogen sensing, P_II (Ikeda et al., 1996; Jiang et al., 1998a, b). GlnD directly senses the level of glutamine in the cell, while P_II binds both -KG and ATP (Jiang et al., 1998b; Kamberov et al., 1995; Ninfa & Jiang, 2005). Under nitrogen-deficient conditions (low glutamine and high -KG) GlnD acts as uridylyltransferase to uridylylate P_II. P_II-UMP then binds to a set of appropriate receptor proteins and affects their activities. In nitrogen-excess conditions (high glutamine and low -KG), GlnD acts as a uridylyl-removing enzyme to deuridylylate P_II, which interacts with a different but overlapping set of receptor proteins to regulate them appropriately. In many bacteria the receptor proteins for P_II interaction include NtrB, which is part of a two-component regulatory system (Ninfa et al., 1995; Reitzer, 2003), and GlnE, which controls the reversible adenylylation of GS (Jiang et al., 1998a; Stadtman, 2001). In diazotrophs P_II homologues interact with either NifA or NifL, which directly or indirectly regulates nif expression (He et al., 1998; Jack et al., 1999; Little et al., 2002, 2000; Rudnick et al., 2002; Stips et al., 2004). Two P_II homologues, GlnB and GlnK, have been identified in E. coli and Klebsiella pneumoniae (Jack et al., 1999; van Heeswijk et al., 1996); however, the number of homologues varies according to the organism (Arcondéguy et al., 2001; Ninfa & Atkinson, 2000; Zhang et al., 2003).

AmtB has been shown to be another receptor for P_II interaction (Blauwkamp & Ninfa, 2003; Coutts et al., 2002; Javelle et al., 2004). In a variety of diverse bacteria, AmtB interacts directly with unmodified GlnK to form a membrane-bound complex under -excess conditions (Coutts et al., 2002; Detsch & Stülke, 2003; Javelle et al., 2004; Klopprogge et al., 2002; Strösser et al., 2004). This interaction is reversible and is thought to block the NH₃-transporting function of AmtB (Coutts et al., 2002; Javelle et al., 2004), and a docking model for the interaction of these two proteins from A. fulgidus has been proposed (Andrade et al., 2005). However, overexpression of AmtB affects other P_II targets in E. coli, such as NtrB (Blauwkamp & Ninfa, 2003), which strongly suggests that the AmtB-P_II interaction can affect P_II function as well. It has been proposed that the co-production of GlnK and AmtB could prevent AmtB from sequestering GlnB under nitrogen-deficient conditions, allowing GlnB to play its necessary roles in the cell (Blauwkamp & Ninfa, 2003). Mutations in amtB of Rhodobacter capsulatus and Azoarcus also show dramatic effects on the regulation of nitrogenase activity by , and it has been proposed that AmtB may be an ammonium sensor (Javelle et al., 2004; Martin & Reinhold-Hurek, 2002; Yakunin & Hallenbeck, 2002), but the effect might be indirect, since mutants lacking P_II also showed an altered response (Drepper et al., 2003; Martin & Reinhold-Hurek, 2002). Given the centrality of P_II to nitrogen regulation, the notion that AmtB might exert its effects through P_II interaction is certainly attractive. It is notable that the darkness response to regulate nitrogenase activity is normal in these amtB mutants of R. capsulatus (Yakunin & Hallenbeck, 2002).

The issue of P_II interacting with different receptor proteins raises another basic, but understudied question. If P_II functions by interacting with different receptor proteins, including AmtB, under different conditions, is there any competition for available P_II among these receptors, which must in turn affect P_II function? This competition would certainly be influenced by the level of each protein in the cell and by the relative affinity between each P_II protein and each receptor. While different interaction specificities of GlnB and GlnK in E. coli have been suggested (Atkinson & Ninfa, 1998, 1999; Atkinson et al., 2002; Blauwkamp & Ninfa, 2002), there are only two clear examples of P_II interaction specificity. In K. pneumoniae, GlnK but not GlnB interacts with NifL to relieve its inhibition of NifA activity (He et al., 1998; Jack et al., 1999). The substitution of residues 43 and 54 allows GlnB of K. pneumoniae to perform the ‘GlnK function’ of interacting with NifL when the altered GlnB is overexpressed (Arcondéguy et al., 2000). In Rhodospirillum rubrum, GlnB-UMP, but not the other two P_II homologues, interacts directly with NifA to activate it. Specific residues of GlnB that define this specific interaction have been analysed (Zhang et al., 2004).

R. rubrum has three P_II homologues, GlnB, GlnK and GlnJ, which play both distinct and overlapping functions by interacting with multiple target proteins in the cell (Zhang et al., 2005, 2001a). All of these homologues can properly regulate GlnE to regulate GS activity (Zhang et al., 2001a). However, GlnB is more effective in the regulation of NtrB activity than are the other P_II homologues (Zhang et al., 2005). R. rubrum strains lacking both glnB and glnJ grow very poorly on a variety of media, irrespective of the presence of a functional glnK (Zhang et al., 2001a).

Another role of P_II homologues in R. rubrum is to regulate the reversible mono-ADP ribosylation of dinitrogenase reductase to control nitrogenase activity (Zhang et al., 2003). Under -excess or energy-limitation (cells are shifted from light to dark) conditions, dinitrogenase reductase is ADP-ribosylated and thereby inactivated by DRAT (dinitrogenase reductase ADP-ribosyl transferase, the gene product of draT). However, when is exhausted or cells are returned to light, the ADP-ribose group can be removed by DRAG (dinitrogenase reductase activating glycohydrolase, the gene product of draG), thus restoring nitrogenase activity. The activities of DRAT and DRAG are themselves subject to post-translational regulation (Zhang et al., 1997). Either GlnB or GlnJ is sufficient for approximately normal function of this regulatory system, though we cannot assume that their roles are mechanistically similar (Zhang et al., 2001a).

In R. rubrum there are two amtB homologues: amtB₁ is linked to glnJ, while amtB₂ is linked to glnK (Zhang et al., 2001a). The goal of this work was to better understand the role of these AmtB homologues in R. rubrum and their possible interactions with P_II homologues.

