JOURNAL OF BACTERIOLOGY, June 2010, p. 2711–2721 Vol. 192, No. 110021-9193/10/$12.00 doi:10.1128/JB.01674-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Mutagenesis and Functional Characterization of the Four Domains ofGlnD, a Bifunctional Nitrogen Sensor Protein�

Yaoping Zhang, Edward L. Pohlmann, Jose Serate, Mary C. Conrad,‡ and Gary P. Roberts*Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 22 December 2009/Accepted 25 March 2010

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be theprimary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays animportant role in nitrogen assimilation and metabolism by reversibly regulating the modification of PIIprotein; PII in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: anN-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidineand aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulatedenzymes in which this domain is found. Here we report the functional analysis of these domains of GlnDfrom Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NTdomain and show that the UR activity is a property specifically of the HD domain: substitutions in thisdomain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. Thedeletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability ofglutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. Thedeletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditionstested, but some of these effects are due to the competition of UTase activity with unregulated UR activityin these variants.

GlnD is a bifunctional uridylyltransferase/uridylyl-removingenzyme (UTase/UR; gene product of glnD) that regulates PII

proteins by uridylylation or deuridylylation. In enteric bacteria,GlnD is believed to be a primary sensor of intracellular nitro-gen status, determined by the level of glutamine in the cell (29,34–36). PII is one of the most broadly distributed regulatoryproteins in nature and directly or indirectly senses nitrogen andcarbon signals in the cell. Multiple PII homologs, mainlytermed GlnB and GlnK, have been found in many bacteria,and they play overlapping but distinct roles in the cell (7, 46,54, 79).

In Escherichia coli, PII proteins can interact with a variety ofreceptor proteins, including NtrB, a sensor protein of the two-component NtrB/NtrC regulatory system. NtrB acts as a histi-dine kinase that phosphorylates NtrC under nitrogen-limitingconditions and can also act as a phosphatase to dephosphory-late NtrC under conditions of nitrogen excess (55). Undernitrogen excess conditions, PII proteins are deuridylylated byGlnD so that they can interact with NtrB to stimulate itsphosphatase activity, resulting in the dephosphorylation ofNtrC. However, under nitrogen-limiting conditions, PII pro-teins become uridylylated, and this uridylylation prevents theirinteraction with NtrB, so that NtrB is dominated by its kinaseactivity to phosphorylate NtrC (33). The phosphorylated formof NtrC acts as a transcriptional activator of glnK amtB, glnA,and other operons involved in nitrogen assimilation. PII, to-

gether with adenylyltransferase (ATase, encoded by glnE), alsocontrols glutamine synthetase activity by reversible adenylyla-tion (1, 32). AmtB, a gas channel for NH3, is another receptorfor PII (11, 16, 31).

In the photosynthetic bacterium Rhodospirillum rubrum,three PII homologs, named GlnB, GlnK, and GlnJ, have beenidentified (37, 77). Although the amino acid sequences of thesethree homologs are very similar, they show both distinct andoverlapping functions in the cell. They are involved in theregulation of NifA and NtrB activities, the covalent modifica-tion of glutamine synthetase, and modulation of the posttrans-lational regulation of nitrogenase (77). In R. rubrum, NifAactivity is tightly controlled through the direct interaction be-tween NifA and the uridylylated form of GlnB in response toNH4

� (78).Although GlnD is central to the global nitrogen regulatory

(Ntr) system, it has not been as well studied as NtrB/NtrC andPII. In 1971, Brown et al. first reported the UTase activity ofpartially purified GlnD by showing the conversion of PII toPII-UMP in the presence of ATP, 2-oxoglutarate, or �-keto-glutarate (�-KG) and UTP, and this activity was inhibited byglutamine (14). UR activity was reported by Mangum et al. in1973 (49), and this was followed by several other studies (1, 20,22, 24). It was found that only Mn2� supports UR activity,while both Mg2� and Mn2� support UTase activity (1, 49).ATP and �-KG are necessary for UTase activity but not forUR activity (14, 20, 49). Glutamine stimulates UR activitywhile inhibiting UTase activity (20). The most extensive enzy-mological characterization of E. coli GlnD was done by Jianget al. in 1998 and the detailed regulation of both UTase andUR activities by small effectors, such as ATP, �-KG, and glu-tamine, as well as metal ions, was investigated (34). It appearsthat the uridylylation state of PII is largely dependent on the

* Corresponding author. Mailing address: Department of Bacteriol-ogy, University of Wisconsin—Madison, Madison, WI 53706. Phone:(608) 262-3567. Fax: (608) 262-9865. E-mail: groberts@bact.wisc.edu.

‡ Present address: Cubist Pharmaceuticals, Inc., Lexington, MA02421.

� Published ahead of print on 2 April 2010.

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level of glutamine in the cell (34). Recently, in vitro studiesof GlnD activities in other bacteria, such as Azospirillumbrasilense, Herbaspirillum seropedicae, and R. rubrum, were alsoreported (2, 3, 10, 13, 38).

GlnD has at least 4 domains, based on a Pfam search (http://pfam.sanger.ac.uk) (9), as well as on previous domain analy-ses (4, 5, 15, 27). There is an N-terminal nucleotidyltransferase(NT) domain, a central HD domain, named after the con-served histidine and aspartate residues (6), and two C-terminalACT domains, named after three of the allosterically regulatedenzymes in which this domain is found: aspartokinase, choris-mate mutase, and TyrA (prephenate dehydrogenase) (5).

