This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zalieckas, J. M.
Right arrow Articles by Fisher, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zalieckas, J. M.
Right arrow Articles by Fisher, S. H.

 Previous Article  |  Next Article 

Journal of Bacteriology, April 2006, p. 2578-2585, Vol. 188, No. 7
0021-9193/06/$08.00+0     doi:10.1128/JB.188.7.2578-2585.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA

Jill M. Zalieckas,{dagger} Lewis V. Wray Jr., and Susan H. Fisher*

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118

Received 7 December 2005/ Accepted 17 January 2006


arrow
ABSTRACT
 
Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which lies immediately upstream of the –35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the –35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the –35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 µM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.


arrow
INTRODUCTION
 
Nitrogen regulation in Bacillus subtilis is mediated by regulatory mechanisms distinct from those seen in enteric bacteria (12, 31). In Bacillus subtilis, two transcriptional factors, TnrA and GlnR, control gene expression in response to nitrogen availability (34, 40). The amino acid sequences of the N-terminal DNA binding domains of TnrA and GlnR are highly similar, and both proteins bind to DNA sequences (GlnR/TnrA sites) with a common consensus sequence (TGTNAN7TNACA) (18, 28, 35, 40). In contrast, the C-terminal signal transduction domains of TnrA and GlnR have no homology, and each protein is active under different growth conditions. TnrA activates and represses gene transcription when nitrogen is limiting for growth, while GlnR represses gene expression during growth with excess nitrogen (34, 40). Genetic studies have shown that glutamine synthetase (GS) is required for transduction of the nitrogen signal to both GlnR and TnrA (34, 40). Although the molecular mechanism by which GS controls GlnR repression is not known, the feedback-inhibited form of GS regulates the activity of TnrA through a protein-protein interaction where GS sequesters TnrA and prevents TnrA from binding to DNA (44).

The dicistronic glnRA operon, which encodes GlnR and GS, contains two GlnR/TnrA sites in its promoter region (6, 18, 36). The transcription of glnRA was previously shown to be repressed by both GlnR and TnrA (6, 34, 40). Mutational analysis has demonstrated that the GlnR/TnrA site positioned upstream of the –35 element of the glnRA promoter is required for nitrogen source-dependent regulation of glnRA expression (35). The tnrA gene has two GlnR/TnrA sites located upstream of its promoter, and only one of these sites is required for TnrA-dependent nitrogen regulation of tnrA expression (32). The studies reported in this communication demonstrate that tnrA expression is regulated by GlnR and define the cis-acting sites that control the GlnR- and TnrA-dependent regulation of glnRA and tnrA expression.


arrow
MATERIALS AND METHODS
 
Bacterial strains, cell growth, media, and enzyme assays. Table 1 lists B. subtilis strains used in this study. Derivatives of these strains containing lacZ fusions were constructed with plasmid DNA as previously described (41). The methods used for bacterial cultivation in the minimal medium of Neidhardt et al. (29) have been described previously (2). Glucose was added to a final concentration of 0.5%. All nitrogen sources were added to a final concentration of 0.2%.


View this table:
[in this window]
[in a new window]
  		TABLE 1. B. subtilis strains used in this study

Extracts for enzyme assays were prepared from cells grown to mid-log growth phase (70 to 90 Klett units). ß-Galactosidase activity was assayed in crude extracts as previously described (2). ß-Galactosidase activity was corrected for the endogenous ß-galactosidase activity present in B. subtilis cells containing the promoterless lacZ gene from pSFL6 integrated at the amyE site.

Plasmids and lacZ fusions. pTNR4 was constructed by inserting an EcoRI-BsaBI DNA fragment from pJVK75 (22) containing the tnrA promoter region between the EcoRI and StuI sites of pMTL21P (7). pTNR6 contains the EcoRI-HindIII tnrA promoter DNA fragment from pTNR4 cloned into the lacZ transcriptional fusion vector pSFL7 (39). Plasmid pTNR30 contains the EcoRI-HindIII fragment from pTNR4 cloned into pALTER-1 (Promega Corp.). Oligonucleotide mutagenesis with pTNR30 was performed using a protocol designed by the supplier of pALTER-1 (Promega Corp.). EcoRI-HindIII DNA fragments of the mutated promoters were cloned into pSFL7 to construct lacZ fusions for in vivo analysis.

