The Streptococcus mutans GlnR protein exhibits an increased affinity for the glnRA operon promoter when bound to GlnK

The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.

Most bacteria regulate nitrogen uptake via AmtB, a well-conserved ammonium transport membrane protein (9).Intracellularly, nitrogen is assimilated by the glutamine synthetase/glutamate synthetase (GS/GOGAT) pathway or the glutamate dehydrogenase (GDH) pathway (9).The regulation of nitrogen metabolism is coordinated by the PII-type signal transduction proteins, GlnB and GlnK (formerly NrgB), which control the activities of enzymes, membrane transport proteins, and transcription factors (9,10).Although the GS/GOGAT assimilatory enzymes and PII signal transduction proteins are widely conserved in Gram-positive bacteria, other modes of regulating nitrogen metabolism also exist (11).
The regulation of nitrogen metabolism in S. mutans is poorly understood.The best characterized Gram-positive bacterium is Bacillus subtilis, which is used as a model organism for low G+C Gram-positive bacteria.B. subtilis expresses two DNA-binding proteins of the MerR family, GlnR and TnrA, which cooperate in the transcriptional repression or activation of nitrogen metabolism genes (12,13).GlnR represses the glnRA and ureABC operons, which encode GlnR and glutamine synthetase and urea utilization enzymes, respectively, and the tnrA gene (14)(15)(16).TnrA activates the transcription of the amtB-glnK (ammonium transport), ureABC, nasBC and nasDEF (nitrate and nitrite utilization) operons and represses glnRA and gltAB, which encodes glutamate synthase (17)(18)(19)(20).Feedback-inhibited GS regulates the activities of both proteins by stabilizing the GlnR-DNA complex and preventing TnrA from binding to DNA (21,22).A study of the B. subtilis GlnK protein identified a GlnK-TnrA interaction; however, the physiological significance of this interaction remains unknown (23).
Studies of nitrogen metabolism in S. mutans may provide insight into the virulence mechanisms of this pathogen.In this report, we demonstrate that S. mutans GlnR binds to the promoter regions of amtB-glnK and glnRA operons.In addition, we show for the first time a stable interaction between the GlnR and GlnK proteins.GlnK within this complex enhances the affinity of GlnR for DNA.

Bacterial strains and growth conditions
Escherichia coli BL21-AI was grown at 37°C in Luria broth (LB) or Luria agar (LA) medium supplemented with kanamycin (50 µg/mL) as appropriate (Table 1).

Cloning, expression and purification of recombinant GlnR
The S. mutans UA159 glnR gene was amplified by PCR with primers N-GlnR and C-GlnR (Table 2) using S. mutans chromosomal DNA as a template.PCR products were digested with NdeI and BamHI and cloned into pET28b+ (Novagen, EMD4 Biosciences, USA) to produce the plasmid pET28R, which expresses GlnR-(His) 6 .The same digested PCR products were cloned into pET29b+ (Novagen, EMD4 Biosciences) to produce the plasmid pET29R, which expresses native GlnR (Table 1).These plasmids were then transformed into E. coli BL21-AI and transformants were selected on LA supplemented with kanamycin.
Bacteria transformed with pET29R were cultured on 1 L LB medium at 16°C for 16 h after induction with 0.2% arabinose.Cells were pelleted by centrifugation, resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.1 mM EDTA, 10% glycerol), and lysed by sonication.The soluble fraction of the cell extract was purified by ion exchange chromatography on a Q-Sepharose column (GE Healthcare Ltd., UK), and proteins were eluted with an NaCl gradient (0.2-1 M).Fractions containing GlnR were selected after SDS-PAGE visualization.These fractions were mixed and diluted in 50 mM Tris-HCl, pH 8.0, and 10% glycerol to a final concentration of 0.2 M NaCl and loaded onto a heparin column (GE Healthcare Ltd.).Proteins then were eluted through a second NaCl gradient (0.2-2 M).
GlnR fused to an N-terminal hexahistidyl tag was overexpressed in E. coli BL21-AI transformed with pET28R and grown on LA at 30°C.Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 40 mM imidazole, 10% glycerol), and disrupted by sonication.The GlnR-(His) 6 protein was purified from the soluble cell fraction by chromatography on an immobilized nickel ion affinity HisTrap HP column (GE Healthcare Ltd.).Proteins were eluted with an imidazole gradient (40 µM to 1 M).
S. mutans GlnK and GlnK-(His) 6 proteins were expressed and purified as described by Portugal et al. (27).