It is clear that UTase activity is localized to the N-terminalNT domain. This domain has a distinct amino acid residuepattern with conserved glycine (G) and aspartate (D) residues(Fig. 1) (4, 27). Enzymes with this conserved domain belong tothe eukaryotic DNA polymerase � nucleotidyltransferase su-perfamily (Pol� superfamily), and they display nucleotidyl-transferase activity (4). The structures of this domain havebeen solved for several family members and are very similar toeach other (17, 18, 27, 50, 58, 59, 65, 73). These structures alsoshow that the two conserved aspartate residues (aspartate andglutamate residues in some members) are involved in the di-rect or indirect binding of metal ions, which are essential forsubstrate catalysis (18, 62, 65). In kanamycin nucleotidyltrans-ferase (Kan-NT) and DNA polymerase �, conserved glycineand serine residues are located in the helical-turn motif, con-necting the first �-sheet and a short helix. The serine residuesin some family members contact the phosphate of nucleotides(50, 59, 65).

Another member of this family is GlnE, which is also abifunctional enzyme and controls glutamine synthetase activityby adenylylation and deadenylylation. It is interesting thatGlnE has two NT domains (Fig. 1), one at the C terminus thatis responsible for an adenylyltransferase activity, and anotherat the N terminus that represents an adenylyl-removing (AR)activity (30). In contrast, GlnD has a single NT domain at theN terminus (Fig. 1). This raised a question about the locationof the UR active site in GlnD: does it share the active site withUTase, or is it localized in a different portion of the protein?Based on kinetic analysis and mutational studies, Jiang et al.

proposed that UTase and UR reactions likely occur at a singleactive center on GlnD (34, 56).

In contrast to the wealth of information about NT domains,the roles of the HD domain in the center of GlnD and of thetwo ACT domains at the C terminus are poorly understood.HD domains have been found in a superfamily of metal-de-pendent phosphohydrolases (6). A number of HD domainproteins have been characterized biochemically (12, 25, 26, 43,52, 53, 60, 61, 63, 69, 74). The structures of this domain inseveral family members have been solved and show that theconserved HD residues chelate metal ions and constitute thecatalytic center (26, 28, 44, 82). Based on sequence compari-sons, it had been suggested that the HD domain in GlnD mightrepresent the UR activity (6).

The ACT domain consensus sequence has been identified inthe sequences of very diverse proteins, and some are involvedin amino acid and purine synthesis (5). It has been proposedthat the ACT is a ligand-binding domain (5, 15). Though onlya few proteins containing this domain have been crystallized(19, 23, 41, 42, 51, 68, 71), their domain structures show asimilar fold and a direct involvement in the binding of aminoacids and other effectors (19, 41, 51, 71). Given the role ofGlnD in sensing glutamine, one supposes that these ACT do-mains might be relevant to that sensing, though this has notbeen experimentally tested.

To better understand the role of the uridylylation of the PII

proteins in nitrogen fixation in R. rubrum, we previously con-structed insertion and deletion mutations in glnD and charac-terized the roles of GlnD in the regulation of NifA and NtrCactivities and the posttranslational regulation of nitrogenaseactivity (80). Here, we report further functional analysis of thedomains in GlnD from both R. rubrum and E. coli.

MATERIALS AND METHODS

Bacterial growth conditions. E. coli was grown in LC medium (similar toLuria-Bertani medium but with 5 g/liter NaCl) (64). R. rubrum was grown in yeastextract-supplemented malate-NH4

� (SMN) rich medium (21, 57). For derepres-sion of nitrogenase activity, SMN cultures of R. rubrum were inoculated intomalate-glutamate (MG) medium (45). The whole-cell nitrogenase activity assayand darkness/NH4Cl treatments have been described previously (75). Antibioticswere used at the following concentrations (mg/liter): for R. rubrum, streptomycin(Sm), 100; kanamycin (Km), 12.5; tetracycline (Tc), 1; gentamicin (Gm), 10; andfor E. coli, ampicillin (Ap), 100; Km, 25; Gm, 5; Cm, 25; and Tc, 12.5.

Construction of R. rubrum glnD mutants. About 3 kb of a BamHI-HindIIIfragment containing R. rubrum glnD was cloned into pUX19 (48), yieldingpUX1000. A QuikChange method (Stratagene, La Jolla, CA) was used accordingto the manufacturer’s instructions to generate localized random mutagenesis ofthe G110 or D123 residue, using pUX1000 as a template and synthetic oligonu-cleotides with a randomized codon at G110 or D123 as a pair of primers. AfterPCR, the DNA fragment was digested with BamHI and HindIII and then clonedinto pUX19. After transformation with E. coli DH5�, plasmids were isolatedfrom transformants and sequenced to identify the mutation. Plasmids with mu-tated glnD were transferred into E. coli S17-1 (70) and then transferred into anR. rubrum �glnD mutant (UR1325) by mating as described previously (47). Smr

Gmr Kmr R. rubrum colonies were selected such that these plasmids were inte-grated into the chromosome of UR1325 at the 5� or 3� end of glnD, yieldingstrains UR1551 to UR1564.

