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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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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-PII regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by PII is proposed. Other results demonstrate a degree of specificity in interaction of AmtB1 with the different PII homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which PII proteins regulate processes involved in nitrogen acquisition and utilization.
-KG, 2-oxoglutarate; MSX, methionine sulfoximine; Gm, gentamycin; Km, kanamycin; Sm, streptomycin; Tc, tetracycline |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; 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, PII (Ikeda et al., 1996; Jiang et al., 1998a, b). GlnD directly senses the level of glutamine in the cell, while PII 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 PII. PII-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 PII, which interacts with a different but overlapping set of receptor proteins to regulate them appropriately. In many bacteria the receptor proteins for PII 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 PII 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 PII 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 PII 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 PII targets in E. coli, such as NtrB (Blauwkamp & Ninfa, 2003), which strongly suggests that the AmtB-PII interaction can affect PII 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 PII also showed an altered 
response (Drepper et al., 2003; Martin & Reinhold-Hurek, 2002). Given the centrality of PII to nitrogen regulation, the notion that AmtB might exert its effects through PII 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 PII interacting with different receptor proteins raises another basic, but understudied question. If PII functions by interacting with different receptor proteins, including AmtB, under different conditions, is there any competition for available PII among these receptors, which must in turn affect PII function? This competition would certainly be influenced by the level of each protein in the cell and by the relative affinity between each PII 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 PII 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 PII 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 PII 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 PII 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 PII 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 PII homologues.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. 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).
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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 Kmr 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. Smr Gmr (Kmr) Tcr R. rubrum colonies were selected. New strains were designated UR1183, UR1184, UR1185, UR1186, UR1203 and UR1204.
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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 PII 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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, but little effect is seen in a mutant lacking AmtB2
stimuli (Zhang et al., 2001a). To test the hypothesis that the absence of AmtB1 might affect PII 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 PII, 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).
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response in amtB1 mutants that also lack GlnB, GlnJ or both), as done previously (Zhang et al., 2000). The K. pneumoniae NifA does not itself perturb DRAT-DRAG regulation, as shown by a comparison of strains UR794 and UR1115 (Table 2
) and other previous data (Zhang et al., 2000).
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 PII 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 PII 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 PII 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 PII proteins.
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amtB1 background on the darkness response. As controls, strains lacking glnB, glnJ or both were also examined. As shown in Table 3 and reported previously (Zhang et al., 2001a), the glnB or glnJ mutations alone do not show any significant effect on the darkness response (UR720 and UR806), although the darkness response was abolished in the glnB glnJ double mutant (UR824). In a amtB1 strain the presence or absence of glnJ was largely irrelevant (UR794 and UR1147 in Table 3), but the absence of glnB completely eliminated the response to darkness in the amtB1 background (UR1117 and UR1153 in Table 3).
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 PII 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).
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or dark treatments in R. rubrum. Previous reports showed that MSX completely blocked the effect of 
on nitrogenase activity, while the effect of MSX on the darkness response has been controversial in R. rubrum (Kanemoto & Ludden, 1984; Li et al., 1987; Sweet & Burris, 1981; Yoch & Gotto, 1982). We again used a draG mutant (UR832) to obtain more interpretable results. After pre-treatment with MSX, UR832 (draG) showed a diminished rate of loss of nitrogenase activity after addition of 
, with about 60 % activity remaining after 60 min of treatment, similar to that seen in cells treated with MSX alone (data not shown). In contrast, after pre-treatment with MSX, UR832 still showed a rapid loss of nitrogenase activity in response to darkness shifts. Similarly, MSX treatment had no significant effect on the darkness response in wild-type (data not shown). These results indicate that glutamine is not the signal for the darkness response. Consistent with this, a rapid and drastic increase in the glutamine pool has been seen after addition of 
, but only a very small change was seen after the darkness shift in R. rubrum (Kanemoto & Ludden, 1987; Li et al., 1987). These data suggest that the modification of GS and the regulation of nitrogenase activity are two independent events, and both are regulated by PII in response to 
and darkness stimuli.
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 PII protein to the cell membrane (Coutts et al., 2002; Detsch & Stülke, 2003; Javelle et al., 2004; Strösser et al., 2004). If the PII 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 PII 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 PII 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 PII 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.
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). The additional elimination of the third PII homologue, glnK, has little effect, which we assumed was because its level of expression is very low under all conditions examined. However, in the course of constructing some of the mutants described above, we found that the elimination of amtB1 allows normal growth in a glnB glnJ background, but not in a glnB glnJ glnK background. To further examine this, we measured the growth rates of a set of different mutants. UR1149 (glnB glnJ-amtB1) had a wild-type growth rate, while UR1240 (glnB glnK glnJ-amtB1) grew slowly in SMN, similar to the growth of glnBJ (UR808) and glnBJK (UR812) mutants (Table 4). This result has two implications. First, it shows that GlnK apparently has the biochemical properties necessary to support normal growth. As we will show elsewhere, these properties probably reflect the ability of GlnK to properly regulate NtrB and GlnE to modulate GS activity levels to a satisfactory degree (Y. Zhang & others, unpublished results). Second, the result implies that the presence of AmtB1 interferes with that ability. We attempted to test this hypothesis directly, but we were unable to reliably detect GlnK in our analyses (data not shown). GlnK is the least abundant PII protein in R. rubrum (Zhang et al., 2005), and glnK : : lacZ expression studies showed that glnK is expressed at background levels in otherwise wild-type backgrounds (D. M. Wolfe & others, unpublished data).