Polyclonal antibody production
Three Wistar albino rats (140 ± 20 g) were immunized subcutaneously with 20 µg GlnR or GlnK, both in native form, in complete Freund's adjuvant.Three booster injections were given at 4-week intervals and rats were bled 4 weeks after the last injection.Whole blood was centrifuged and the serum was used in the experiments.The antibody specificity was tested in dot blot experiments.
The effect of GlnK on the binding affinity of GlnR to PglnRA DNA was determined by adding various concentrations of native GlnK to the incubation buffer.
Approximated apparent dissociation constants (K 0.5 ) were calculated by fitting the data to a double-reciprocal plot as described by Levitzki (28).K 0.5 corresponds to the concentration of GlnR necessary for 50% of DNA binding sites to be occupied.

Cross-linking experiments
The interaction of GlnR and GlnK was further analyzed via chemical cross-linking with glutaraldehyde.GlnK and GlnR, both in native form, were incubated in HGNED buffer (1 mM DTT, 100 mM NaCl, 0.2 M EDTA, 0.05% [v/v] NP-40, 10% glycerol, 25 mM HEPES, pH 8.0) at room temperature for 15 min.Glutaraldehyde was added to a final concentration of 0.1% (v/v) and the reactions were quenched after 7 min by the addition of 0.1 volume of 2 M Tris.Proteins were fractionated by 15% SDS-PAGE (25) and transferred to polyvinylidene difluoride membranes (Millipore, USA) (29).Membranes were probed with mouse antiserum against GlnK or GlnR, and proteins were visualized by chemiluminescence using horseradish peroxidase-labeled secondary antibodies and luminol (ECL Western Blotting System, GE Healthcare Ltd.).
In these experiments, the Prestained Protein Molecular Weight Marker (Fermentas, Canada) was used to verify the Western transfer efficiency and to estimate the protein weight through relative mobility (30).
All experiments were performed at least twice.

GlnR binds DNA corresponding to glnK-amtB and glnRA promoters in vitro
GlnR binding to glnK-amtB and glnRA promoter regions was examined by EMSA.The K 0.5 values for PglnRA and PamtB-glnK were 50 and 24 nM, respectively (Figure 1A and  B).The PglnRA DNA fragment used in this study includes a putative palindromic binding site (ATGTNAN 7 TNACAT) (8) at -58 bp relative to the possible transcriptional start point (Figure 1C).The DNA fragment with the PamtB-glnK region contains the putative GlnR binding site at -18 bp relative to the possible transcriptional start point and a 6-base conserved repeat at -79 bp (Figure 1C).The presence of two band shifts in GlnR concentrations over 50 nM suggests that these binding sites are recognized by GlnR in vitro (Figure 1B).

GlnR interacts with GlnK in vitro
The oligomeric states of GlnR and GlnK and complex formation between these proteins were studied using chemical cross-linking with glutaraldehyde (Figure 2A and  B).GlnR existed as a dimer independently of the presence of glnRA promoter DNA (Figure 2A, lanes 2 and 3).After chemical cross-linking, GlnK protein samples were visualized as five bands with different migration characteristics (Figure 2B, lane 5).Although this result may be explained as a cross-linking artifact, it is also possible that GlnK exists as five different oligomers under the tested conditions.The observed bands possibly correspond to many oligomeric forms of GlnK.GlnR interacts directly with different putative GlnK oligomers (Figure 2A, lane 7).
Complex formation between native GlnR and GlnK-(His) 6 was examined using pull-down assays in the absence or presence of glnRA promoter DNA (Figure 2C and D).

C D
of DNA or in the presence of 15 nM glnRA promoter DNA (Figure 2), implying that DNA is not required for GlnR-GlnK complex formation.