	METHODS

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Bacterial strains, growth conditions and whole-cell nitrogenase activity assay.
The strains and plasmids used in this study are listed in Table 1. R. rubrum was grown in SMN (rich) medium, malate-glutamate (MG, minimal medium with glutamate as nitrogen source) medium or MN^– (nitrogen-free minimal) medium as described previously (Fitzmaurice et al., 1989; Lehman & Roberts, 1991; Zhang et al., 2001a). Antibiotics were used as necessary at the levels described previously (Zhang et al., 2000). Whole-cell nitrogenase activity assay and darkness/NH₄Cl treatments have also been described previously (Zhang et al., 1995).

Construction of amtB₁ and amtB₂ mutants.
A 5 kb EcoRI fragment of the glnJ-amtB₁ region from pUX268 (Zhang et al., 2001a) was subcloned into pUX19 (Lies, 1994), yielding pUX277. To construct amtB₁ mutants, aacC1 from pUCGM (Schweizer, 1993) was inserted into XcmI sites, resulting in the deletion of 1.3 kb of the 3' portion of amtB₁ and the adjoining region, yielding pUX283. pUX283 was conjugated into R. rubrum wild-type (UR2) and a glnB mutant (UR717) as described previously (Liang et al., 1991). Mutants resulting from a double-crossover recombination event were identified by drug phenotype and verified by PCR. Two amtB₁ mutants were designated UR794 (amtB₁1 : : aacC1) and UR795 (glnB3 amtB₁1 : : aacC1).

Similarly, a 3 kb BglII–HindIII fragment containing glnK amtB₂ was cloned into pSUP202, yielding pUX1664. The kan gene from pUC4K (Tanigawa et al., 2002) was inserted into a PstI site in amtB₂, yielding pUX1974. This plasmid was transferred into R. rubrum wild-type and an amtB₁ mutant and mutants created by double crossovers were identified: UR1635 (amtB₂1 : : kan) and UR1790 (amtB₁1 : : aacC1 amtB₂1 : : kan).

Construction of glnJ amtB₁ deletion mutants.
An 8 kb EcoRI fragment containing glnJ-amtB₁ was amplified by PCR and cloned into pBSKS(–) yielding pUX281. The Km^r gene from pUC4K (Vieira & Messing, 1982) was inserted into a BglII site, yielding pUX952, so that about 2.2 kb of glnJ-amtB₁ region was deleted and replaced with the kan gene. The mutated glnJ-amtB₁ region was subcloned into pSUP202 (Simon et al., 1983), yielding pUX954, where kan is transcribed in the same direction as glnJ. pUX954 was conjugated into R. rubrum wild-type (UR2), a glnB mutant (UR717) and a glnB glnK mutant (UR757) to create glnJ-amtB₁2 : : kan (UR1147), glnB3 glnJ-amtB₁2 : : kan (UR1149) and glnB3 glnK1 : : aacC1 glnJ-amtB₁2 : : kan (UR1240). Strains with the opposite orientation of kan were constructed, but behaved identically, so that only data from the first set of mutants is reported here.

Construction of glnJ amtB₁ draG and glnB glnJ amtB₁ draG mutants.
A 1.7 kb BamHI–HindIII fragment of R. rubrum draTG was cloned into pSUP202, yielding pUX343. aacC1 from pUCGM was inserted into draG (same orientation) at EcoRV sites, yielding pUX347. This plasmid was conjugated into R. rubrum wild-type (UR2), glnJ-amtB₁ (UR1147) and glnB glnJ-amtB₁ (UR1149) to create three draG mutants: draG11 : : aacC1 (UR832), glnJ-amtB₁2 : : kan draG11 : : aacC1 (UR1406) and glnB3 glnJ-amtB₁2 : : kan draG11 : : aacC1 (UR1410). As above, the orientation of aacC1 was not important and only results from the first set are presented.

To restore nitrogenase activity, pCK3 carrying K. pneumoniae nifA (Kennedy & Drummond, 1985) was transferred into R. rubrum glnB mutants by the tri-parental mating method described previously (Grunwald et al., 1995).

Construction of amtB₁ expression plasmids and strains.
A 6 kb EcoRI–HindIII fragment containing glnJ amtB₁ from pUX282 (which is similar to pUX281, but with different orientation of insert) was cloned into pRK404 (Ditta et al., 1985), yielding pUX1109 (Fig. 1). pUX1109 was digested with BamHI and religated, deleting a 231 bp fragment internal to glnJ, yielding pUX1139. This in-frame deletion of glnJ allows amtB₁ to be expressed from both the glnJ promoter and its own promoter. A 2.4 kb BglII fragment containing glnJ' amtB₁ from pUX282 was cloned into pRK404, yielding pUX1138. These plasmids were transformed into E. coli DH5 and then conjugated into R. rubrum glnB glnK (UR757) and glnJ glnK (UR810) mutants. Sm^r Gm^r (Km^r) Tc^r R. rubrum colonies were selected. New strains were designated UR1183, UR1184, UR1185, UR1186, UR1203 and UR1204.

View larger version (8K): [in this window] [in a new window]   Fig. 1. Physical map of plasmids for amtB1 expression. In pUX1109, amtB1 is expressed from both its own promoter and (with glnJ) from the glnJ promoter. In pUX1138, amtB1 is expressed only from its own promoter, since the glnJ promoter was deleted. In pUX1139, amtB1 is expressed from both its own promoter and that of glnJ, though glnJ itself has been deleted.

Immunoblotting of GlnJ and GS.
Trichloroacetic acid precipitation was used to extract and concentrate protein from whole-cell, cytoplasmic and membrane fractions (Zhang et al., 1993). A Tricine SDS-PAGE gel was used (Schägger & von Jagow, 1987), but with a low-cross-linker acrylamide/bisacrylamide mix (40 % T and 0.23 % C) in the resolving gel to increase separation of the modified and unmodified subunits of P_II proteins. Proteins were electrophoretically transferred to a nitrocellulose membrane, probed with polyclonal antibody against R. rubrum GlnJ and visualized with enhanced chemiluminescence (ECL) detection reagents (Amersham Biosciences).

GS immunoblotting was detected with horseradish peroxidase colour detection reagents (Bio-Rad) as described previously (Zhang et al., 1993, 2000).

