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Journal of Bacteriology, November 2005, p. 7481-7491, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7481-7491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biochemical and Mutational Analysis of Glutamine Synthetase Type III from the Rumen Anaerobe Ruminococcus albus 8

Kensey R. Amaya,¹ Svetlana A. Kocherginskaya,¹ Roderick I. Mackie,^1,2 and Isaac K. O. Cann^1,2*

Department of Animal Sciences,¹ Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801²

Received 23 April 2005/ Accepted 8 August 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the -glutamyl transferase (-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glutamine synthetase (GS) converts glutamate to glutamine through the incorporation of ammonia (NH₃). This reaction is driven by ATP hydrolysis and requires a cationic group for activity (2, 34). Glutamine is responsible for donating the amino group, from assimilated NH₃, for the synthesis of amino acids and other nitrogen-containing metabolites such as purines and pyrimidines. In many organisms, ammonia assimilation by GS leads to synthesis of glutamate from glutamine by glutamate synthase (GOGAT). Consequently, this pathway is referred to as the GS/GOGAT pathway. GS is one of the most important enzymes in nitrogen metabolism, and there is phylogenetic evidence suggesting the gene coding for GS to be one of the oldest existing and functioning genes (17).

The GS family of proteins can be classified into three types. There is GS type I (GSI), a dodecameric (M_r 55,000 subunit) protein arranged into two superimposed hexagonal rings. This type of GS is widely distributed among bacteria (26, 34). GSII is an octameric (M_r 36,000 subunit) protein derived from two superimposed tetrahedrons. The type II proteins are found in bacterial symbionts of plants as well as in eukaryotes (10, 19). Then there is GSIII, a hexameric (M_r 75,000 subunit) protein that was first identified in the obligate anaerobe Bacteroides fragilis (16). Genes encoding GS type III proteins have subsequently been identified in a few more anaerobic bacteria and cyanobacteria (9, 15, 29).

The obligate anaerobe Ruminococcus albus, along with Ruminococcus flavefaciens and Fibrobacter succinogenes, is the primary cellulolytic bacteria in ruminants (7). These bacteria play a major role in the hydrolysis of the ß-1,4-linked glucose residues that make up cellulose, a major structural component of plant cell wall and fiber (7, 14). In addition, the fermentation end products of these bacteria contribute to the pool of short-chain fatty acids that is used directly by the ruminant as a source of energy (24, 31). Ammonia assimilation, which is intimately associated with carbohydrate metabolism, in these organisms is therefore of great importance and interest, since NH₃ is the primary N source for these cellulolytic ruminal bacteria (28).

Based on bioinformatics analysis, two genes encoding two different GS-like polypeptides were identified in the genome of R. albus 8. One of the genes coded for a GSI (a putative glnA gene product) and the other a GSIII (a putative glnN product). The cyanobacterium Synechocystis sp. strain PCC 6803 also contains genes encoding two GS proteins (types I and III), and the native form of the type III protein is one of the few GSIII proteins that have been well characterized biochemically (15). It has been previously reported that the GSIII proteins have four signature motifs, characterized by the sequences AEKHDxFI, GEALD, EQEYFLxD, and HRLGxNEAPPAI, where x is any amino acid (9). The contribution of these motifs to the structure-function of GSIII, however, has yet to be elucidated.

In the present study, biochemical methods were used to demonstrate that the recombinant form of the R. albus 8 GSIII-like polypeptide exhibits its predicted enzymatic activity, and mutational analysis was used to study, for the first time, the contribution of the four GSIII motifs to the structure-function of this homolog. Furthermore, we used R. albus 8 cells grown under ammonia-limiting and -excess conditions to study the regulation of GSIII activity in this bacterium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organism and culture conditions. R. albus 8 was obtained from the culture collection of the Department of Animal Sciences at the University of Illinois at Urbana-Champaign. For the isolation of genomic DNA from R. albus 8, cells were cultured under anaerobic conditions in a chemically defined medium containing 2 mM NH₄Cl and 10 mM cellobiose at 37°C in crimped butyl-rubber-stoppered vials saturated with CO₂:H₂ (95:5 [vol/vol]) (12, 13).

Construction of expression vector. The glnN gene, encoding the GSIII-like protein, was amplified by PCR from R. albus 8 genomic DNA. Isolation of genomic DNA was based on a protocol described elsewhere (30). The PCR amplification was carried out with GSIIIF, as the forward primer, and GSIIIR, as the reverse primer. To facilitate cloning of the PCR fragment, NdeI and XhoI sites were introduced into the forward and reverse primers, respectively (Table 1). The PCR conditions were 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min. All PCR amplifications were done with ExTaq PCR kit according to the manufacturer's instructions (TaKaRa Bio., Inc.). The amplified glnN gene was cloned into pGEM-T, a TA-cloning vector (Promega), and clones with inserts were sequenced to verify the integrity of the gene (W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign). The plasmid with the correct sequence was designated pGEMT-GSIII. The glnN gene was subsequently released via digestion with NdeI and XhoI and inserted into a modified pET28a vector. The construct was designated pET28-GSIII. The modification in the vector was a replacement of the kanamycin resistance gene with that for ampicillin resistance (6).