pGLN16 contains a DraI-HpaI glnRA promoter fragment from pSF42 (13) cloned into the StuI site of pLEW424 (41). This DNA fragment contains promoter sequences extending from –77 to +80 with respect to the transcriptional start site (+1). The previously reported sequence of glnRA contains two cytosine residues in the nontemplate strand at positions –67 and –68 relative to the transcriptional start site (36). The sequence obtained in this study contained only one cytosine residue at this location. Plasmid pGLN17 is a transcriptional fusion between the glnRA promoter and lacZ gene that was constructed by cloning the EcoRI-HindIII glnRA promoter from pGLN16 into pSFL6 (11). pGLN30 contains the EcoRI-HindIII DNA fragment from pGLN16 cloned into pALTER-1. Following oligonucleotide mutagenesis of pGLN30, mutated glnRA promoter fragments were cloned into pSFL6 to generate transcriptional lacZ fusions. The tnrA-lacZ and glnRA-lacZ fusions were integrated into 168 strains (Table 1) as single copies at the amyE locus.

Electrophoretic mobility shift assay for TnrA DNA binding. TnrA protein purification was done according to a published procedure (43). Gel mobility shift assays were performed as previously described (43) with the modifications that 100 µg/ml of poly(dG-dC) · poly(dG-dC) was used as the nonspecific competitor DNA and Triton X-100 was replaced with 3-(N,N-dimethyloctylammonio)-propanesulfonate. Gels were dried and exposed to a phosphor storage screen. Band intensities were determined with the volume measurement function in ImageQuant software (Molecular Dynamics). For DNA fragments with a single TnrA binding site, the fraction of bound DNA was calculated as the volume of the protein-bound DNA band divided by the sum of the free and bound bands. The equilibrium dissociation binding constant (KD) was determined by using nonlinear regression analysis to fit the data to the equation {theta} = Pt/(Pt + KD), where {theta} is the fraction of bound DNA and Pt is the total protein concentration. For DNA fragments with two TnrA binding sites, the fraction of bound DNA was calculated as the sum of the volume of the band with a single TnrA molecule bound and twice the volume of the band with two TnrA molecules bound divided by the sum of the volumes of all three bands. These data were fit to the equation {theta} = Pt/(Pt + KD1) + Pt/(Pt + KD2), which describes the binding isotherm for two independent binding sites (9).

Primer extension analysis. RNA was isolated from B. subtilis cells grown to mid-log growth phase (70 to 90 Klett units) by extraction with guanidine thiocyanate and CsCl centrifugation (14). Primer extensions were performed as previously described (14) with 2 µg of RNA and oligonucleotide primers TNR8 (5'-TCAATTCACTCACAATTCCG) and TNR9 (5'-ATACCTGATCTGTCTTACGG) that are complementary to the 5' end of the tnrA coding region.

Bioinformatics analysis. The GlnR and TnrA protein sequences used in this analysis were taken from bacterial strains that encode both of these proteins. The accession numbers for the GlnR and TnrA protein sequences, respectively, are: M22811 and AAC44335 (B. subtilis); YP_079161 and YP_078674 (Bacillus licheniformis); EAM86191 and EAM88231 (Exiguobacterium sp. strain 2515-15); BAC13606 and BAC12906 (Oceanobacillus iheyensis); BAD75611 and BAD76145 (Geobacillus kaustophilus). The Geobacillus stearothermophilus GlnR and TnrA sequences were obtained from the web site for the Geobacillus stearothermophilus genome sequencing project (http://www.genome.ou.edu/bstearo.html). Multiple-protein sequence alignments were generated with ClustalW (37). Consensus sequence logos were produced with WebLogo (10, 33).


arrow
RESULTS AND DISCUSSION
 
Identification of the tnrA transcriptional start site. Primer extension analysis was used to identify the apparent transcriptional start site for tnrA and to determine whether tnrA transcription is regulated in response to nitrogen availability. A ladder of products was obtained in primer extension experiments using RNA isolated from cells grown on glucose minimal medium containing the limiting nitrogen source glutamate (Fig. 1). The same pattern of cDNA products was obtained in experiments using two different oligonucleotide primers as well as with RNA preparations isolated from independent cultures (data not shown). Previous studies have shown that this type of heterogeneity can result from the addition of extra residues to the 5' end of RNA transcripts during initiation at promoters which contain homopolymeric sequences at the start site (17, 26, 45). Indeed, the +1 position for tnrA transcription, which corresponds to the most abundant cDNA product, is located at the beginning of four consecutive adenine residues (Fig. 2).