	RESULTS

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The absence of AmtB₁ dramatically perturbs the post-translational regulation of nitrogenase activity in response to , but little effect is seen in a mutant lacking AmtB₂
Previous studies showed that both GlnB and GlnJ are involved in the regulation of the DRAT-DRAG system in response to darkness and stimuli (Zhang et al., 2001a). To test the hypothesis that the absence of AmtB₁ might affect P_II function and then perturb the regulation of DRAT and DRAG activities, we constructed amtB₁ and amtB₂ mutants and determined the roles of these AmtB homologues in the regulation of nitrogenase activity. MN^–-grown cultures were used to study the response, since they show a more drastic response than do MG-grown cultures (Zhang et al., 2001a). As shown in Table 2, UR2 (wild-type) had moderate nitrogenase activity in MN^– medium before the addition of , and this activity decreased 95 % after addition. The amtB₁ single mutant (UR794) showed slightly lower initial nitrogenase activity, but it lost only about 15 % of nitrogenase activity after addition. However, the amtB₂ single mutant (UR1635) showed rapid loss of nitrogenase activity after addition, similar to that seen in wild-type (UR2). The amtB₁ amtB₂ double mutant (UR1190) also showed an altered response, similar to that seen in the amtB₁ single mutant (UR794). These results indicate that the regulation of the DRAT-DRAG system is dramatically perturbed in mutants lacking AmtB₁. In contrast, AmtB₂ appears to play a minor role in the regulation of the DRAT-DRAG system in response to . An obvious possibility is that NH₃ transport is impaired in the amtB₁ mutant, preventing the signal to the DRAT-DRAG regulatory system. However, as shown below, the effect of AmtB₁ on DRAT-DRAG is more complicated, and it is more likely that AmtB₁ is sequestering P_II, rather than directly regulating DRAT-DRAG. The fact that the amtB₁ amtB₂ double mutants had substantial initial nitrogenase activity indicates that these AmtB homologues are not necessary for the uridylylation of GlnB, which is absolutely necessary for activating NifA for nif expression (Zhang et al., 2000).

View this table: [in this window] [in a new window]   Table 2. Nitrogenase activity and response in MN–-grown R. rubrum wild-type, amtB1, amtB2 and other mutants

response in amtB₁ mutants that also lack GlnB, GlnJ or both

As controls, strains lacking glnB, glnJ or both were also examined. As shown in Table 2 and reported previously (Zhang et al., 2001a), the glnJ mutation alone (UR806) does not show any significant effect on the DRAT-DRAG regulatory system. The glnB mutant with K. pneumoniae nifA (UR720) responded to , but with slightly higher residual activity, indicating that ADP-ribosylation of dinitrogenase reductase was altered slightly in this mutant. As seen previously, a poor response to was seen in the glnB glnJ double mutant (UR824) (Zhang et al., 2001a). All tested amtB₁ strains, including glnB amtB₁, glnJ amtB₁ and glnB glnJ amtB₁ mutants, completely failed to respond to addition (Table 2). Strains lacking amtB₁ and either glnB or glnJ appeared to be even more perturbed than strains lacking only amtB₁. All of these results show that the absence of AmtB₁ dramatically perturbs the regulation of the DRAT-DRAG system irrespective of the presence or absence of P_II homologues. This suggests that proper DRAT-DRAG regulation requires the presence of AmtB₁ and either GlnB or GlnJ, consistent with a functional interaction between AmtB₁ and the P_II homologues. The data also show that the glnB glnJ amtB₁ mutant (UR1153) is even more perturbed than is the glnB glnJ strain (UR824). We believe this reflects the availability of GlnK, as discussed below.

The absence of AmtB₁ also perturbs the post-translational regulation of nitrogenase activity in response to darkness
We also examined the impact of AmtB₁ on the response of the DRAT-DRAG system to energy depletion. Unlike the slow response to addition to MG medium (Zhang et al., 1995), wild-type (UR2) grown in MG medium showed a dramatic decline in nitrogenase activity upon removal of light and an equally dramatic recovery of activity upon its restoration (Table 3). The MG medium contains the poor nitrogen source glutamate, which supports higher nitrogenase activity than does the nitrogen-free medium used in Table 2. The amtB₁ mutants, with or without nifA of K. pneumoniae, had slightly lower initial nitrogenase activities, but only a partial response to the removal of light, followed by a complete recovery of activities after the return of light (UR794 and UR1115 in Table 3). The amtB₂ single mutant (UR1635) also showed a normal darkness response, with only slightly higher residual activity remaining under darkness conditions than that seen in wild-type (UR2), while the amtB₁ amtB₂ double mutant (UR1790) showed a partial response to darkness shifts, similar to that seen in the amtB₁ single mutant (UR794). This result indicates that AmtB₁, but not AmtB₂, is necessary for the proper response of DRAT-DRAG to energy depletion. This is important because such an energy response defect is very difficult to explain based on the known role of AmtB₁ in NH₃ transport. Because of our previous results implicating P_II proteins in this light/dark regulation of nitrogenase activity, it was an obvious possibility that the effect of the absence of AmtB₁ might be mediated through the P_II proteins.

These results have two implications. First, the interaction of AmtB₁ and GlnB/J is important not only to the response, but also to the darkness response. We note, however, that mutants lacking AmtB₁ and/or GlnB/J showed differences in the response to these different stimuli, indicating that darkness and might have different mechanisms for regulation of DRAT-DRAG. Second, the result shows that GlnB and GlnJ are not equivalent in a amtB₁ background, which might reflect a differential interaction between AmtB₁ and these two P_II homologues. These two issues will be discussed below.

Finally, we were curious to know if the absence of AmtB₁ affected DRAT, DRAG or both, because the perturbed regulation seen in these amtB₁ mutants might result from perturbation of the activation of DRAT or inactivation of DRAG. As mentioned in the Introduction, DRAT modifies dinitrogenase reductase by ADP-ribosylation under -excess or energy-limiting conditions, resulting in inactivation of that enzyme. DRAG restores dinitrogenase reductase activity by removing the ADP-ribose group. We therefore examined strains lacking draG, so that we could focus only on the activity of DRAT without the complication of the competing DRAG activity. In an otherwise wild-type background, a draG mutant has fairly normal initial nitrogenase activity because DRAT is very tightly regulated until nitrogen addition or energy depletion (UR832 in Table 3), which is consistent with a previous report of other draG mutants (Liang et al., 1991). However, all draG mutants lacking amtB₁, glnJ amtB₁ or glnB glnJ amtB₁ show low initial nitrogenase activities (UR1550, UR1406 and UR1436 in Table 3), indicating that some DRAT had already escaped normal down-regulation and was able to modify dinitrogenase reductase in these amtB₁ mutant backgrounds even before treatment. Therefore, the failure of the DRAT-DRAG system to turn off nitrogenase activity in response to or darkness in amtB₁ mutant backgrounds cannot be attributed to the failure to activate DRAT activity, but must instead be the result of the mutant's inability to inactivate DRAG under these conditions.