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TABLE 1. Primers used in this studya

Gene expression and protein purification. We used approximately 100 ng of plasmid DNA to transform Epicurian Escherichia coli BL21-CodonPlus (DE3) RIL competent cells (Stratagene) by the heat shock method. The cells were spread on LB plates, supplemented with ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml), and incubated at 37°C overnight. A single colony was picked and incubated in 500 ml of LB broth supplemented with the two antibiotics as described above. The cells were cultured at 37°C on a rotary shaker until the optical density at 600 nm reached 0.3. The expression of glnN was then induced by adding isopropyl-ß-D-thiogalactopyranoside (IPTG) to the culture at a final concentration of 1 mM. After a further incubation for 5 h, the cells were harvested, resuspended in a lysis buffer (50 mM sodium phosphate [pH 7.0], 300 mM NaCl), and lysed by a French pressure cell (American Instrument Company). The cell debris was pelleted by centrifugation at 4,000 x g for 15 min at 4°C and, since the gene was placed in-frame with the hexahistidines (His₆) encoded by the pET28 vector, the supernatant was applied to a cobalt-charged affinity resin to immobilize the His₆-tagged GSIII. The host proteins were removed by washing with 50 column volumes of lysis buffer. The recombinant GSIII protein was eluted with the lysis buffer containing imidazole at 150 mM concentration (50 mM sodium phosphate [pH 7.0], 300 mM NaCl, 150 mM imidazole). The fractions containing the His₆-tagged GSIII were dialyzed against buffer A (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol [DTT], 10% glycerol). Further purification of the GSIII was carried out by applying the dialysate to a buffer A-equilibrated anion-exchange column (HiTrap Q, 5 ml; Amersham Biosciences) fitted to a high-pressure liquid chromatography apparatus (AKTA Explorer 10; Amersham Biosciences). The proteins that bound to the column were eluted with buffer A containing 1 M NaCl (50 mM Tris-HCl [pH 8.0], 1 M NaCl, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol). Fractions containing >90% pure GSIII protein were pooled and dialyzed against buffer A. For prolonged storage, R. albus 8 GSIII was dialyzed against a storage buffer (buffer A containing 1 mM DTT and 50% glycerol) and stored at –80°C.

Size exclusion chromatography. The purified GSIII protein was dialyzed against a buffer composed of 50 mM imidazole-HCl (pH 6.5), 50 mM NaCl, and 50 mM MgCl₂ and injected into a Superose 12 HR 10/30 gel filtration column (Amersham Biosciences) already equilibrated with the same buffer. The chromatography was developed with the same buffer at a flow rate of 0.4 ml/min. Fractions were collected with an automated fraction collector and aliquots were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The column was calibrated by running a set of protein standards (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; ovalbumin, 43 kDa; RNase A at 13.7 kDa) under the same conditions, and a standard curve was derived for the analysis.

Site-directed mutagenesis. Using a site-directed mutagenesis kit (QuikChange Multi Site-Directed Mutagenesis Kit; Stratagene), point mutations were introduced into the glnN gene in pGEMT-GSIII. The primers for the mutations E77A, E152A, E192A, E194A, K308A, K318A, and E380A are shown in Table 1. Mutagenesis was carried out with 100 ng of plasmid DNA, 100 ng of the appropriate primer, and other ingredients according to the manufacturer's recommendation (Stratagene). PCR conditions were as follows: 95°C for 5 min and then 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 65°C for 10 min 24 s. The parental plasmid, which was already methylated in E. coli cells, was digested with 10 U of DpnI for 1.5 h at 37°C, and the newly (PCR) synthesized plasmids were used in transforming E. coli JM109 cells by the heat shock method. Cells were then incubated for 1 h in 500 µl of prewarmed SOC medium at 37°C with shaking. A total of 100 µl of cell culture was spread onto LB plates supplemented with 100 µg of ampicillin/ml and, after overnight incubation, colonies were picked and screened by DNA sequencing for plasmids with the correct mutations. Fragments containing the mutations were shuffled into a pET28-GSIII vector with the same fragment removed. The plasmid constructs were sequenced to confirm the presence of each mutation, and the correct constructs were designated pET28/E77A, pET28/E152A, pET28/E192A, pET28/E194A, pET28/K308A, and pET28/E380A.

Enzymatic characterization of R. albus 8 recombinant GSIII protein. The characterization of R. albus 8 GSIII was done through the modified -glutamyl transferase (-GT) and forward (mimics the biosynthetic assay) assays according to the method of Bender et al. (4). The assay mixture for the transferase activity contained 135 mM imidazole-HCl (pH 6.4), 18 mM hydroxylamine-HCl, 25 mM K-arsenate, 1 mM MnCl₂, 0.36 mM sodium-ADP, and 3 µg of GSIII. The mixture was equilibrated at 37°C for 5 min, and the reaction was initiated by adding 50 µl of 200 mM L-glutamine (final concentration of 20 mM) in a final assay volume of 500 µl. After 10 min, the reaction was quenched with 1 ml of "stop solution" (5.5% FeCl₃ · 6H₂O [wt/vol], 2% trichloroacetic acid [wt/vol], 0.25 N HCl) and then centrifuged for 5 min at 10,000 x g to remove precipitates. The formation of -glutamyl-hydroxamate was measured by the absorbance at 540 nm, where 1 µmol of -glutamyl-hydroxamate had an absorbance of 0.6857. A reaction mixture without GSIII enzyme served as a blank. All measurements were within the linear range of the -glutamyl-hydroxamate standard curve.

The assay mixture for the forward reaction consisted of 94 mM imidazole (pH 6.2), 47 mM hydroxylamine-HCl, 168 mM L-glutamate, and 50 mM MgCl₂, to which approximately 3 µg of GSIII protein was added, and equilibrated at 37°C for 5 min. The reaction was initiated by adding 60 µl of 200 mM ATP (24 mM final concentration) in a total reaction volume of 500 µl. The reaction was terminated with 1 ml of "stop solution" after 60 min, and the products were centrifuged for 5 min at 10,000 x g to remove precipitates. The absorbance reading at 540 nm was then determined against a blank without GSIII enzyme.