Different primers were designed to construct deletions of the ACT region in R.rubrum GlnD by PCR, using pUX1236 (80) as a template. One plasmid(pUX1614) has a second ACT domain and the C terminus deleted (�T839-A936), and another (pUX1615) has both ACT domains and the C terminusdeleted (�S723-A936). Similarly, two deletions in the central HD domain werealso constructed: one plasmid (pUX1617) has an in-frame deletion of the centralregion from an NruI site to the first ACT domain (�E394-A721), and anotherone (pUX1620) has a 66-base pair (bp) deletion in the HD domain (�A530-

FIG. 1. Alignment of nucleotidyltransferase (NT) domains of kana-mycin nucleotidyltransferase (Kan-NT), rat DNA polymerase �, RNA-editing terminal uridylyl transferase 2 (TUTase 2) from Trypanosomabrucei, bovine poly(A) polymerase, and GlnD and GlnE from R.rubrum (Rr) and E. coli (Ec). Conserved glycine (serine or lysine insome members) and aspartate (glutamate in Kan-NT) residues are inbold. A gap in the DNA polymerase � sequence is indicated by anasterisk.

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V551). These plasmids were transformed into E. coli S17-1 and then conjugatedinto an R. rubrum �glnD mutant (UR1325). Smr Gmr Kmr R. rubrum colonieswere selected such that these plasmids were integrated into the chromosome ofUR1325, yielding strains UR1446, UR1447, UR1448, UR1449, and UR1658.

Overexpression and purification of R. rubrum GlnD variants. To overexpressR. rubrum GlnD in E. coli, the wild-type (wt) and truncated glnD were amplifiedby PCR with a set of primers with NdeI and EcoRI sites at each end and thencloned into pJAL503 (67) at the NdeI and EcoRI sites, yielding pUX1815,pUX1816, pUX1817, pUX1818, and pUX1819. Together with the vectorpJAL503, these plasmids were transferred into E. coli strain BD (glnB glnDmutant) (39), yielding strains UQ4091 to UQ4096.

We also constructed an R. rubrum wt GlnD-MalE fusion protein by subcloningwt glnD into pMAL-C2 (New England Biolabs, Inc., Beverly, MA), yieldingstrain pUX1655. This MalE-GlnD fusion protein was purified using an amyloseresin column (New England Biolabs, Inc., Beverly, MA) according to the man-ufacturer’s instructions and then desalted using a G-25 column.

Construction, overexpression and purification of E. coli GlnD variants. E. coliGlnD variants with substitutions at the G93, G94, D107, H514, and D515 resi-dues and HD or ACT deletions were constructed in the same way as the R.rubrum GlnD variants except using different numbers for these conserved resi-dues. Two truncated GlnD variants with the N terminus only or without the Nterminus were also constructed. These mutated glnD genes were cloned intopET-15b (Novagen/EMD Chemicals, Gibbstown, NJ), and His-tagged GlnDvariants were purified with a His-Bind resin column (Novagen) and then desaltedwith G-25 column. Most proteins were at least 90% pure.

UTase and UR activity assay. The UTase activity of E. coli GlnD was assayedunder two different conditions: in the presence of Mg2� only or in the presenceof both Mn2� and Mg2�, as described previously (34). For Mg2�-UTase activity,the reaction mixture contained 100 mM Tris (pH 7.5), 100 mM KCl, 25 mMMgCl2, 1 mM dithiothreitol (DTT), 0.3 mg/ml bovine serum albumin (BSA), 1mM ATP, 10 mM �-KG, 60 �M E. coli GlnB, and various concentrations ofGlnD as indicated below, in a volume of 50 �l. The reaction mixture waspreincubated at 30°C for 2 min, and the reaction was started by adding 1 �l of 50mM UTP. After incubation at 30°C for 60 min, the reaction was stopped byadding 2.5 �l of 0.5 M EDTA. For Mg2�-Mn2�-UTase activity, the reactionconditions were the same as described above except that 1 mM MnCl2 wasadded.

The UR activity of E. coli GlnD was also assayed under two different condi-tions: in the presence of Mn2� or in the presence of Mg2�, as described previ-ously. For Mg2�-UR activity, the reaction mixture contained 100 mM Tris (pH7.5), 100 mM KCl, 50 mM MgCl2, 1 mM DTT, 0.3 mg/ml BSA, 0.5 mM ATP, 0.5mM �-KG, 2.5 mM glutamine, 40 �M E. coli GlnB-UMP, and various concen-trations of GlnD as indicated below, in a volume of 50 �l. The reaction wascarried out at 30°C for 60 min, and was stopped by adding 2.5 �l of 0.5 M EDTA.For Mn2�-UR activity, the reaction conditions were the same as described aboveexcept that 1 mM MnCl2 was added, the glutamine concentration was increasedfrom 2.5 mM to 10 mM, and MgCl2, ATP, and �-KG were omitted.