Effects of darkness do not seem to be mediated through glutamine pools
The effects of darkness on GS activity have been reported for different organisms (Johansson & Gest, 1977; Margués et al., 1992; Reyes & Florencio, 1995; Reyes et al., 1995), including R. rubrum where GS activity decreased when cells were shifted to dark and recovered on re-illumination (Nordlund et al., 1985). We tested the notion that changes in nitrogen regulation by a darkness shift might result from direct or indirect effects on pools of nitrogen metabolites, such as glutamine. However, this hypothesis seems not to be the case for the darkness effect in R. rubrum for several reasons. First, we examined the effect of both darkness and on the timing of DRAT activation. We reasoned that the response to should certainly be at least as rapid as to darkness if the latter treatment led indirectly to changes in the nitrogen status. We used a draG mutant (UR832) to avoid complications with DRAG. In fact, when UR832 was derepressed for nitrogenase in MG medium, the response to darkness was significantly faster than to , implying that darkness is not signalled through an indirect effect on nitrogen status (Fig. 2). Consistent with the efficacy of treatment, GS was rapidly modified in response to addition, but showed only a very slow response to darkness (Fig. 3).

View larger version (9K): [in this window] [in a new window]   Fig. 2. Regulation of nitrogenase activity by () and darkness () in an R. rubrum draG mutant (UR832). At time zero, NH4Cl was added to derepressed cultures of UR832 to a final concentration of 10 mM or cultures were shifted to darkness and then returned to light at 60 min. At the times indicated, 1 ml portions of cells were withdrawn and assayed for nitrogenase activity anaerobically under illumination conditions for 2 min. Initial nitrogenase activities (100 %) in UR832 wereabout 600 nmol ethylene produced h–1 (ml cells at OD600=1)–1. Each point represents a mean of at least three replicate assays.

View larger version (17K): [in this window] [in a new window]   Fig. 3. The modification of GS in an R. rubrum draG mutant (UR832) after (a) and darkness (b) treatments. Derepressed cultures of UR832 were shifted to darkness at time zero. At the times indicated, 1 ml portions of the same culture were rapidly extracted by the trichloroacetic acid precipitationmethod. Protein samples were loaded on low cross-linker SDS-PAGE gels and immunoblotted with antibody against R. rubrum GS. Arrow M indicates the position of the modified subunit and arrow U indicates the position of the unmodified subunit.

AmtB₁ is required to sequester unmodified GlnJ to the membrane in R. rubrum
A number of reports have been published demonstrating that AmtB homologues can sequester P_II protein to the cell membrane (Coutts et al., 2002; Detsch & Stülke, 2003; Javelle et al., 2004; Strösser et al., 2004). If the P_II proteins sequestered by AmtB₁ were altered in their ability to regulate receptor proteins, this would be an obvious mechanism for explaining the physiological roles of AmtB₁ in response to and darkness. There are, however, particular technical challenges in testing this with a photosynthetically grown organism: abundant intracytoplasmic membranes make separation of membrane and cytoplasm challenging, and the very act of harvesting the cells by centrifugation allows for a darkness response. We therefore grew wild-type (UR2) and amtB₁ (UR794) strains in MG medium to early exponential phase and then treated them with NH₄Cl or darkness shifts, or left them untreated. Cultures were chilled before harvesting, as described in Methods, to reduce the rate of response to darkness. The extracts were analysed by Western blotting developed with antibody to GlnJ. Whole-cell, cytoplasmic and membrane extracts were loaded such that both protein and P_II levels in the cytoplasmic and membrane lanes added up to the levels in the original whole-cell extract. This GlnJ antibody is significantly more responsive to GlnJ than to GlnB, and GlnJ is the most abundant P_II homologue in R. rubrum under these conditions (Zhang et al., 2005). We therefore interpret the results below in terms of GlnJ location, but cannot exclude the additional presence of lesser amounts of other P_II proteins.

As shown in Fig. 4, in the wild-type grown in the light under nitrogen-deficient conditions, GlnJ is mostly uridylylated and primarily found in the cytoplasm. There is some GlnJ associated with the membrane under these conditions, but this may reflect the difficulty of stopping the physiological response to darkness during cell harvest. It is notable that the majority of cytoplasmic GlnJ is uridylylated while membrane-associated GlnJ is deuridylylated, suggesting that unmodified GlnJ preferentially binds to AmtB₁, consistent with previous reports in E. coli (Coutts et al., 2002; Javelle et al., 2004). In response to darkness or the addition of 10 mM NH₄Cl, GlnJ is completely deuridylylated and the ratio of GlnJ found in the membrane to that found in the cytoplasm is reversed from the initial sample, consistent with a significant increase in membrane association. The samples from the amtB₁ strain are roughly similar to those of the wild-type in terms of the uridylylation state of GlnJ, but the striking difference is the apparently undetectable levels of GlnJ in the membrane fraction under any conditions tested. Thus, AmtB₁ does not appear to be necessary for the approximately normal regulation of GlnJ modification by GlnD under the tested conditions, but is necessary for GlnJ membrane association.

View larger version (32K): [in this window] [in a new window]   Fig. 4. AmtB1-dependent sequestration of GlnJ to the membrane in R. rubrum. Cells of wild-type (UR2) and an amtB1 mutant (UR794) were collected and separated into cytoplasmic and membrane fractions, as described in Methods, after 60 min of darkness treatment, 30 min after addition of NH4Cl to a final concentration of 10 mM, or with no treatment. Samples were resuspended in identical final volumes of loading buffer and equal volumes of whole-cell (WC), cytoplasmic (C) and membrane (M) samples were loaded on low cross-linker Tricine SDS-PAGE gels and immunoblotted with antibody against R. rubrum GlnJ. M indicates the position of modified GlnJ and U indicates the position of unmodified GlnJ.