A biosynthetic assay that measures the amount of phosphate released in the forward reaction, as described elsewhere (32), was also used to further compare the activity in the wild-type recombinant GSIII with those of its mutants. In brief, the assay mixture contained 7.6 mM ATP, 0.1 M L-glutamic acid, 0.05 M NH₄Cl, 0.05 M MgCl₂, 0.05 M imidazole buffer (pH 6.2), and 3 µg of protein in a total reaction volume of 0.2 ml. The reaction was stopped by adding 1.8 ml of a freshly prepared solution containing 0.8% FeSO₄ · 7H₂O in 0.15 N H₂SO₄, followed by 0.15 ml of a solution containing 6.6% (NH₄)₆Mo₇O₂₄ · 7H₂O in 7.5 N H₂SO₄. After color development, free phosphate was measured by determining the absorbance at 600 nm, where 1 µmol of free phosphate had an absorbance of 0.3628. A reaction mixture without GSIII protein served as the blank.

ATPase activity. The ATP hydrolysis assay was carried out by using the ATP-driven forward assay in a final volume of 20 µl. The final concentrations of substrates were the same as in the forward colorimetric assay, and 0.85 µM nonradioactive ATP and 0.085 mM [-³²P]ATP were supplied to each reaction. The reaction was equilibrated at 37°C for 5 min and initiated by adding 1 µg of GSIII. After 20 min, 2 µl of a 0.5 M EDTA (pH 7.5) solution was added to terminate the reaction, and 1 µl of the reaction mixture was spotted onto a polyethyleneimine-cellulose thin-layer plate (Merck, Darmstadt, Germany). The products were resolved in 1 M LiCl and 0.5 M formic acid, and a phosphorimager (BAS-1800 II; Fuji) was used for visualization and quantitation of the results.

CD. Far-UV circular dichroism (CD) spectra of the recombinant GSIII, and its mutants were measured at room temperature from 190 to 250 nm by using a JASCO J-720 spectropolarimeter (Japan Spectroscopic Co., Inc., Tokyo, Japan) and a cuvette (Starna) with a path length of 0.1 cm. The spectra were collected at a scanning rate of 50 nm/min. Buffer runs were carried out to determine baseline readings, and all samples were baseline corrected before calculations. The buffer used was 10 mM Tris-HCl (pH 8.0). The proteins were at a concentration of 0.2 mg/ml, and the molar ellipticity () was calculated by using the equation:

, where _obs is the observed ellipticity, MW is the molecular weight, C is the concentration (mg/ml), l is the path length of the cuvette in centimeters, and n is the number of residues (11). The protein concentrations were determined by the Bradford method using a commercially available kit (Bio-Rad) with bovine serum albumin (New England Biolabs) as the standard.

Growth of R. albus 8 under excess-ammonia and ammonia-limiting conditions. To determine the presence or absence of GSIII transcripts under different ammonia concentrations, R. albus 8 was grown under different carbon/nitrogen ratios. Two 250-ml cultures of R. albus 8 were grown in a chemically defined medium (3, 12, 13). One culture was grown under ammonia-limiting conditions (20 mM cellobiose and 1 mM NH₄Cl), and the other was grown under non-ammonia-limiting conditions (10 mM cellobiose and 10 mM NH₄Cl). The cultures were grown to late log phase, and the cells were harvested by centrifugation at 4,700 x g for 30 min at 4°C.

RNA extraction and purification. All solutions and buffers were made RNase free by diethyl pyrocarbonate treatment. Equipment and glassware were made RNase free by overnight soaking in 3% H₂O₂ and a rinse in diethyl pyrocarbonate-treated water. The total RNA isolation was based on a previous protocol with some modification (1, 22). The RNA samples were made DNA-free by using RNase-free DNase (Promega) according to the manufacturer's instructions. To prevent RNase activity, a RNase inhibitor (Invitrogen) was used during the DNase treatment. Total RNA concentration was estimated by measuring the absorbance at 260 nm (DU 7500; Beckman).

Northern blot analysis. Northern blot analysis was carried out by using the Whatman 3MM filter paper wick method as described elsewhere (5). For electrophoresis, 10 µg of DNase-treated RNA, determined by a spectrophotometer (DU 7500; Beckman), was run on a 1% formaldehyde agarose gel. Included in the electrophoresis were two sets of RNA molecular size standards (0.16- to 1.77-kb and 0.24- to 9.5-kb RNA ladders; Invitrogen). The PCR primers GSIIIF and GSIIIR (Table 1) were used to generate probes for detection of the glnN gene expression levels. To determine the relationship between the regulation of GS and the ammonium transporter (AmtB), the two primers, AmtBF and AmtBR (targeting the entire amtB/glnK gene), were designed to amplify by PCR a product for detection of amtB/glnK transcripts (Table 1). The primers 006F and 1518R were used to generate the probe for detection of 16S rRNA, which was used as a total RNA load control (Table 1). Probes were generated by PCR amplification of the specific gene from R. albus 8 genomic DNA. Each probe was gel purified (QIAquick gel extraction kit; QIAGEN) and suspended in 30 µl of sterile distilled H₂O. Each probe was then radiolabeled by using an internal random primer labeling kit (Invitrogen) and 50 µCi of [-³²P]dCTP (Perkin-Elmer) per 100 ng of DNA in a reaction outlined by the manufacturer (Invitrogen). The labeling reaction was terminated with a "stop buffer," and unincorporated nucleotides were removed by using the NENSORB 20 nucleic acid purification cartridge (NEN Life Science Products) according to the manufacturer's instructions. The final probe activity was between 1.00 x 10⁸ cpm/µg and 1.00 x 10⁹ cpm/µg as determined by a liquid scintillation counter (Beckman LS 5000TD). Prehybridization was carried out for 1 h in 7 ml of hybridization solution (Sigma) at 42°C. After prehybridization, the solution was replaced with another 7 ml of hybridization solution, and then purified labeled probe was added. Hybridization was performed at 42°C for 16 h, and the membrane was exposed to an imaging plate (Fuji), which was next scanned with a phosphorimager (BAS-1800 II; Fuji).