For the assay of R. rubrum GlnD activity, UTase activity was monitored underthe same conditions as described above for E. coli GlnD in the presence of bothMn2� and Mg2�. However, the UR activity was monitored using different con-ditions than those described above for E. coli GlnD, as suggested previously (38).The reaction mixture of 50 �l contained 100 mM Tris (pH 7.5), 100 mM KCl, 25mM MgCl2, 1 mM MnCl2, 1 mM DTT, 0.3 mg/ml BSA, 2 mM ATP, and 10 mMglutamine. R. rubrum or E. coli GlnB-UMP (20 to 60 �M) was used as asubstrate. Different levels of �-KG (0 to 1 mM) were added to the mixture.

Quantitation of the modification of GlnB on native gels. Samples from theUTase or UR assay were mixed with nondenaturing sample buffer, and thedegree of uridylylation of GlnB was detected using nondenaturing polyacryl-amide gel electrophoresis (PAGE) analysis, as described previously (8). All 4forms of GlnB (with 0 to 3 UMP groups attached) were well separated. Thebands in the gel were visualized using an Alpha Imager (Alpha Innotech Co., SanLeandro, CA) and quantified using ImageQuant software (GE Healthcare,Piscataway, NJ).

The modified states of GlnB were determined using the following formulaaccording to the amount of modified and unmodified forms of the protein:[GlnB-UMP � 2 � GlnB-(UMP)2 � 3 � GlnB-(UMP)3] � [GlnB � GlnB-UMP � GlnB-(UMP)2 � GlnB-(UMP)3]. GlnB represents the unmodified form,while GlnB-UMP, GlnB-(UMP)2, and GlnB-(UMP)3 represent forms with one,two, and three modified subunits, respectively.

SDS-PAGE and immunoblotting of R. rubrum GlnB. A trichloroacetic acidprecipitation method was used to quickly extract protein (76), and a low-cross-linker (ratio of acrylamide to bisacrylamide, 172/1) tricine gel was used to sep-arate modified and unmodified GlnB, as described previously (66). Proteins were

electrophoretically transferred onto a nitrocellulose membrane, immunoblottedwith polyclonal antibody against R. rubrum GlnB, and visualized with horseradishperoxidase color detection reagents (Bio-Rad, Richmond, CA).

RESULTS

In vivo and in vitro studies of R. rubrum GlnD variants.Previous mutagenesis and complementation studies showedthat the N-terminal region of R. rubrum GlnD is responsiblefor UTase activity (80). GlnD has an NT domain in the Nterminus with several conserved glycine and aspartate residues(Fig. 1). To determine the role of these residues in UTaseactivity, we performed random mutagenesis on two of theconserved residues in the NT domain, G110 and D123, andmonitored nitrogenase activity, which indirectly measuresUTase activity. In R. rubrum, GlnD modifies GlnB under ni-trogen-limiting conditions, and GlnB-UMP then activatesNifA, which leads to the synthesis of nitrogenase (78, 80, 81).Little nitrogenase activity is detected in a �glnD mutant, sinceGlnB remains in the unmodified form regardless of nitrogenstatus (80). As shown in Table 1, most substitutions at residuesG110 and D123 eliminated nitrogenase activity, suggesting theabsence of UTase activity. Only the G110A substitution sup-ported normal nitrogenase activity, implying substantial UTaseactivity. This is surprising since this glycine (G110) and theadjacent one (G109) are highly conserved in GlnD. As wereported previously, the activation of NifA requires lowerUTase activity than does the activation of other receptors, suchas NtrC (80). Nevertheless, these data suggest that the NTmotif is important for UTase activity.

GlnD also has a central HD domain and two ACT domainsat the C terminus, but the roles of these domains in UTase/URare unknown. To study the roles of the HD and ACT domains,we constructed four R. rubrum glnD mutants with a deletion ofthe second ACT domain (GlnD-�ACT2, strain UR1447), adeletion of both ACT domains (GlnD-�ACT1&2, strainUR1448), a deletion of the central region including the entireHD domain (�central, strain UR1449), or a deletion of 22amino acids including the core HD residues (�HD-22aa, strainUR1658). As shown in Table 1, these deletions in R. rubrumglnD showed no significant effects on the activation of NifA,indicating that these HD and ACT domains are not essentialfor UTase activity. We also monitored two other regulatorysystems that have previously been shown to be affected byGlnD activity (80), the DRAT/DRAG regulatory system, bymonitoring nitrogenase activity in response to dark/light shiftsor ammonium addition, and the NtrB-NtrC regulatory system,by monitoring the accumulation of GlnJ, whose expression isregulated by NtrC. Overall, the regulation of nitrogenase ac-tivity, reflecting DRAT/DRAG regulation, was normal in thesemutants (data not shown). GlnJ levels were slightly decreasedin strains UR1448 (GlnD-�ACT1&2) and UR1658 (GlnD-�HD-22aa), but very low in UR1449 (GlnD-�central) (datanot shown). This implies that there are different levels of un-modified PII in these mutants, which would interact with NtrBand thereby inactivate NtrC. These results reflect alteration ofUTase and/or UR activities in these mutants.