GlnK enhances DNA-binding activity of GlnR in vitro
The effect of GlnK on the ability of GlnR to bind glnRA promoter DNA was determined using EMSA (Figure 3).Various concentrations of GlnK were added to 50 nM GlnR (protomer).In the presence of 150 nM GlnK (protomer), which corresponds to a 6-fold excess over dimeric GlnR (25 nM), binding of GlnR to DNA was increased 5-fold as estimated by band densitometry (Figure 3A, lane 5).This result suggests that two GlnK trimers or, possibly, the hexameric form of GlnK increases the affinity of the GlnR dimer for DNA.
The EMSA analyses detected the formation of a stable ternary complex between GlnR, GlnK and DNA and confirmed the results of the pull-down experiments in the presence of DNA.Long-term EMSA indicated that the ternary complex exhibited a considerably lower mobility than the GlnR-DNA complex (Figure 3B, lane 3).
The effect of GlnK on the affinity of GlnR binding to glnRA promoter DNA was also examined by EMSA (Figure 3C and D).GlnK did not bind DNA fragments under these conditions (Figure 3D, lane 2).In the presence of a 6-fold excess of GlnK protomer relative to the GlnR dimer, the K 0.5 was 13 nM (Figure 3D).When only GlnR was present in the reaction mixture, the K 0.5 was 50 nM (Figures 1A and  3C).These results indicate that GlnK increases GlnR-DNA binding activity by a factor of approximately 4.

Discussion
We report here the cloning, expression, and purification of the S. mutans GlnR protein.Our results demonstrate that S. mutans GlnR binds to glnRA promoter DNA, as described for B. subtilis GlnR (12), and that S. mutans GlnR binds the amtB-glnK promoter.These results agree with the in vivo results obtained by Chen et al. (8), which showed that S. mutans GlnR regulates its own transcription and the transcription of the amtB-glnK operon.
The present study describes for the first time an interaction between S. mutans GlnK and GlnR in vitro (Figure 2).EMSA analyses identified stable GlnR-GlnK-DNA complexes (Figure 3B).In B. subtilis, GlnK binds to the TnrA protein, which is functionally and structurally analogous to GlnR (23).However, no studies have examined the modulation of TnrA or GlnR activity by GlnK.
Compared to GlnR alone, S. mutans GlnR bound to GlnK has a 4-fold higher affinity for glnRA promoter DNA (Figure 3C and D), suggesting that GlnK enhances GlnR-mediated transcriptional repression of the glnRA promoter.
Interactions between GlnK and various transcription factors have been described for the Gram-positive Corynebacterium glutamicum.In this microorganism, adenylylated GlnK interacts with AmtR, a transcriptional repressor, leading to de-repression of genes involved in the control of nitrogen metabolism (31).In cyanobacteria, GlnK regulates gene expression by binding to NtcA and PipX proteins.NtcA is a global transcriptional activator, and PipX binds to and enhances the activity of NtcA during nitrogen starvation.Under nitrogen excess, GlnK interacts with PipX to impair NtcA activation (32)(33)(34).
Almost all PII-type proteins are trimeric; however, gel filtration chromatography results suggest that the GlnK protein of S. mutans has the molecular weight of a hexamer in solution (27).Supporting this, an S. mutans GlnK structure deposited in the Protein Data Bank (PDB ID: 3L7P) is described as a biological assembly of six domains (Fan X-X, Wang K-T, Su X-D; Protein Data Bank, 2011).Our results suggest that a 6-fold excess of GlnK promoter increases the affinity of the GlnR dimer for glnRA operon promoter DNA.This implies that two trimers of GlnK protein, or possibly its hexameric form, are responsible for the increase in GlnR DNA-binding activity (Figure 3A and D).
GlnR-DNA binding was confirmed in vitro for the amtB-glnK and glnRA promoters.The in vitro formation of a stable complex between GlnR and GlnK is described here for the first time.Once bound to GlnK, GlnR exhibits a higher affinity for glnRA promoter DNA.Information regarding nitrogen availability is likely to be transmitted to GlnR via GlnK binding in S. mutans.
Future in vivo studies should permit the determination of the physiological conditions in which the GlnR-GlnK interaction occurs.

Table 2 .
Primers used in the present study.
Restriction enzyme cleavage sites are underlined.