Phylogenetic analysis of GS proteins. All of the protein sequences used for the analysis, except for R. albus 8 sequences, were retrieved from GenBank. Sequences were aligned by using the multiple sequence alignment program CLUSTALX, and this program was also used to generate an unrooted neighbor-joining tree (25).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of genes encoding GS-like proteins in R. albus 8. The presence of two genes encoding GS-like proteins in R. albus 8 led us to further investigate each of the predicted polypeptides. Our analysis suggested that one of the polypeptides was similar to a GSI (GenBank accession no. DQ011853), whereas the other was similar to a GSIII (GenBank accession no. DQ010164). Although we expressed both GS proteins from R. albus 8, failure to detect activity in R. albus 8 GSI together with a dearth of research addressing, in detail, the biochemistry of GSIII proteins, prompted us to concentrate our investigation on the GSIII-like protein. An alignment of the putative GSIII from R. albus 8 with other GSIII proteins revealed that it shares the highest identity (64%) with a partially characterized GSIII from Ruminococcus flavefaciens FD-1 (S. A. Kocherginskaya, S. R. Wallace, R. I. Aminov, D. A. Antonopoulos, M. A. Pfister-Genskow, P. A. Duncan, I. K. O. Cann, B. A. White, and R. I. Mackie, unpublished data). The predicted polypeptide also exhibited 46 to 49% identities with its homologs from Pseudanabaena sp. strain PCC 6903, Synechococcus sp. strain PCC 7942, Prevotella bryantii, and Synechocystis sp. strain PCC 6803. Furthermore, the alignment enabled us to identify conserved motifs found in all GS proteins (motifs A, B, C, D, and E) in addition to those (motifs I, II, III, and IV) ascribed specifically to GSIII proteins (Fig. 1) (9). These findings suggested the likelihood of a functional GSIII protein in R. albus 8. On the other hand, the R. albus 8 GSI was not active. An alignment of the amino acid sequence of R. albus 8 GSI with those of other glutamine synthetases showed that in this GSI polypeptide, there are several mutations at amino acid positions that are known to be invariant. One of these changes is a serine-to-alanine mutation in the second serine of D(G/A)SS, which occurs in motif A (Fig. 1). Another example is the conversion of a conserved glutamic acid in motif E to isoleucine. This glutamic acid is recognized as a ligand for Mn²⁺. It is quite reasonable to assume that one or more of these changes led to a nonfunctional R. albus 8 GSI. It is also possible that this protein requires a unique cofactor that was lacking in our GS activity assay. Future experiments may lead to resolution of this surprising finding.

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FIG. 1. Amino acid sequence alignment of biochemically characterized and hypothetical GSIII proteins. Motifs A, B, C, D, and E, are conserved throughout the GS proteins (types I, II, and III). Motifs I, II, III, and IV are identified as signature motifs that are unique to the type III GS proteins. Amino acid sequences with black background are conserved, and those that are similar are shaded gray. Superscripts a, b, and c denote GSIII that have been biochemically characterized, partially characterized proteins, and hypothetical GSIII proteins, respectively.

Phylogenetic analysis of GS proteins. We investigated the relationship of the putative GSIII protein of R. albus 8 to other GS proteins through phylogenetic analysis. The phylogenetic tree showed three distinct branches corresponding to GS type I, II, and III proteins (Fig. 2). The GSI branch included sequences from both bacteria and archaea. The archaeal GSIs clustered with similar proteins from mainly low-G+C gram-positive bacteria (B. subtilis, L. johnsonii, and R. albus 8), although a sequence from a high-G+C gram-positive organism (Mycobacterium leprae) is also found in this cluster. Thus, this sub-branch consisted mainly of representatives from outside the proteobacteria. The rest of the GSI proteins clustered together on a sub-branch composed of bacterial sequences, primarily from proteobacteria and also cyanobacteria, which are gram-negative nonproteobacteria. Also, note that within this branch is a unique cluster of two sequences from high G+C gram-positive bacteria (M. tuberculosis and C. glutamicum). The GSII branch includes eukaryotes and bacterial species mainly -proteobacteria that are plant symbionts. However, a unique cluster of proteins from high-G+C gram-positive bacteria (S. viridochromogenes) is also found. In the GSIII branch, we find a clustering of proteins from cyanobacteria, and another cluster of proteins from low-G+C gram-positive bacteria. In addition, we see proteins from P. bryantii and B. fragilis, which belong to the Cytophaga-Flavobacter-Bacteroides phylum. Thus, in this cluster no proteobacteria are represented. Interestingly, bacteria with two GS proteins normally harbor the GSI and GSIII types of protein.

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FIG. 2. An unrooted phylogenetic tree of GS proteins. The phylogenetic tree was constructed by using CLUSTAL X and viewed using a neighbor-joining plot. The phylogenetic tree was developed from amino acid sequences of characterized (in boldface) and hypothetical glutamine synthetase proteins representing the three GS types (I, II, and III).