To test this hypothesis more directly, we created expression systems that overexpressed AmtB₁ at two different levels and examined the effects with or without the simultaneous overexpression of GlnJ. We took advantage of our observation that there appear to be both NtrC- and RpoN-binding sites 5' of glnJ and amtB₁ (as well as glnB and a number of other genes). Based on the DNA sequence alignment in Fig. 5, we believe that amtB₁ can be expressed from both the glnJ promoter and its own, and the following results are also consistent with this hypothesis.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Sequence alignment of RpoN- and NtrC-binding sites from the promoter regions of R. rubrum nifH, nifB, glnB, glnJ and amtB1. Consensus bases, thought to be essential for these binding sites, are shown in bold. Highly conserved bases are inbold type, and less conserved or non-conserved bases are labelled as N. The number indicates the distance in nucleotides between the binding site and the start codon of translation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We do not know the basis of the darkness effect, but speculate that ATP or -KG might be the direct signal. It is recognized that -KG and ATP bind synergistically to P_II homologues of both the enterics and the cyanobacteria, though there are differences in the specific details (Forchhammer, 2004; Ninfa & Jiang, 2005). Because -KG is thought to have effects on P_II function in vivo, it is clear that ATP binding could at least have indirect effects through -KG. Given the clear role of ATP on P_II function in vitro (Jiang et al., 1998b; Kamberov et al., 1995), it is somewhat surprising that an in vivo role for ATP in the process has not been proposed. Indeed, there are a number of observations that might be explained by fluctuations in ATP pools. In the cyanobacteria there is a clear effect of redox state on P_II function (Hisbergues et al., 1999), which has been interpreted as an effect on the -KG pool, though without direct support (Forchhammer, 2004). It was also shown many years ago by the Magasanik group (Bender et al., 1977) that the method of breaking Klebsiella aerogenes cells affected the GS adenylylation state and again this has been assumed to reflect a direct perturbation of pools of nitrogen, though this was not demonstrated and perturbation of ATP pools is a possibility. The notion that ATP levels can fluctuate and have important biological effects has been clearly demonstrated in the regulation of rRNA expression in E. coli (Paul et al., 2004; Schneider et al., 2002). However, the adenylate and pyridine nucleotide pools have been analysed in R. rubrum in response to darkness and treatments (Nordlund & Höglund, 1986; Paul & Ludden, 1984) and only a slight transient decrease of the ATP pool was found after a shift to darkness (Paul & Ludden, 1984). Because of the obvious role of ATP in modulating P_II function in vitro, we favour the general model that energy deprivation (darkness in our system) creates an ATP-deficient form of P_II that behaves sufficiently like unmodified P_II, irrespective of the actual presence of UMP, at least in terms of regulation of nitrogenase activity. It is possible that the modest ATP effects previously detected in R. rubrum are sufficient to affect P_II function.

We still lack some critical information in sorting out exactly how GlnB and GlnJ effect DRAT and DRAG regulation, but the following model is consistent with the above argument as well as previous and recent data on the system (Huergo et al., 2005; Wang et al., 2005; Zhang et al., 2005, 2003, 2001b; Zhu et al., 2006). P_II binds DRAT and probably activates its activity under nitrogen-excess conditions. However, P_II-UMP binds and inhibits DRAT and also activates DRAG under nitrogen-deficient conditions. The addition of causes P_II to be deuridylylated, eliminating DRAT inhibition and inactivating DRAG. The inhibition of DRAG activity is actually the result of the sequestration of DRAG by membrane-associated AmtB₁ binding the P_II-DRAG complex.

This model is consistent with data from two recent papers on the membrane sequestration of DRAG in mutants lacking P_II or AmtB (Huergo et al., 2006; Wang et al., 2005). Wang et al. (2005) reported the different location of DRAG in R. rubrum mutants lacking P_II proteins. Substantial amounts of DRAG were found in the cytosolic fraction in glnB glnJ and glnB glnJ glnK mutants, but very little DRAG was seen in the cytosolic fraction in a glnB mutant and much less in a glnJ mutant. Similar to our results, they found that an R. rubrum amtB₁ mutant showed an altered response to treatment in regulating nitrogenase activity and the modification of dinitrogenase reductase. Interestingly, DRAG was found predominantly in the cytosol in this amtB₁ mutant (Wang et al., 2005). Similarly, Huergo et al. (2006) reported that, in Azospirillum brasilense, P_II proteins (GlnB and GlnZ) and DRAG were also found to be associated with the membrane after treatments, and this membrane-sequestration was AmtB-dependent. In an amtB mutant, the inability of DRAG to be associated with the membrane altered the regulation of its activity, resulting in a poor response to (Huergo et al., 2006). These results indicated that the inhibition of DRAG activity is actually the result of the sequestration of DRAG by membrane-associated AmtB₁, probably binding the P_II-DRAG complex. Thus the elimination of either GlnB/GlnJ or AmtB₁ abolishes the turn-off of nitrogenase activity in response to or darkness because DRAG cannot be properly regulated by sequestration in the absence of either P_II protein. Darkness might create an ATP-deficient form of P_II that is in the nitrogen-sufficient state. This model is also consistent with the previous data on the system: (i) DRAG has been detected in association with the membrane fraction of R. rubrum cells in which DRAG activity is downregulated (Norén & Nordlund, 1997); (ii) in A. brasilense, the reactivation of DRAG activity was altered in a mutant lacking GlnZ, a GlnK-like protein (Klassen et al., 2001); (iii) in R. capsulatus, DRAT interacts with both GlnB and GlnK in the yeast two-hybrid system (Pawlowski et al., 2003) and we have detected similar interactions between DRAT and all three P_II homologues of R. rubrum (Zhu et al., 2006). Thus, although we specifically propose this scheme for the R. rubrum system, it is consistent with results seen with Azoarcus, R. capsulatus and Azospirillum brasilense as well, in which the DRAT-DRAG regulation is altered in mutants lacking P_II or AmtB (Drepper et al., 2003; Klassen et al., 2001; Martin & Reinhold-Hurek, 2002; Yakunin & Hallenbeck, 2002). We note that rather different results have been seen in the case of R. capsulatus, where mutants lacking two AmtB homologues (AmtB and AmtY) affected the DRAT-DRAG response to , but not to darkness; the basis for this difference is unclear (Yakunin & Hallenbeck, 2002).