We also examined in vivo GlnD activity more directly bymonitoring the modification of GlnB in response to the addi-tion of NH4

�. We monitored the modification of GlnB be-

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cause its expression in R. rubrum is not tightly regulated bynitrogen status, unlike that of glnJ (37). In the presence of wtGlnD (UR1446), GlnB was partially modified under nitrogen-limiting conditions and became completely unmodified afterthe addition of NH4

� (Fig. 2). This is caused by the stimulationof UR activity and the inhibition of UTase of GlnD by theNH4

� signal (i.e., the glutamine pool). In GlnD-�ACT2(UR1447) and GlnD-�ACT1&2 (UR1448), GlnB was alsopartially modified before NH4

� treatment, but only a fractionof GlnB became unmodified after the addition of NH4

�, im-plying that these mutants have lower UR activities than doesthe wild type. Under nitrogen-limiting conditions, GlnB wasalso partially modified in the GlnD-�central variant (UR1448)and almost completely modified in the GlnD-�HD-22aa vari-ant (UR1658), but these two variants showed no significant

changes in GlnB modification in response to NH4�, suggesting

that these deletion mutants lacking the HD domain might havelost UR activity.

However, it is difficult to interpret these data obtained fromin vivo experiments. Poor deuridylylation of GlnB in responseto NH4

� might be due to either low UR activity or inappro-priately high (unregulated) UTase activity, which in turn couldcompete with the UR activity. It is impossible to separate thesetwo GlnD activities in vivo, so we attempted to examine UTaseand UR activities separately in vitro.

We overexpressed R. rubrum wt and four truncated GlnDproteins with deletions of ACT or HD domains in E. coli, usingthe heat-inducible pJAL503 (67). Unlike wt GlnD, most of thetruncated R. rubrum GlnD proteins were insoluble. However,GlnD variants with deletions of ACT2, ACT1&2, central re-gion, and HD-22aa had substantial amounts of UTase activityin crude extracts, but no UR activity was detected in any GlnDvariants, including wt GlnD (data not shown). To rule out thepossibility of an inhibitor of UR activity in crude extracts, wepurified wild-type GlnD; it showed high UTase activity but noUR activity (data not shown). We also constructed and puri-fied a MalE fusion to wt GlnD, which again showed highUTase activity but no UR activity (data not shown). We notethat Jonsson and Nordlund did report UR activity in R. rubrumGlnD expressed with a glutathione S-transferase fusion thatwas later removed, but the activity seems to have been ratherlow and detected only with a very sensitive [�-32P]UMP assayusing excess GlnD (38). Our inability to assay in vitro URactivity of R. rubrum GlnD forced us to consider another ho-molog, E. coli GlnD.

Construction, expression, and purification of E. coli glnDvariants. As shown in Fig. 3, we constructed a number ofmutants of E coli GlnD, with substitutions at the G93, G94,and D107 residues (corresponding to the G109, G110, andD123 residues in R. rubrum GlnD), double substitutions at

TABLE 1. Nitrogenase activity in R. rubrum glnD mutants

Strains Chromosomal genotype with gene integration Nitrogenaseactivitya

UR2 wt 750UR1446 �glnD mutant with wt glnD 650UR1556 �glnD mutant with glnD encoding GlnD-G110L 20UR1557 �glnD mutant with glnD encoding GlnD-G110D 20UR1558 �glnD mutant with glnD encoding GlnD-G110R 20UR1559 �glnD mutant with glnD encoding GlnD-G110I 20UR1560 �glnD mutant with glnD encoding GlnD-G110A 660UR1561 �glnD mutant with glnD encoding GlnD-G110V 20UR1562 �glnD mutant with glnD encoding GlnD-G110W 20UR1563 �glnD mutant with glnD encoding GlnD-G110N 30UR1564 �glnD mutant with glnD encoding GlnD-G110Q 20UR1551 �glnD mutant with glnD encoding GlnD-D123A 20UR1552 �glnD mutant with glnD encoding GlnD-D123G 20UR1553 �glnD mutant with glnD encoding GlnD-D123V 20UR1554 �glnD mutant with glnD encoding GlnD-D123L 20UR1555 �glnD mutant with glnD encoding GlnD-D123Y 20UR1447 �glnD mutant with glnD-�ACT2 (�T839-A936) 680UR1448 �glnD mutant with glnD-�ACT1&2 (�S723-A936) 700UR1449 �glnD mutant with glnD-�central region (�E394-A721) 450UR1658 �glnD mutant with glnD-�HD-22aa (�A530-V551) 650

a Each unit of nitrogenase activity is expressed as nmol of ethylene produced per h per ml of cells at an optical density of 1 at 600 nm. Each activity value is fromat least five replicate assays from different individually grown cultures. The standard deviations were between 5 and 15%.

FIG. 2. Modification of GlnB in R. rubrum strains UR1446 (wtGlnD), UR1447 (GlnD-�ACT2), UR1448 (GlnD-�ACT1&2),UR1449 (GlnD-�central), and UR1658 (GlnD-�HD-22aa) in re-sponse to NH4

� treatment. R. rubrum strains were grown in MGmedium for 2 days, and protein samples were collected before (N) or60 min after (�N) the addition of 20 mM NH4Cl by trichloroaceticacid precipitation, loaded on low-cross-linker tricine SDS-PAGE gels,and immunoblotted with antibody against R. rubrum GlnB. The posi-tions of the modified (M) and unmodified GlnB subunits (U) areindicated by arrows.

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residues H514 and D515 (HD-AA and HD-QN) in the HDdomain, deletions of the entire central and C-terminal region(GlnD-N), �ACT1&2, �ACT2, �HD (22 amino acids), anddeletion of the N-terminal NT domain (GlnD-�NT). All ofthese GlnD proteins were His tagged and expressed in E. coli,purified using a His-bind resin column, and desalted using aG-25 column.