Other proteins of ammonia assimilation. To investigate whether other genes involved in nitrogen regulation occurred in the vicinity of the putative glnN gene in R. albus 8, we analyzed its flanking region for protein coding sequences. Interestingly, 121 nucleotides downstream of the glnN gene was a single open reading frame encoding a putative ammonium transporter (amtB) fused to its usually cotranscribed glnK gene (Fig. 3) (8). Further analysis of the intergenic region showed that the amtB/glnK gene occurs in a different frame from that of the glnN gene, and it also appeared to be under its own promoter (Fig. 3).

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FIG. 3. Arrangement of the glnN and the amtB/glnK genes in R. albus 8 genome. Indicated in boldface letters are the putative –35 and –10 regions of a promoter sequence. The start codons (ATG) are also shown.

Expression and purification of recombinant GSIII of R. albus 8. To determine whether the gene product (GSIII) of the glnN gene in R. albus 8 exhibits its predicted biological activity, the gene was expressed as a fusion protein containing an N-terminal His₆ tag. By using affinity chromatography and anion-exchange chromatography, we obtained a highly purified (>90% purity) protein as demonstrated by SDS-PAGE in Fig. 4. The cells from a 2-liter culture yielded about 9.5 mg of purified protein (Table 2). There was a slight decrease in the specific activity of the protein after anion-exchange chromatography (1 U), and this could be due to some denaturation of the protein. However, this is unclear since the GSIII protein remained stable for approximately 3 to 4 weeks when stored in buffer A at 4°C and, unlike previous reports, exclusion of cations from the purification steps did not seem to affect the activity or stability of the R. albus 8 GSIII (23, 26, 33).

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FIG. 4. Purification of R. albus 8 recombinant GSIII expressed in E. coli BL 21-CodonPlus (DE3) RIL. A Coomassie blue-stained SDS-PAGE gel (7.5%) of fractions from successive purification steps is shown. Lane 1, protein molecular mass markers; lane 2, crude extract; lane 3, post-Co2+ affinity resin; lane 4, post-Hi-Trap Q anion exchange. A total of 1 µg of protein was loaded in each lane.

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TABLE 2. Purification of recombinant R. albus 8 GSIII from E. coli cellsa

Subunit organization of recombinant GSIII of R. albus 8. The purified GSIII protein was subjected to gel filtration analysis to estimate its subunit organization in solution. Samples were taken from the beginning to the end of each peak for SDS-PAGE analysis, and for biochemical analysis proteins from peak elutions were used. Two peaks were observed for R. albus 8 GSIII (Fig. 5), and fractions from each peak exhibited GS activity. The first peak occurred before that of thyroglobulin, suggesting a protein larger than 669 kDa. Although outside of the range of our injected standards, the protein from this peak was estimated to be approximately 978.0 ± 133.0 kDa. The protein that eluted in the second peak was estimated to be 81.0 ± 15.0 kDa. The variations are the standard deviations estimated from three injections. A monomer of R. albus 8 GSIII was estimated from its translated sequence to be 77.0 kDa. The results, therefore, suggested that, in solution, R. albus 8 GSIII exists as dodecamers and monomers, if we assume that the relative molecular mass of a monomer is 81.0 kDa. The dodecameric protein could represent a protein complex existing in solution as two superimposed hexagonal structures. A shoulder that was estimated to be 162.0 ± 20.0 kDa was also observed (Fig. 5), and this suggests the presence of also GSIII homodimers in solution. Note that in this case also aliquots taken from the shoulder exhibited GS activity.

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FIG. 5. Size exclusion chromatography of the R. albus 8 GSIII. (A) The chromatograph represents 100 µl (8 mg/ml) of a >90%-purified recombinant R. albus 8 GSIII dialyzed against 50 mM imidazole-HCl (pH 6.5)-50 mM NaCl-50 mM MgCl2, injected into a Superose 12 HR 10/30 gel filtration column (Amersham Biosciences), and eluted at a rate of 0.4 ml/min. Elution volumes of the standards are represented by arrows 1 to 6. 1, thyroglobulin 669 kDa; 2, ferritin 440 kDa; 3, catalase 232 kDa; 4, aldolase 158 kDa; 5, ovalbumin 43 kDa; 6, RNase A 13.7 kDa. (B) SDS-PAGE of 5-µl aliquots of fractions collected during size exclusion chromatography analysis. All bands correspond in size to the R. albus 8 GSIII monomeric subunit (77 kDa).

Optimization of recombinant GSIII activity. Buffers other than Tris-HCl were reported to cause a dramatic decrease in the activity of Synechocystis sp. strain PCC 6803 GSIII (15). However, initial analysis of R. albus 8 GSIII in either Tris-HCl or imidazole buffer did not show any difference in activity of the protein (data not shown). Thus, in all activity assays, the imidazole-based buffer was used.