Figure 1
View larger version (72K):
[in this window]
[in a new window]
  		FIG. 1. Primer extension analysis of the tnrA promoter. B. subtilis RNA was isolated from wild-type cells grown in glucose minimal medium containing either glutamate plus ammonium (lane 3) or glutamate (lane 2) as the nitrogen source. Saccharomyces cerevisiae RNA was used in the primer extension shown in lane 1. The TNR8 oligonucleotide primer was used for dideoxy sequencing of pTNR4 (lanes A, C, G, T) and the primer extension reactions.


Figure 2
View larger version (10K):
[in this window]
[in a new window]
  		FIG. 2. Nucleotide sequence of the tnrA promoter region. The proposed –35 and –10 promoter elements are overlined (19, 21). The GlnR/TnrA consensus sequence for the two binding sites is shown above the nucleotide sequence. The transcriptional start site is indicated by an asterisk. The nucleotide substitutions in TNR26, TNR35, TNR37, and TNR38 are indicated below the nucleotide sequence. The upstream endpoint of the deletion in TNR142 is indicated by an EcoRI restriction site (GAATTC).

Examination of DNA sequences upstream of the apparent tnrA transcriptional start site reveals that the –10 region (TATACT) has only one mismatch with the {sigma}A consensus sequence (TATAAT), while there are five mismatches between the –35 region (AGGTTT) and the {sigma}A consensus sequence (TTGACA) (19, 21). This suggests that the tnrA promoter is a nonoptimal {sigma}A-dependent promoter with a low level of intrinsic transcriptional activity. Transcription from the tnrA promoter is nitrogen regulated because only very low levels of the primer extension products were obtained with RNA isolated from cells grown with excess nitrogen (glutamate plus ammonium) (Fig. 1). The regulation of tnrA expression observed in these primer extension experiments is consistent with the results of the transcriptional lacZ fusion studies obtained by Robichon et al. (32) and described below.

Nitrogen regulation of tnrA expression. Regulation of tnrA expression was examined in vivo using the (tnrA-lacZ)6 transcriptional fusion. This lacZ fusion contains a tnrA promoter fragment extending from –222 to +66 with respect to the transcriptional start site and contains two GlnR/TnrA binding sites (Fig. 2). One site, designated GlnR/TnrA site 2, is centered 94 bp upstream of the tnrA transcriptional start site and was previously shown to be required for high-level expression of the divergently transcribed ykzB gene (32). The second site, designated GlnR/TnrA site 1, is centered 51 bp upstream of the tnrA transcriptional start site. The location of GlnR/TnrA site 1 in the tnrA promoter is similar to the position of TnrA binding sites in other TnrA-activated promoters (15, 27, 28, 42).