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). The simplest interpretation is that both GlnB and GlnJ are involved in the darkness response, but that AmtB1 is only important for GlnJ function. In this case, the absence of glnJ in a amtB1 background has little effect because GlnJ is already rendered non-functional by the absence of AmtB1. In contrast, GlnB apparently has a substantial effect even in the absence of AmtB1, since its removal from a amtB1 strain has a striking effect. This suggests a greater specificity of interaction of AmtB1 with GlnJ than with other PII proteins.
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.
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). When glnJ and amtB1 were both overexpressed from their normal promoters on a multi-copy plasmid (pUX1109) in a background with a wild-type copy of glnJ (but lacking both glnB and glnK), growth appeared normal (UR1183 in Table 5). However, when only amtB1 was overexpressed from its normal promoter on a multi-copy plasmid (pUX1138), it caused slow growth in UR1203. This suggests that the excess AmtB1 is sequestering the normal levels of GlnJ and creating a cell that is phenotypically GlnB– GlnJ–, which grows poorly as we have already shown (Zhang et al., 2001a). We then used an in-frame deletion within glnJ to create a plasmid that expressed amtB1 from both the glnJ and normal amtB1 promoters (pUX1139). This is expected to provide even higher levels of AmtB1, and indeed caused an even more severe growth defect in the glnBK mutant background (UR1184). The poorly growing colonies produced fast-growing suppressor-containing strains, as we have seen with glnB glnJ mutants (Zhang et al., 2001a). In contrast, the same expression plasmids in a strain with normal levels of GlnB, but lacking GlnJ and GlnK, resulted in perturbed growth only at the highest levels of AmtB1 expression, and then only slightly (UR1186). The same background strains overexpressing glnJ-amtB1 (UR1185) or amtB1 from its normal promoter (UR1204) showed wild-type growth. We interpret this to mean that AmtB1 is much more effective at sequestering GlnJ than GlnB, since the presence of either is sufficient to promote good growth. Because we lack pure AmtB1 protein and antibody against it, we are unable to quantify the exact level of AmtB1 and PII in these different strains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Coutts et al., 2002; Javelle et al., 2004). While this is reasonable, it does not appear to explain the results reported here. Indeed, if regulation of AmtB activity is so important, it is odd that elimination of AmtB has such a modest effect on growth in most organisms under most tested conditions. The exception is the reduced growth at low NH3 concentration seen in several organisms lacking amtB homologues (Soupene et al., 1998; Van Dommelen et al., 1998). The other mutant phenotype reported for amtB mutants is the failure to transport methylammonium (Meletzus et al., 1998; Michel-Reydellet et al., 1998; Soupene et al., 1998; Van Dommelen et al., 1998; Yakunin & Hallenbeck, 2002). Rather, it appears that the model of a direct interaction between AmtB and PII to affect PII activities (Blauwkamp & Ninfa, 2003) is more consistent with our data. We therefore favour the notion that interaction between AmtB1 and PII is a regulated phenomenon with the effect of removing PII from the cytoplasm and decreasing its effective concentration for some targets. Because PII apparently acts by protein–protein interactions with various target proteins, a decrease in PII concentration would potentially affect all of these interactions. Indeed, the model is easiest to understand in the enteric bacteria where the gene for the ‘extra’ PII (termed GlnK, but analogous to GlnJ and GlnK of R. rubrum) is only occasionally expressed and is also closely linked to amtB. In this case, growth conditions that demand higher levels of PII lead to GlnK synthesis, but that decision might be rapidly reversed by sequestration of GlnK by AmtB, restoring PII levels to those seen prior to glnK expression. This close linkage between amtB and the gene for the extra PII protein is found in most prokaryotic genomes, suggesting that interactions between the protein products of the two genes might be a common theme (Thomas et al., 2000a). The co-transcription of a glnK-amtB operon could prevent titration of GlnB when production of AmtB is elevated under nitrogen-deficient conditions (Blauwkamp & Ninfa, 2003).
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 PII 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 PII function in vivo, it is clear that ATP binding could at least have indirect effects through -KG. Given the clear role of ATP on PII 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 PII 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 PII function in vitro, we favour the general model that energy deprivation (darkness in our system) creates an ATP-deficient form of PII that behaves sufficiently like unmodified PII, 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 PII 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). PII binds DRAT and probably activates its activity under nitrogen-excess conditions. However, PII-UMP binds and inhibits DRAT and also activates DRAG under nitrogen-deficient conditions. The addition of 
causes PII 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 PII-DRAG complex.
This model is consistent with data from two recent papers on the membrane sequestration of DRAG in mutants lacking PII 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 PII 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, PII 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 PII-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 PII protein. Darkness might create an ATP-deficient form of PII 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 PII 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 PII 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 PII 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 PII interactions is clearly of great potential importance, since it affects the differential PII 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 PII proteins.
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ACKNOWLEDGEMENTS |
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Received 7 February 2006;
revised 10 March 2006;
accepted 14 March 2006.
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response in MN–-grown R. rubrum wild-type, amtB1, amtB2 and other mutants
(
) 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.
(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.