In addition to catalyzing biosynthesis of glutamine from ammonia, glutamate, and ATP (biosynthetic reaction), GS may catalyze glutamine hydrolysis into glutamate and ammonia in the presence of ADP (-GT reaction). The effects of pH, type of cation, cation concentration, and temperature on the forward and -GT activity assays were determined. The optimal enzymatic activity of R. albus 8 GSIII using the -GT assay occurred at pH 6.4, with activity decreasing until no apparent product, -glutamyl-hydroxamate, was formed below pH 5.0 or above pH 7.5 (results not shown). In the -GT assay, the R. albus 8 GSIII showed a clear preference for Mn²⁺ as the cation, and Mg²⁺, Ca²⁺, Co²⁺, or Fe²⁺ produced little to no detectable activity. The optimal concentration of Mn²⁺ for the -GT assay was 1 mM, and concentrations less than 0.5 mM or greater than 1 mM inhibited enzymatic activity. The R. albus 8 GSIII enzymatic activity exhibited a broad temperature optimum in the -GT assay with the most constant activity occurring between 25 and 37.5°C. A drastic decrease in activity, however, occurred at 40°C, and little or no activity was detected at 45°C or above (results not shown). Using the forward activity assay, the GSIII showed maximum activity at pH 6.2. The pH range for activity was narrower, compared to the -transferase assay, and no product was detected at pH values below 5.5 or above 7.0. Optimal enzymatic activity was with Mg²⁺ as the cation at a concentration of 50 mM, and activity remained stable between the concentrations of 40 and 100 mM. However, at concentrations of less than 40 mM, a drastic decrease in the rate of product formation occurred (results not shown). Substitution of Mg²⁺ with Mn²⁺ or Ca²⁺ resulted in GSIII activity decreasing by 30% and 90%, respectively. Furthermore, cation substitution with Co²⁺ or Fe²⁺ resulted in no detectable activity. Thus, in the forward assay, R. albus 8 GSIII showed a clear preference for Mg²⁺ as the cation. Enzymatic activity changed very little between 35 and 40°C, although a slight increase was discernible at 37.5°C. At 30°C, activity decreased to little or undetectable, and at 25 or 45°C and above no activity was detected.

Kinetic properties of recombinant GSIII of R. albus 8. Kinetic parameters of R. albus 8 GSIII were studied by using the -GT and forward assays. Substrate affinities were calculated by varying the concentration of one substrate, with the other substrates at saturating concentration. The apparent K_m values were determined by a double reciprocal Lineweaver-Burk plot for reactions that followed Michaelis-Menten kinetics. In the -GT assay, the calculated apparent K_m for ADP was 0.09 mM, with hydroxylamine-HCl and glutamine having apparent K_m values of 4.7 and 6.8 mM, respectively (Table 3). The estimated K_m values for the substrates in the forward assay were 4.5 mM for ATP and 1.14 mM for hydroxylamine-HCl. A slight inhibitory effect on GSIII was detected when the concentrations of ATP were greater than 24 mM. Glutamate did not follow Michaelis-Menten kinetics. Instead, various glutamate concentrations between 0.05 and 400 mM produced a linear graph. Therefore, a K_m value could not be determined by the Lineweaver-Burk method for glutamate. Ammonium chloride, as a substrate, did not follow Michaelis-Menten kinetics. Instead, a linear graph was obtained when ammonium chloride concentration was varied between 2.5 and 70 mM with the other substrates at saturating concentrations. Changes in enzyme concentrations and lengths of reaction time did not improve the results.

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TABLE 3. Apparent Kms of R. albus 8 GSIII and mutant E380A

Enzymatic properties of R. albus 8 recombinant GSIII mutants. In order to gain insight into the contribution of the four GSIII signature motifs to structure and function, site-directed mutagenesis was used to replace conserved glutamic acids, found in each motif, with alanine. In addition, we investigated the effect of mutating conserved lysines in motif C on the activity of R. albus 8 GSIII. Motif C-like sequences were identified previously as ATP-binding motifs (27). Seven different mutants were generated: E77A in motif I, E152A in motif II, E192A and E194A in motif III, E380A in motif IV, and K308A and K318A in motif C. Three assays (the -GT, forward, and biosynthetic assays) were used to compare the activities of the mutants with those of wild-type GSIII under the optimum conditions determined for the wild-type protein in the present experiment.

In the -GT assay, all mutant GSIII proteins showed decreased enzymatic activity, excluding mutant E77A, which exhibited no difference in activity within the margin of error (Fig. 6). The mutation E152A and K308A resulted in 25 and 65% decrease in activity, whereas E192A, E194A, E380A, and K318A exhibited no detectable activity (Fig. 6). In the forward activity assay, all mutations (E77A, E152A, E192A, E194A, K308A, and K318A) except E380A exhibited drastic decreases in activity compared to the wild-type GSIII. Unexpectedly, the E380A mutation in motif IV yielded 20% more product than the wild-type GSIII under the same conditions. Using the biosynthetic assay, the results obtained for the mutants in comparison with those of the wild-type proteins were quite similar to those obtained with the forward assay. Although in this assay the E380A mutant showed higher activity, the standard deviation was also large. Similar mutations in other GSIII homologs are necessary for a clearer conclusion.

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FIG. 6. Comparison of wild-type R. albus 8 GSIII activity with those of its mutants using the -transferase assay (), the forward assay (), and a biosynthetic assay (). The results are grouped as wild-type GSIII (wt GSIII), E77A (motif I), E152A (motif II), E192A and E194A (motif III), K308A and K318A (motif C, putative nucleotide-binding site), and E380A (motif IV). In the -transferase and forward assays 4 µg of purified GSIII protein was used, whereas in the biosynthetic assay 3 µg of protein was used. The results are the means and standard deviations of four replicates.

The increased enzymatic activity observed for mutant E380A led us to determine some of its kinetic properties using the forward assay. The apparent K_ms for ATP and hydroxylamine-HCl were 6.45 and 1.5 mM, respectively. Thus, both values were slightly higher than those of wild-type GSIII (Table 3). A K_m for ammonium chloride could not be estimated for the E380A mutant since the results did not follow Michaelis-Menten kinetics, as described above for the wild-type recombinant GSIII.