As expected, expression of the (tnrA-lacZ)6 fusion is nitrogen regulated. In a wild-type strain, ß-galactosidase levels are 367-fold higher in cells grown with limiting nitrogen (glutamate) than in cells grown with excess nitrogen (glutamine) (Table 2). Examination of the expression of the (tnrA-lacZ)6 fusion in wild-type and mutant cells revealed that both TnrA and GlnR are involved in nitrogen regulation of tnrA expression. In glutamate-grown cultures, the (tnrA-lacZ)6 fusion was expressed at 55-fold lower levels in a tnrA mutant than in a wild-type strain, while no difference in expression levels were seen between wild-type and tnrA mutant cells grown with glutamine as the nitrogen source (Table 2). These results are consistent with previous observations that TnrA functions as a transcriptional regulator only during nitrogen-limited growth (40). Interestingly, tnrA expression retains some nitrogen regulation in a tnrA mutant. ß-Galactosidase levels from the (tnrA-lacZ)6 fusion in a tnrA mutant are 10-fold higher in glutamate-grown cells than in glutamine-grown cells (Table 2). The residual nitrogen regulation seen in tnrA cells is mediated by GlnR because the (tnrA-lacZ)6 fusion is expressed constitutively in a {Delta}glnR tnrA double mutant (Table 2). Expression of the (tnrA-lacZ)6 fusion in glutamine-grown cultures was sevenfold higher in a {Delta}glnR mutant than in a wild-type strain (Table 2). Since GlnR only regulates gene expression during growth with excess nitrogen, repression of tnrA expression by GlnR should not be observed under nitrogen-limited conditions. Indeed, similar levels of (tnrA-lacZ)6 expression were observed in {Delta}glnR and wild-type strains grown with glutamate (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. ß-Galactosidase expression of tnrA-lacZ fusions in wild-type and mutant strains

To determine whether GlnR/TnrA site 1 is required for nitrogen regulation of tnrA expression, this site was inactivated by introduction of symmetrical mutations (TNR38) in the highly conserved C and G bases in the GlnR/TnrA consensus sequence (Fig. 2). The TNR38 mutation would be expected to inactivate this GlnR/TnrA site because the same alteration in the amtB TnrA binding site severely reduced the in vivo expression of amtB (42). The observation that the (tnrA-lacZ)38 fusion expressed ß-galactosidase at low constitutive levels (Table 2) indicates that this site is required for regulation of tnrA expression by both TnrA and GlnR. Double mutations (TNR35 and TNR37) (Fig. 2) in each half site of GlnR/TnrA site 1 completely relieved GlnR-dependent repression but allowed low-level TnrA-dependent nitrogen regulation (Table 2).

Mutational analysis demonstrated that GlnR/TnrA site 2 is not required for regulation of tnrA expression by TnrA or GlnR. No defect in nitrogen regulation of tnrA expression by TnrA or GlnR was observed with tnrA-lacZ fusions containing either a mutationally inactivated [(tnrA-lacZ)26] or deleted [(tnrA-lacZ)142] GlnR/TnrA site 2 (Fig. 2; Table 2). Surprisingly, ß-galactosidase levels in glutamate-grown wild-type cells containing lacZ fusions lacking a functional GlnR/TnrA site 2 [(tnrA-lacZ)26 and (tnrA-lacZ)142] were 2- to 2.5-fold higher than that seen with the (tnrA-lacZ)6 fusion (Table 2). This suggests that TnrA bound at the GlnR/TnrA site 2 inhibits transcription from the tnrA promoter. Interestingly, expression of the ilv-leu operon is also inhibited by the binding of TnrA to a site located 200 bp upstream of the transcriptional start site (38). The molecular mechanism for this mode of transcription inhibition of these two promoters is not known. The observation that only GlnR/TnrA site 1 is required for regulation of tnrA expression by TnrA was also made by Robichon et al. (32).

Since the GlnR/TnrA site 1 is located immediately upstream of the –35 element of the tnrA promoter (Fig. 2), TnrA bound at this site most likely activates transcription by directly interacting with RNA polymerase. In contrast, GlnR bound to the same site represses tnrA transcription. GlnR may repress tnrA expression by sterically hindering the binding of RNA polymerase or by masking an UP element that activates the basal level of transcription when TnrA is not bound at this site. The observation that GlnR does not activate transcription of the tnrA promoter indicates that GlnR lacks the transcriptional activation region that is present in TnrA.

Interaction of TnrA with the tnrA promoter region. Gel mobility shift experiments were used to examine the interaction of TnrA with wild-type and mutated tnrA promoter DNA fragments. poly(dG-dC) · poly(dG-dC) was used as the nonspecific competitor DNA in these experiments. This was necessary to prevent nonspecific binding of TnrA to glnRA promoter DNA when high TnrA concentrations were used (see below). In previous studies where the binding of TnrA to the amtB promoter DNA was measured with poly(dI-dC) · poly(dI-dC) as the competitor DNA, a KD of 7.7 ± 1.0 nM was obtained (43). In contrast, when poly(dG-dC) · poly(dG-dC) was used as the nonspecific competitor, a KD of 66 ± 5 nM was obtained (data not shown). The difference in these binding constants is most likely a reflection of the competition between the amtB DNA fragment and the nonspecific DNA for the binding of TnrA. These results indicate that TnrA has a higher affinity for poly(dG-dC) · poly(dG-dC) DNA than for poly(dI-dC) · poly(dI-dC) DNA.