The results presented here also imply some degree of specificity between AmtB₁ and the different P_II species in R. rubrum. Such specificity has not been well documented previously. In E. coli, AmtB can sequester both GlnB and GlnK, although not identically (Coutts et al., 2002). GlnB and GlnK antagonize AmtB similarly (Blauwkamp & Ninfa, 2003). However, Javelle et al. (2004) suggested that GlnK and GlnB have quite distinct functions in E. coli, GlnK being concerned with AmtB regulation and GlnB with control of NtrB. The situation in R. rubrum is apparently different. In response to energy deprivation, for example, a glnB amtB₁ mutant is significantly more perturbed than a glnJ amtB₁ mutant. The overexpression experiments (Table 5) also show an apparent preference of AmtB₁ for GlnJ. In contrast, the differential growth rates of glnBJ, glnBJ amtB₁ and glnBJK amtB₁ mutants strongly imply a significant degree of affinity of AmtB₁ for GlnK as well. Such specificity in P_II interactions is clearly of great potential importance, since it affects the differential P_II effects on the various receptor proteins.

In summary, these results show that AmtB₁ is critical for the response of the DRAT-DRAG system to both addition and energy deprivation. The latter role is difficult to attribute to the role of AmtB₁ as either an NH₃ channel or an NH₃ sensor. Lastly, the results strongly imply selectivity in the interaction of AmtB₁ with specific P_II proteins.

	ACKNOWLEDGEMENTS

 
This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and NIGMS grant GM65891 to G. P. R. We thank Wally C. von Heeswijk for his advice about Tricine SDS-PAGE electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Andrade, S. L., Dickmanns, A., Ficner, R. & Einsle, O. (2005). Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus. Proc Natl Acad Sci U S A 102, 14994–14999.[Abstract/Free Full Text]

Arcondéguy, T., Lawson, D. & Merrick, M. (2000). Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression. J Biol Chem 275, 38452–38456.[Abstract/Free Full Text]

Arcondéguy, T., Jack, R. & Merrick, M. (2001). PII signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev 65, 80–105.[Abstract/Free Full Text]

Atkinson, M. R. & Ninfa, A. J. (1998). Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli. Mol Microbiol 29, 431–447.[CrossRef][Medline]

Atkinson, M. R. & Ninfa, A. J. (1999). Characterization of the GlnK protein of Escherichia coli. Mol Microbiol 32, 301–313.[CrossRef][Medline]

Atkinson, M. R., Blauwkamp, T. A. & Ninfa, A. J. (2002). Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli. J Bacteriol 184, 5364–5375.[Abstract/Free Full Text]

Bender, R. A., Janssen, K. A., Resnick, A. D., Blumenberg, M., Foor, F. & Magasanik, B. (1977). Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol 129, 1001–1009.[Abstract/Free Full Text]

Blakey, D., Leech, A., Thomas, G. H., Coutts, G., Findlay, K. & Merrick, M. (2002). Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry. Biochem J 364, 527–535.[CrossRef][Medline]

Blauwkamp, T. A. & Ninfa, A. J. (2002). Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation. Mol Microbiol 46, 203–214.[CrossRef][Medline]

Blauwkamp, T. A. & Ninfa, A. J. (2003). Antagonism of PII signalling by the AmtB protein of Escherichia coli. Mol Microbiol 48, 1017–1028.[CrossRef][Medline]

Coutts, G., Thomas, G., Blakey, D. & Merrick, M. (2002). Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB. EMBO J 21, 536–545.[CrossRef][Medline]

Detsch, C. & Stülke, J. (2003). Ammonium utilization in Bacillus subtilis: transport and regulatory functions of NrgA and NrgB. Microbiology 149, 3289–3297.[Abstract/Free Full Text]

Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.-W., Finlay, D. R., Guiney, D. & Helinski, D. R. (1985). Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13, 149–153.[CrossRef][Medline]

Drepper, T., Gross, S., Yakunin, A. F., Hallenbeck, P. C., Masepohl, B. & Klipp, W. (2003). Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiology 149, 2203–2212.[Abstract/Free Full Text]

Fitzmaurice, W. P., Saari, L. L., Lowery, R. G., Ludden, P. W. & Roberts, G. P. (1989). Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum. Mol Gen Genet 218, 340–347.[CrossRef][Medline]

Forchhammer, K. (2004). Global carbon/nitrogen control by P_II signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol Rev 28, 319–333.[CrossRef][Medline]

Grunwald, S. K., Lies, D. P., Roberts, G. P. & Ludden, P. W. (1995). Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase. J Bacteriol 177, 628–635.[Abstract/Free Full Text]

He, L., Soupene, E., Ninfa, A. & Kustu, S. (1998). Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions. J Bacteriol 180, 6661–6667.[Abstract/Free Full Text]

Hisbergues, M., Jeanjean, R., Joset, F., Tandeau de Marsac, N. & Bédu, S. (1999). Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803. FEBS Lett 463, 216–220.[CrossRef][Medline]

Howitt, S. M. & Udvardi, M. K. (2000). Structure, function and regulation of ammonium transporters in plants. Biochim Biophys Acta 1465, 152–170.[Medline]

Huergo, L. F., Souza, E. M., Steffens, M. B., Yates, M. G., Pedrosa, F. O. & Chubatsu, L. S. (2005). Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense. Arch Microbiol 183, 209–217.[CrossRef][Medline]

Huergo, L. F., Souza, E. M., Araujo, M. S., Pedrosa, F. O., Chubatsu, L. S., Steffens, M. B. R. & Merrick, M. (2006). ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG. Mol Microbiol 59, 326–337.[CrossRef][Medline]

Ikeda, T. P., Shauger, A. E. & Kustu, S. (1996). Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation. J Mol Biol 259, 589–607.[CrossRef][Medline]

Jack, R., De Zamaroczy, M. & Merrick, M. (1999). The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae. J Bacteriol 181, 1156–1162.[Abstract/Free Full Text]

Javelle, A., Severi, E., Thornton, J. & Merrick, M. (2004). Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation. J Biol Chem 279, 8530–8538.[Abstract/Free Full Text]

Javelle, A., Thomas, G., Marini, A. M., Kramer, R. & Merrick, M. (2005). In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase. Biochem J 390, 215–222.[CrossRef][Medline]

Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998a). The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 37, 12802–12810.[CrossRef][Medline]

Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998b). Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein. Biochemistry 37, 12782–12794.[CrossRef][Medline]

Johansson, B. C. & Gest, H. (1977). Adenylylation/deadenylylation control of the glutamine synthetase of Rhodopseudomonas capsulata. Eur J Biochem 81, 365–371.[Medline]

Kamberov, E. S., Atkinson, M. R. & Ninfa, A. J. (1995). The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J Biol Chem 270, 17797–17807.[Abstract/Free Full Text]

Kanemoto, R. H. & Ludden, P. W. (1984). Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe protein modification in Rhodospirillum rubrum. J Bacteriol 158, 713–720.[Abstract/Free Full Text]

Kanemoto, R. H. & Ludden, P. W. (1987). Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity. J Bacteriol 169, 3035–3043.[Abstract/Free Full Text]

Kennedy, C. & Drummond, M. H. (1985). The use of cloned nif regulatory elements from Klebsiella pneumoniae to examine nif regulation in Azotobacter vinelandii. J Gen Microbiol 131, 1787–1795.[Abstract/Free Full Text]

Khademi, S., O'Connell, J., 3rd, Remis, J., Robles-Colmenares, Y., Miercke, L. J. & Stroud, R. M. (2004). Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305, 1587–1594.[Abstract/Free Full Text]

Klassen, G., de Souza, E. M., Yates, M. G., Rigo, L. U., Inaba, J. & Pedrosa Fde, O. (2001). Control of nitrogenase reactivation by the GlnZ protein in Azospirillum brasilense. J Bacteriol 183, 6710–6713.[Abstract/Free Full Text]

Klopprogge, K., Grabbe, R., Hoppert, M. & Schmitz, R. A. (2002). Membrane association of Klebsiella pneumoniae NifL is affected by molecular oxygen and combined nitrogen. Arch Microbiol 177, 223–234.[CrossRef][Medline]

Lehman, L. J. & Roberts, G. P. (1991). Identification of an alternative nitrogenase system in Rhodospirillum rubrum. J Bacteriol 173, 5705–5711.[Abstract/Free Full Text]

Li, J. D., Hu, C. Z. & Yoch, D. C. (1987). Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by or darkness. J Bacteriol 169, 231–237.[Abstract/Free Full Text]

Liang, J. H., Nielsen, G. M., Lies, D. P., Burris, R. H., Roberts, G. P. & Ludden, P. W. (1991). Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation. J Bacteriol 173, 6903–6909.[Abstract/Free Full Text]

Lies, D. P. (1994). Genetic manipulation and the overexpression analysis of posttranslational nitrogen fixation regulation in Rhodospirillum rubrum. PhD thesis, University of Wisconsin-Madison, Madison, WI, USA.

Little, R., Reyes-Ramirez, F., Zhang, Y., van Heeswijk, W. C. & Dixon, R. (2000). Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein. EMBO J 19, 6041–6050.[CrossRef][Medline]

Little, R., Colombo, V., Leech, A. & Dixon, R. (2002). Direct interaction of the NifL regulatory protein with the GlnK signal transducer enables the Azotobacter vinelandii NifL-NifA regulatory system to respond to conditions replete for nitrogen. J Biol Chem 277, 15472–15481.[Abstract/Free Full Text]

Margués, S., Mérida, A., Candau, P. & Florencio, F. J. (1992). Light-mediated regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. PCC 6301. Planta 187, 247–253.

Marini, A. M., Urrestarazu, A., Beauwens, R. & Andre, B. (1997). The Rh (Rhesus) blood group polypeptides are related to transporters. Trends Biochem Sci 22, 460–461.[CrossRef][Medline]

Martin, D. E. & Reinhold-Hurek, B. (2002). Distinct roles of P_II-like signal transmitter proteins and amtB in regulation of nif gene expression, nitrogenase activity, and posttranslational modification of NifH in Azoarcus sp. strain BH72. J Bacteriol 184, 2251–2259.[Abstract/Free Full Text]

Meletzus, D., Rudnick, P., Doetsch, N., Green, A. & Kennedy, C. (1998). Characterization of the glnK-amtB operon of Azotobacter vinelandii. J Bacteriol 180, 3260–3264.[Abstract]

Michel-Reydellet, N., Desnoues, N., de Zamaroczy, M., Elmerich, C. & Kaminski, P. A. (1998). Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans. Mol Gen Genet 258, 671–677.[CrossRef][Medline]

Ninfa, A. J. & Atkinson, M. R. (2000). PII signal transduction proteins. Trends Microbiol 8, 172–179.[CrossRef][Medline]

Ninfa, A. J. & Jiang, P. (2005). PII signal transduction proteins: sensors of -ketoglutarate that regulate nitrogen metabolism. Curr Opin Microbiol 8, 168–173.[CrossRef][Medline]

Ninfa, A. J., Atkinson, M. R., Kamberov, E. S., Feng, J. & Ninfa, E. G. (1995). Control of nitrogen assimilation by the NR_I-NR_II two-component system of enteric bacteria. In Two-Component Signal Transduction, pp. 147–158. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC: American Society for Microbiology.

Nordlund, S. & Höglund, L. (1986). Studies of the adenylate and pyridine nucleotide pools during nitrogenase ‘switch-off’ in Rhodospirillum rubrum. Plant Soil 90, 203–209.[CrossRef]

Nordlund, S., Kanemoto, R. H., Murrell, S. A. & Ludden, P. W. (1985). Properties and regulation of glutamine synthetase from Rhodospirillum rubrum. J Bacteriol 161, 13–17.[Abstract/Free Full Text]

Norén, A. & Nordlund, S. (1997). Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP. J Bacteriol 179, 7872–7874.[Abstract/Free Full Text]

Paul, T. D. & Ludden, P. W. (1984). Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity. Biochem J 224, 961–969.[Medline]

Paul, B. J., Ross, W., Gaal, T. & Gourse, R. L. (2004). rRNA transcription in Escherichia coli. Annu Rev Genet 38, 749–770.[CrossRef][Medline]

Pawlowski, A., Riedel, K. U., Klipp, W., Dreiskemper, P., Grob, S., Bierhoff, H., Drepper, T. & Masepohl, B. (2003). Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus. J Bacteriol 185, 5240–5247.[Abstract/Free Full Text]

Reitzer, L. (2003). Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 57, 155–176.[CrossRef][Medline]