The central HD domain of GlnD encodes UR activity. Tocompare UR activity, two assay conditions were used, as de-scribed in Materials and Methods; one is for Mg2�-UR activ-ity, and the other is for Mn2�-UR activity. Three differentconcentrations of GlnD were used in the assays: 0.1, 0.5, and 2�M. Similar to previous reports (34), E. coli wt GlnD showedmore than 5-fold higher Mn2�-UR activity than Mg2�-URactivity (Fig. 4). The majority of the mutant proteins showedUR activity that was comparable to or only modestly lowerthan that of wt GlnD under both assay conditions. The NTdeletion mutant (GlnD-�NT) and the G93L and G93V sub-stitution mutants displayed Mn2�-UR activities similar tothat of wt GlnD but had low Mg2�-UR activity. Only theGlnD-N, �HD, and two HD substitutions lacked UR activityunder both conditions. These results indicate that the cen-tral HD domain is essential for UR activity and likely rep-resents the active site of UR. The N-terminal NT domain isnot essential for UR activity but might be involved in Mg2�

binding to support Mg2�-UR activity, based on the resultwith GlnD-�NT.

The N-terminal NT domain is essential for UTase activitybut not UR activity. To compare UTase activities, two assayconditions were also used for the UTase assay, as described inMaterials and Methods: Mg2�-UTase activity and Mg2�-Mn2�-UTase activity. wt GlnD had slightly higher Mg2�-UTase activity than Mg2�-Mn2�-UTase activity (Fig. 5), butthe difference between these two activities was less than thatseen previously (34). As expected, GlnD-N showed substantialUTase activity in both conditions, although it is much lowerthan wt GlnD in the presence of Mg2�; GlnD-�NT lacked

UTase activity. These data are consistent with the hypothesisthat the NT domain is the active site for UTase. All substitu-tions at G93, G94, and D107, except G94L, also caused almostcomplete loss of UTase activity under both conditions. Thesedata confirm an important role for these NT domain residuesin UTase activity, as predicted by the structural analysis of thisdomain from other family members (17, 18, 58, 62, 65).

Importantly, the HD substitution mutants (HD-AA andHD-QN) shown above to lack UR activity (Fig. 4) had UTaseactivities similar to that seen in wt GlnD in both assays (Fig. 5),indicating that these variants are not simply dead GlnD pro-teins. This argues that UTase and UR activities do not sharethe same active site. Both the GlnD-�ACT1&2 and the GlnD-�ACT2 variant appear to have lower UTase activities than thatseen in wt GlnD in both assays, though to different degrees,suggesting that ACT might play some role in the regulation ofUTase activity. However, as described below, the apparentreduced Mg2�-Mn2�-UTase activities in these ACT deletionmutants might actually be due to the altered regulation of URactivity, which then competes with UTase activity to decreasethe modification of GlnB.

Competition of UTase and UR activities in in vitro assays. Ina UR activity assay, UTase does not interfere with UR activity,since there is no UTP in the assay for GlnD to use for PII

modification. In contrast, under UTase assay conditions, inap-propriately high UR activity in a mutant protein could competewith the UTase activity by deuridylylating GlnB. This wouldhave the appearance of altered UTase activity, though thedirect effect would actually be on UR activity. The reducedUTase activity in ACT mutants could therefore be caused byunregulated UR activity. To test this, we measured UR activityunder the Mg2�-Mn2�-UTase assay conditions in these vari-ants and in wt GlnD. Under these assay conditions and in theabsence of glutamine, GlnD-�ACT1&2 and GlnD-�ACT2showed substantial UR activity—even higher than that of wtGlnD (Fig. 6B). This result strongly suggests that there iscompetition of UTase and UR activities under Mg2�-Mn2�-

FIG. 3. Substitutions and deletions of E. coli GlnD mutants. The location of each domain is indicated by the amino acid residue numbers. Thepositions of the replaced conserved residues in the NT and HD domains are indicated by the dots shown on the top line of the figure. E. coli strainsare indicated in parenthesis.

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UTase assay conditions in these ACT mutants and, probably,in the wild type as well, resulting in a futile cycle of the uridy-lylation and deuridylylation of GlnB. We also measured URactivity under Mg2�-UTase assay conditions, and the ACTmutants and wt GlnD showed low UR activities (Fig. 6A).Unlike Mg2�-Mn2�-UTase, the poor Mg2�-UTase activities inthese ACT variants (Fig. 5A) do not seem to be due to thecompetition of UR activity, for the following reasons: (i)the Mg2�-UR activity in these variants is much lower than theMn2�-UR activity (Fig. 4); (ii) very low UR activity was alsoseen under Mg2�-UTase assay conditions (Fig. 6B); and (iii)low Mg2�-UTase activity was also seen in GlnD-N and GlnD-�HD, which have no UR activity (Fig. 5A). In summary, thereare substantial UTase and UR activities in the presence ofMn2�, causing a futile cycle of the uridylylation and deuridy-lylation of GlnB. The ACT domains might be important for theregulation of UR activity under these conditions to avoid thisfutile cycle.