When the binding of TnrA to a tnrA promoter DNA fragment that contained both GlnR/TnrA sites (TNR6) was examined, two shifted bands were observed (Fig. 3A). Nonlinear regression was used to fit the binding data from this experiment to an equation for two independent binding sites (see Materials and Methods). This analysis revealed that the KDs for the binding of TnrA to these two sites were 52 ± 7 and 750 ± 100 nM. Experiments that examined the binding of TnrA to DNA fragments that contained mutations in either GlnR/TnrA site 1 or site 2 revealed that TnrA binds more tightly to the GlnR/TnrA site 1 than to site 2. The KD of TnrA for the TNR26 DNA fragment, which contains only a functional GlnR/TnrA site 1, was 55 ± 5 nM (Fig. 3B). The affinity of TnrA for the TNR38 DNA fragment, which only has an active GlnR/TnrA site 2, was 800 ± 80 nM (Fig. 3C). The observation that TnrA has a higher affinity for site 1 than site 2 is most likely explained by the fact that site 1 has a perfect match with the consensus sequence, while site 2 contains two mismatches (Fig. 2). TnrA binds independently to the two sites upstream of the tnrA promoter because the TnrA binding affinities obtained with the DNA fragment containing both sites (TNR6) are essentially identical to the individual affinities for the DNA fragments that contained only a single functional site (TNR26 and TNR38).


Figure 3
View larger version (120K):
[in this window]
[in a new window]
  		FIG. 3. Gel mobility shift analysis of the binding of TnrA to wild-type and mutant tnrA promoter fragments. The binding reactions contained the DNA fragments TNR6 (A), TNR26 (B), and TNR38 (C). For each diagram, the lowest row of bands corresponds to free DNA, the middle row to DNA having TnrA bound to one site, and the top row to DNA having TnrA bound to two sites. Concentrations of TnrA used in each binding reaction are as follows: no TnrA (lane 1), 4 nM (lane 2), 8 nM (lane 3), 16 nM (lane 4), 32 nM (lane 5), 64 nM (lane 6), 128 nM (lane 7), 256 nM (lane 8), 512 nM (lane 9), 1,024 nM (lane 10), and 2,048 nM (lane 11).

Regulation of glnRA expression. Expression of the glnRA promoter is repressed by both GlnR and TnrA (Table 3) (6, 34, 40). The glnRA promoter region contains two GlnR/TnrA sites (Fig. 4). The glnRAo1 site, which is centered 50 bp upstream of the glnRA transcriptional start site, lies immediately upstream of the –35 promoter element and was previously shown to be required for nitrogen source-dependent regulation of glnRA expression (35). The glnRAo2 site is centered 26 bp upstream of the glnRA transcription site and overlaps the –35 region of the glnRA promoter. The function of the glnRAo2 operator in nitrogen regulation has not been previously examined.


View this table:
[in this window]
[in a new window]
  		TABLE 3. ß-Galactosidase expression of glnRA-lacZ fusions in wild-type and mutant strains


Figure 4
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 4. Nucleotide sequence of the glnRA promoter region. The proposed –35 and –10 promoter elements are overlined (19, 21). The GlnR/TnrA consensus sequence is shown above the operator sites (6, 18, 35). The transcriptional start site is indicated by an asterisk. The nucleotide substitutions in GLN34, GLN37, and GLN38 are indicated below the nucleotide sequence. The upstream endpoint of the deletion in GLN51 is indicated by an EcoRI restriction site (GAATTC).