ATPase activity. The forward activity assay of GSIII requires ATP hydrolysis. Therefore, we compared the wild-type GSIII with the two mutants harboring mutations in motif C (K308A and K318A) and E380A, which exhibited an increased rate of product formation in the forward activity assay. Hydrolysis of ATP by the K308A mutant revealed that this particular mutation in the putative nucleotide-binding site (motif C) does not entirely abolish ATPase activity, although it decreased activity with respect to the wild-type protein (Fig. 7). Mutant K308A hydrolyzed about half the amount of ATP hydrolyzed by the wild type under the same conditions. On the other hand, little to no hydrolysis of ATP was observed for mutant K318A. Interestingly, mutant E380A hydrolyzed about three times the amount of ATP hydrolyzed by the wild-type GSIII under the same conditions (Fig. 7).

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FIG. 7. ATPase activities of R. albus 8 wild-type GSIII and its mutant forms. Hydrolyzed products were resolved by thin-layer chromatography and quantified with a phosphorimager. The results presented are for wild-type GSIII (wt), K308A and K318A (mutations in GS signature motif C), and E380A (GSIII signature motif IV). All assays were performed in replicates of four, and the means and standard deviations are shown.

CD. Due to the effects observed for the mutant GSIII proteins, we used CD to determine whether mutations generated in the four signature motifs and also in motif C of R. albus 8 GSIII led to gross structural changes in the protein. The CD spectra of recombinant wild-type GSIII and its mutant proteins displayed very similar spectra, which suggests that the mutations carried out in the present study did not grossly impact the secondary structural elements in the protein (results not shown).

Detection of glnN mRNA transcript levels. To investigate whether the glnN gene is regulated under different nitrogen conditions, the levels of glnN mRNA transcript in R. albus 8 cells grown under ammonia-limiting and excess-ammonia conditions were determined. In cells subjected to ammonia limiting growth conditions (1 mM NH₄Cl), a prominent 2.1-kb band, corresponding to the predicted size of glnN mRNA transcript, was detected by Northern blot with labeled glnN gene as the probe (Fig. 8, top panel). RNA transcripts from R. albus 8 cells grown under excess ammonia conditions (10 mM NH₄Cl) resulted in a markedly reduced level of the 2.1-kb mRNA transcript of glnN. In the Northern blots with the glnN gene as the probe, a faint band around 3.8 kb, corresponding to a transcript predicted to encode the glnN and amtB/glnK genes, was observed from R. albus 8 grown under ammonia limiting conditions (Fig. 8, top panel). To verify the presence of a glnN+amtB/glnK transcript, a probe targeting amtB/glnK was generated and a fresh membrane was probed for the amtB/glnK transcript. Under low ammonia levels (1 mM NH₄Cl) and with the amtB/glnK gene as a probe a band corresponding to the size of amtB/glnK (1.8 kb) was observed (Fig. 8, bottom panel). Moreover, in the same lane a band was observed at 3.8 kb, which is about the size of a transcript containing both the glnN and amtB/glnK genes. This result increases the likelihood of a transcript containing the two genes coding for glnN and amtB/glnK (Fig. 8, bottom panel).

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FIG. 8. Northern blot analysis. Detection of mRNA transcripts for glnN and amtB/glnK of R. albus 8 were carried out for cells grown under excess-ammonia (10 mM NH4+) and ammonia-limiting (1 mM NH4+) conditions. (Top panel) Detection of transcripts i and ii was with labeled glnN as the probe. Transcripts representing glnN+amtB/glnK (i) and glnN (ii) were detectable under ammonia-limiting conditions. (Bottom panel) Detection of transcripts i and ii was done with labeled amtB/glnK as the probe. Transcripts representing glnN+amtB/glnK (i) and amtB/glnK (ii) were detectable under ammonia-limiting conditions. In both panels, labeled 16S rRNA gene was used as a probe for total RNA load control (iii).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The limited number of studies on GSIII, along with its unique ecological distribution among different organisms, resulted in our interest to better understand its role in ammonia assimilation in R. albus 8. Initial verification of the identity of the putative GSIII was done through comparison of its predicted amino acid sequence with those of biochemically characterized and hypothetical GSIII proteins (Fig. 1). The five GS motifs (A, B, C, D, and E) shown here were identified previously by others (21). Subsequently, Crespo et al. identified four regions (I, II, III, and IV) of high homology in GSIII proteins, and this led to a hypothesis that these domains are essential for catalytic function (9). As can be clearly seen in Fig. 1, the four regions are highly conserved in the R. albus 8 GSIII, thus providing further support for this hypothesis.

The R. albus 8 glnN gene was highly expressed, and its gene product was purified close to homogeneity (Fig. 4). Using this recombinant protein, the biochemical characteristics of the enzyme were studied by using the biologically similar forward assay and the -GT assay. R. albus 8 is a major cellulolytic bacterium in the rumen, and the GSIII from this ruminal bacterium exhibited pH and temperature optima at 6.2 to 6.4 and 37°C, respectively. These parameters were estimated for a GSIII homolog in a cyanobacterium, Synechocystis sp. strain PCC 6803, to be 8.25 and 42°C, respectively (15). These differences are likely a reflection of the habitats in which these organisms reside. Clearly the optimal pH and temperature for the GSIII from R. albus 8 correlates well with the conditions of the ruminal environment (pH 5.7 to 7.3 and a temperature of 36 to 41°C).