Glutamine stimulates UR activity in wt GlnD but not in theACT deletion mutants. Previous studies showed that glutaminestimulates the UR activity of E. coli GlnD (20, 34). However,as seen in the results described above, GlnD-�ACT1&2 andGlnD-�ACT2 showed high UR activities in the absence ofglutamine. Because of the potential role of the ACT domainsin the binding of glutamine, we further investigated the effect

of glutamine on UR activity in wt GlnD and ACT deletionvariants in the presence of Mn2�, since all strains have verysimilar Mn2�-UR activities. As shown in Fig. 7, glutaminestimulates the Mn2�-UR activity of wt GlnD but has no sig-nificant effect on the Mn2�-UR activities of ACT deletionvariants. This suggests that these ACT domains might playsome regulatory role in the regulation of GlnD activity inresponse to glutamine.

Inhibition of UTase activity by glutamine. Previous studieshave shown that glutamine inhibits the UTase activity of E. coliGlnD (1, 20, 34, 39) but not that of R. rubrum GlnD (38).However, this is complicated by the glutamine stimulation ofUR activity noted above. A decrease in PII modification byglutamine, suggestive of the inhibition of UTase activity, couldtherefore be caused wholly or in part by the elevation of URactivity. Indeed, as shown in the results described above, thisfutile cycle of the uridylylation/deuridylylation of PII clearlyoccurs under our in vitro assay conditions. To avoid the inter-ference in UTase activity by UR, we investigated the effects ofglutamine on UTase activities in the mutants lacking UR ac-tivity. As shown in Fig. 8B, glutamine inhibits the uridylylationof GlnB by wt GlnD under Mg2�-Mn2�-UTase assay condi-tions. However, the glutamine effect was more striking at ahigh protein concentration (2 �M) than at a low concentration(0.5 �M). This is difficult to explain by the “UTase inhibition”

FIG. 4. Mg2�-UR activities (A) and Mn2�-UR activities (B) in E. coli wt GlnD (full-length) and mutant variants. The starting substrate wasfully uridylylated GlnB, and UR activity was measured by the decrease in the average number of UMP groups per GlnB trimer. Each variant hadthree concentrations of GlnD in the assay: 0.1 (open bars), 0.5 (dark gray bars), and 2 (light gray bars) �M. The results for the negative control(without GlnD) are shown by the first (black) bars. After the reaction, protein samples were separated on nondenaturing gels, and the degree ofuridylylation of GlnB was determined as described in Materials and Methods.

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model but is consistent with glutamine stimulating UR activity.With our GlnD variants lacking UR activity (the GlnD-N andHD deletion or substitution mutants), there was little effectof glutamine on the Mg2�-Mn2�-UTase activity. Mutantswith ACT deletions also showed no significant effect ofglutamine, although they have lower Mg2�-Mn2�-UTase ac-tivities than do other variants, due to the high and unregu-lated UR activity under these assay conditions (Fig. 6B).These results suggest that under the Mg2�-Mn2� assay con-ditions, the effect of glutamine is solely on the stimulation ofUR activity and that the ACT domains are important forthis glutamine effect.

In contrast, in the absence of Mn2�, glutamine showed par-tial inhibition of the Mg2�-UTase in wt GlnD and GlnD-HD-AA or QN substitution mutants but had no effect onMg2�-UTase in GlnD-N (Fig. 8A). Clearly this glutamine ef-fect cannot be on the stimulation of UR activity, since thesemutants lack that. Instead, this effect must reflect inhibition ofUTase activity by glutamine under these conditions. TheGlnD-N variant lacks this effect, which might mean eitherthat glutamine inhibition requires a C terminus and/or acentral region of GlnD or that this variant has an alteredstructure that affects regulation. Unfortunately, we are un-able to study the roles of ACT in this inhibition, since ACT

deletion mutants have very low Mg2�-UTase activity (Fig.5A). In summary, these results suggest that glutamine prob-ably inhibits Mg2�-UTase activity but not Mg2�-Mn2�-UTase activity.

DISCUSSION

Role of the N-terminal NT domain. GlnD is a bifunctionaluridylyltransferase/uridylyl-removing enzyme (UTase/UR),and it is clear that the UTase active site is located at the Nterminus of the protein, based on mutational studies (56, 72,80) and domain analysis (4, 27). The results of in vivo analysisof R. rubrum glnD mutants and the in vitro studies of E. coliGlnD variants reported here confirm that the N-terminal NTdomain is critical for UTase activity. Most substitutions forconserved glycine and aspartate residues in this domain com-pletely abolish its UTase activity but have no significant effecton UR activity.

Role of the central HD domain. Although sequence com-parisons suggested that the HD domain might be the active sitefor UR activity, previous mutational and kinetic studies im-plied that UR and UTase of E. coli GlnD might share an activesite (34, 56). Our results clearly indicate that HD is critical forUR activity. Two substitution mutations of HD residues in the

FIG. 5. Mg2�-UTase activities (A) and Mg2�-Mn2�-UTase activities (B) in E. coli wt GlnD and mutant variants. UTase activity was measuredby monitoring the appearance of GlnB-UMP with unmodified GlnB as the substrate. Each variant had three or four concentration of GlnD in theassay: 0.025 (striped bars), 0.1 (open bars), 0.5 (dark gray bars) and 2 (light gray bars) �M. The results for the negative control (without GlnD)are shown by the first (black) bars.