Reyes, J. C. & Florencio, F. J. (1995). Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 27, 789–799.[CrossRef][Medline]

Reyes, J. C., Crespo, J. L., Garcia-Dominguez, M. & Florencio, F. J. (1995). Electron transport controls glutamine synthetase activity in the facultative heterotrophic cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol 109, 899–905.[Abstract]

Rudnick, P., Kunz, C., Gunatilaka, M. K., Hines, E. R. & Kennedy, C. (2002). Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii. J Bacteriol 184, 812–820.[Abstract/Free Full Text]

Saier, M. H., Jr, Eng, B. H., Fard, S. & 15 other authors (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta 1422, 1–56.[Medline]

Schägger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379.[CrossRef][Medline]

Schneider, D. A., Gaal, T. & Gourse, R. L. (2002). NTP-sensing by rRNA promoters in Escherichia coli is direct. Proc Natl Acad Sci U S A 99, 8602–8607.[Abstract/Free Full Text]

Schweizer, H. P. (1993). Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831–834.[Medline]

Simon, R., Priefer, U. B. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: tranposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784–791.[CrossRef]

Soupene, E., He, L., Yan, D. & Kustu, S. (1998). Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein. Proc Natl Acad Sci U S A 95, 7030–7034.[Abstract/Free Full Text]

Soupene, E., Ramirez, R. M. & Kustu, S. (2001). Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH₃ across the cytoplasmic membrane. Mol Cell Biol 21, 5733–5741.[Abstract/Free Full Text]

Soupene, E., Lee, H. & Kustu, S. (2002a). Ammonium/methylammonium transport (Amt) proteins facilitate diffusion of NH₃ bidirectionally. Proc Natl Acad Sci U S A 99, 3926–3931.[Abstract/Free Full Text]

Soupene, E., Chu, T., Corbin, R. W., Hunt, D. F. & Kustu, S. (2002b). Gas channels for NH₃: proteins from hyperthermophiles complement an Escherichia coli mutant. J Bacteriol 184, 3396–3400.[Abstract/Free Full Text]

Stadtman, E. R. (2001). The story of glutamine synthetase regulation. J Biol Chem 276, 44357–44364.[Free Full Text]

Stips, J., Thummer, R., Neumann, M. & Schmitz, R. A. (2004). GlnK effects complex formation between NifA and NifL in Klebsiella pneumoniae. Eur J Biochem 271, 3379–3388.[Medline]

Strösser, J., Lüdke, A., Schaffer, S., Krämer, R. & Burkovski, A. (2004). Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum. Mol Microbiol 54, 132–147.[CrossRef][Medline]

Sweet, W. J. & Burris, R. H. (1981). Inhibition of nitrogenase activity by in Rhodospirillum rubrum. J Bacteriol 145, 824–831.[Abstract/Free Full Text]

Tanigawa, R., Shirokane, M., Maeda Si, S., Omata, T., Tanaka, K. & Takahashi, H. (2002). Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro. Proc Natl Acad Sci U S A 99, 4251–4255.[Abstract/Free Full Text]

Thomas, G., Coutts, G. & Merrick, M. (2000a). The glnKamtB operon. A conserved gene pair in prokaryotes. Trends Genet 16, 11–14.[Medline]

Thomas, G. H., Mullins, J. G. & Merrick, M. (2000b). Membrane topology of the Mep/Amt family of ammonium transporters. Mol Microbiol 37, 331–344.[CrossRef][Medline]

Van Dommelen, A., Keijers, V., Vanderleyden, J. & de Zamaroczy, M. (1998). (Methyl)ammonium transport in the nitrogen-fixing bacterium Azospirillum brasilense. J Bacteriol 180, 2652–2659.[Abstract/Free Full Text]

van Heeswijk, W. C., Hoving, S., Molenaar, D., Stegeman, B., Kahn, D. & Westerhoff, H. V. (1996). An alternative P_II protein in the regulation of glutamine synthetase in Escherichia coli. Mol Microbiol 21, 133–146.[CrossRef][Medline]

Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.[CrossRef][Medline]

Wang, H., Franke, C. C., Nordlund, S. & Norén, A. (2005). Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1. FEMS Microbiol Lett 253, 273–279.[CrossRef][Medline]

Yakunin, A. F. & Hallenbeck, P. C. (2002). AmtB is necessary for -induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus. J Bacteriol 184, 4081–4088.[Abstract/Free Full Text]

Yoch, D. C. & Gotto, J. W. (1982). Effect of light intensity and inhibitors of nitrogen assimilation on inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp. J Bacteriol 151, 800–806.[Abstract/Free Full Text]

Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. (1993). Posttranslational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense. J Bacteriol 175, 6781–6788.[Abstract/Free Full Text]

Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. (1995). Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense. J Bacteriol 177, 2354–2359.[Abstract/Free Full Text]

Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. (1997). Regulation of nitrogen fixation in Azospirillum brasilense. FEMS Microbiol Lett 152, 195–204.[CrossRef][Medline]

Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. (2000). Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 182, 983–992.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. (2001a). Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J Bacteriol 183, 6159–6168.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L., Halbleib, C. M., Ludden, P. W. & Roberts, G. P. (2001b). Effect of P_II and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae. J Bacteriol 183, 1610–1620.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. (2003). Regulation of nitrogen fixation by multiple P_II homologs in the photosynthetic bacterium Rhodospirillum rubrum. Symbiosis 35, 85–100.

Zhang, Y., Pohlmann, E. L. & Roberts, G. P. (2004). Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum. Proc Natl Acad Sci U S A 101, 2782–2787.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L. & Roberts, G. P. (2005). GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum. J Bacteriol 187, 1254–1265.[Abstract/Free Full Text]

Zheng, L., Kostrewa, D., Bernèche, S., Winkler, F. K. & Li, X.-D. (2004). The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli. Proc Natl Acad Sci U S A 101, 17090–17095.[Abstract/Free Full Text]

Zhu, Y., Conrad, M. C., Zhang, Y. & Roberts, G. P. (2006). Identification of Rhodospirillum rubrum GlnB variants that are altered in their ability to interact with different targets in response to nitrogen-status signals. J Bacteriol 188, 1866–1874.[Abstract/Free Full Text]

Received 7 February 2006; revised 10 March 2006; accepted 14 March 2006.

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