The GSIII under study required the same metal cation for optimal biosynthetic activity (Mg²⁺) as its homolog from Synechocystis sp. strain PCC 6803 (15). Furthermore, reduced GSIII activity using the forward assay was demonstrated by substituting Mg²⁺ with Mn²⁺ and Ca²⁺. Only minimal amounts of products were detected with Mn²⁺ and Ca²⁺. This is consistent with published data on other GS proteins, providing further evidence that the GS proteins discriminate against most cations for activity (15, 20, 35). The K_m of the R. albus GSIII for glutamine in the -GT assay was similar to that determined for the cyanobacterium, Synechocystis sp. strain PCC 6803 GSIII. The apparent K_m for ADP was, however, much higher for the R. albus 8 homolog (90 versus 7.0 µM).

The contribution of the proposed GSIII signature motifs (I to IV) to structure and function is not well understood. In order to elucidate the role of these motifs in preserving the integrity of R. albus 8 GSIII, mutational studies targeting glutamic acid residues in each of the GSIII signature motifs were carried out. In addition, conserved lysines found in motif C, which is common to all GS proteins, were also mutated to study their role in ATP hydrolysis and enzyme activity. The glutamic acid residues were chosen for mutational analysis due to their polarity and large side chains, which often contribute to enzyme catalysis and/or proper protein folding. Also interestingly, each of the GSIII signature motifs contained at least one glutamic acid residue (Fig. 1). The residues of interest were mutated to alanine and enzymatic activity of each mutant protein was analyzed by using three different assays. In the -GT assay, mutants E77A and E152A exhibited little or no difference in enzymatic activity compared to the wild type, while all other mutations resulted in drastic decreases in activity. Aside from E380A, all mutations in the GSIII signature motifs almost abolished activity in the forward assay and biosynthetic assays. These results suggest important roles for the signature motifs in the structure and function of R. albus 8 GSIII. The -GT assay in GSI depends on the binding of ADP to the nucleotide-binding site, which induces a conformational change leading to an active protein (18). The forward reaction used in the present experiment and also the biosynthetic reaction are both energy-requiring reactions. Therefore, mutations (K308A and K318A) that replace conserved lysines with alanine in motif C, a putative nucleotide-binding site, may affect the stability of the GSIII/nucleotide complex, leading to an overall reduction in GS activity. The increase in GSIII activity of mutant E380A was surprising. It should be noted, however, that although this mutant hydrolyzed about three times the amount of ATP that was hydrolyzed by the wild-type protein (Fig. 7), we did not observed an equally elevated activity in the forward assay. This may suggest that the mutation uncoupled ATP hydrolysis from end product formation. Although the biochemical analysis for the mutants were carried out under the optimum conditions for the recombinant wild-type GSIII, it is expected that the mutants may have different optimum conditions for GS activity. Further characterization of the mutants, especially E380A, is needed to clarify the importance of the targeted amino acids in GSIII activity. We used CD spectroscopy to determine whether there were any gross changes in the mutant proteins. The results showed that although the mutants exhibited CD spectra that were similar to that of the wild-type protein, their mean molar ellipticities were slightly different, suggesting changes that did not heavily impact the structure. Mutational analysis of other GSIII proteins and more importantly, structural analysis of a GSIII protein will further clarify our observations.

To determine whether the GS type III plays an important role in vivo in R. albus 8 cells, the glnN mRNA transcript levels were analyzed under ammonia-limiting (1 mM NH₄⁺) and excess-ammonia (10 mM NH₄⁺) growth conditions. R. albus 8 cells grown under ammonia-limiting conditions clearly showed an increased level of glnN mRNA transcript, with little to no glnN transcript detected under excess ammonia condition. This is a good indication that the GSIII plays a role in ammonia assimilation during growth under low-nitrogen conditions. Also noted was a faint band at 3.8 kb, which may correspond to a transcript containing the genes encoding the GSIII and the AmtB/GlnK proteins. Note, however, that sequences that might represent distinct promoter sequences for the glnN and amtB/glnK are located upstream of each gene. It is possible that low-level transcripts containing all three genes occur from the single glnN promoter. To verify the presence of a single glnN and amtB/glnK transcript, a new membrane was prepared and probed by using the amtB/glnK gene specific probe, and this yielded an 1.8-kp product corresponding to the amtB/glnK transcript and an 3.8-kb band that may correspond to a transcript containing the glnN and amtB/glnK genes. Thus, the Northern blot data suggest that transcription of the glnN and amtB/glnK genes can occur from their own promoters, and, in addition, from the single glnN promoter site yielding a transcript containing the genes encoding the GSIII and the AmtB/GlnK proteins. We look forward to investigating how GlnK regulates its fused partner, AmtB, at the protein level in the future.

The biochemical and gene transcription evidence presented here suggests that the GSIII from R. albus 8 is a biologically functional protein and may play an important role in ammonia assimilation in this bacterium. The mutational studies provided, for the first time, an insight into the potential roles of the proposed GSIII signature motifs in the structure and function of GSIII proteins. Further studies, including mutational, structural, and biochemical analyses of other GSIIIs, would help elucidate the importance of the GSIII proteins in the microbes in which they are found.

 
We thank the North American Consortium for Genomics of Fibrolytic Ruminal Bacteria. We thank Sean Daugherty for help with bioinformatics analysis and S. Ohene-Adjei for help with CD scans.

We received financial support provided from U.S. Department of Agriculture grants AG99-35206-7950 and AGOSURF 868589.

 
* Corresponding author. Mailing address: Department of Animal Sciences, 1207 W. Gregory Dr., University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-2090. Fax: (217) 333-8804. E-mail: icann{at}uiuc.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, November 2005, p. 7481-7491, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7481-7491.2005
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