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central HD domain, as well as the deletion of the HD domain,completely eliminate UR activity but have little effect onUTase activity. Furthermore, a truncated GlnD lacking theN-terminal NT domain showed high Mn2�-UR activity. Thesedata indicate that the UR and UTase activities do not share anactive site. Given the fact that this HD domain is found inmany proteins with phosphohydrolase activity, this HD domainis likely the active site for the UR reaction.

Role of ACT domains. Based on sequence comparisons andprevious studies of proteins containing ACT domains that bindamino acids or small effectors, it is likely that this domain inGlnD is involved in the binding of glutamine. The results ofprevious studies indicate that glutamine stimulates UR activityand inhibits the UTase activity of GlnD, and it is believed thatthis is the mechanism for nitrogen signal transduction in thecell (1, 20, 34, 39). Our results show that the deletion of ACTdomains eliminates this effect of glutamine on UR activity,strongly supporting this model. Presumably, the ACT domaincauses some inhibitory effect on UR activity, which is elimi-nated when it binds glutamine.

However, in vivo studies of R. rubrum ACT mutants indi-cated that these mutants could still sense a nitrogen signal evenin the absence of the ACT domains of GlnD. As shown in Fig.2, R. rubrum GlnD variants with deletions of the ACT domainswere able to sense NH4

�, resulting in the deuridylylation ofsome portion of GlnB after the addition of NH4

�, although thedeuridylylation of GlnB in these mutants is much less completethan that seen in the wild type. Previously, Tøndervik et al.reported that an E. coli glnD mutant lacking the ACT domainswas still able to sense nitrogen status (72). These authorssuggested that either the N terminus of GlnD senses glutamineor that there is another metabolite or mechanism for nitrogensensing. Given the present results, we favor a model in whichcells also sense the nitrogen signal through PII. PII binds �-KG(40, 54), and a change in the glutamine level would affect the�-KG pool, which would change the PII structure and its mod-

FIG. 6. UR activities in E. coli wt GlnD and mutant variants in the absence of glutamine. The assay was done in the presence of 1 mM ATP,1 mM �-KG, and 50 mM MgCl2 (Mg2�-UTase assay conditions) (A) or in the presence of 1 mM ATP, 10 mM �-KG, 50 mM MgCl2, and 1 mMMnCl2 (Mg2�-Mn2�-UTase assay conditions) (B). A concentration of 0.5 �M GlnD was used for the assay. The results for the negative control(without GlnD) are shown by the first bars.

FIG. 7. Effects of glutamine on Mn2�-UR activities in E. coli wtGlnD and ACT deletion variants. Each variant had 8 different con-centrations of GlnD in the assay: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2�M. Glutamine concentrations were either 0 or 10 mM.

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ification state. This should be sufficient to explain the results ofTøndervik et al. (72) and our own results with the R. rubrumACT mutants.

We have shown that a futile cycle of the uridylylation anddeuridylylation of GlnB occurs under in vitro assay conditions.The ACT domains play important roles for the regulation ofUR activity under these conditions to avoid this futile cycle. Itwould be interesting to know if this futile cycle occurs in vivo aswell. We noticed that the addition of glutamine has significanteffects on both UTase and UR activities, but the inhibition ofUTase activity by glutamine is not very strong, at least underour in vitro assay conditions. If the regulation of GlnD is solelydependent on the glutamine level in the cell, it is likely that thefutile cycles of the uridylylation/deuridylylation of PII occur invivo as well.

The role of metal ions. It is well known that Mn2� and Mg2�

have significant effects on both the UTase and the UR activityof E. coli GlnD (1, 22, 34), though the precise role of thesemetals in the regulation of GlnD activity is still unknown.Mg2� is believed to be the physiologically important metal ioneffector (34), but it is impossible at present to know the effec-tive concentrations of these metal ions in vivo. Thus, whileGlnD appears to have two metal-binding sites, and our resultsindicate that the deletion of one site has a significant effect onthe activity located at the different site, conjectures about therelative impacts of different ions at these sites would be purespeculation.

The structures of kanamycin nucleotidyltransferase (Kan-

NT), DNA polymerase �, and other family members show thatthe NT domain can be directly involved in binding metal ions(18, 62, 65), so it is reasonable to suppose that the NT domainin GlnD is also involved in metal binding for UTase activity.However, variants altered in the NT domain showed robustMn2�-UR activity but low Mg2�-UR activity, indicating thatthere are other metal-binding site(s) for Mn2� to supportMn2-UR activity, likely in the HD domain.

In summary, we confirm that several conserved residues inthe N-terminal NT domain of GlnD are critical for UTaseactivity. Our results indicate that the UR active site is locatedin the central HD domain. The ACT domains at the C termi-nus appear to play regulatory roles in GlnD activity, probablythrough the binding of glutamine.

ACKNOWLEDGMENTS

This work was supported by NIGMS grant GM65891 to G.P.R.We thank Alex Ninfa for generously providing the E. coli glnB glnD

mutant and the GlnB and GlnD overexpression strains. We also thankStefan Nordlund for providing information about in vitro UTase andUR activity assays for R. rubrum GlnD.

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