The specific roles of these operators in the TnrA- and GlnR-dependent regulation of glnRA expression were determined by individually inactivating each site. Two base pair changes are present in the glnRAo1 operator of the (glnRA-lacZ)37 fusion, while the glnRAo1 operator is completely deleted in the (glnRA-lacZ)51 fusion (GLN37 and GLN51, respectively, in Fig. 4). In agreement with previous studies (35), both mutations abolish GlnR-dependent regulation of glnRA expression. In wild-type cells grown with excess nitrogen, where GlnR is active, ß-galactosidase levels from the (glnRA-lacZ)37 and (glnRA-lacZ)51 fusions were 120- and 90-fold higher, respectively, than the levels from the (glnRA-lacZ)17 fusion (Table 3). The level of expression from the (glnRA-lacZ)37 and (glnRA-lacZ)51 fusions in glutamine-grown cells was not altered by {Delta}glnR or tnrA mutations (Table 3).

Expression from the two lacZ fusions lacking a functional glnRAo1 operator is still repressed by TnrA. During nitrogen-limited growth, where TnrA is active, ß-galactosidase expression from the (glnRA-lacZ)37 and (glnRA-lacZ)51 fusions is 2.5- to 3-fold lower in wild-type and {Delta}glnR cells than in tnrA cells (Table 3). Since ß-galactosidase levels in nitrogen-limited wild-type and {Delta}glnR cells containing the wild-type (glnRA-lacZ)17 fusion are also 2.5- to 3-fold lower than in tnrA cells (Table 3), the glnRAo1 operator is not required for TnrA-dependent repression of glnRA expression.

The glnRAo2 operator was mutationally inactivated by the introduction of two base pair changes within one half site so that residues critical for promoter function were not altered (GLN34 in Fig. 4). This mutation had a significant effect on the GlnR-dependent regulation of glnRA transcription. In glutamine-grown wild-type strains, expression of the (glnRA-lacZ)34 fusion was 38-fold higher than that seen with the (glnRA-lacZ)17 fusion (Table 3). This derepression was also observed in tnrA strains under the same growth conditions (Table 3). ß-Galactosidase levels from the (glnRA-lacZ)34 fusion were 2.5-fold higher in {Delta}glnR cells than in wild-type and tnrA cells during growth with excess nitrogen (Table 3), indicating that the GLN34 mutation retained a low level of GlnR-dependent repression.

The GLN34 mutation was also found to relieve TnrA-dependent repression of glnRA expression during nitrogen-limited growth. In glutamate-grown wild-type strains, ß-galactosidase levels from the (glnRA-lacZ)34 fusion were 2.5-fold higher than that seen with the (glnRA-lacZ)17 fusion (Table 3). This derepression of the (glnRA-lacZ)34 fusion was also observed with glutamate-grown {Delta}glnR cells (Table 3), indicating that this difference in expression did not result from the relief of residual GlnR-dependent repression. These observations indicate that only the glnRAo2 operator is required for TnrA-dependent repression of glnRA expression. Since this operator overlaps the –35 region of the glnRA promoter, TnrA most likely represses glnRA transcription by inhibiting RNA polymerase binding to promoter sequences.

Previous studies showing that GlnR binds simultaneously to both operators of the glnRA promoter led to the proposal that GlnR binds cooperatively to these two sites and blocks RNA polymerase binding to promoter sequences (6, 18). The observation that a 5-bp insertion between the two glnRA operators prevents GlnR repression supports this hypothesis (35). Our mutational results showing that both operators are required for GlnR-dependent repression of the glnRA promoter also provides evidence that the binding of GlnR to these two sites is cooperative.

Expression of the ure P3 promoter, which contains two GlnR/TnrA sites, is regulated by GlnR (39). One site is centered 91 bp upstream of the ure P3 transcriptional start site, while the other site is centered 15 bp downstream of the transcriptional start site. The mutational inactivation of either site or the introduction of spacer mutations between the two sites significantly reduced the levels of GlnR repression of ure expression. Thus, GlnR most likely binds cooperatively to the two GlnR/TnrA sites in the ure P3 promoter region (4). Since cooperative GlnR binding occurs at the two GlnR/TnrA sites in the glnRA and ure P3 promoters, it is noteworthy that GlnR-dependent repression of the tnrA promoter only requires a single binding site. No other potential GlnR/TnrA site can be identified in the tnrA promoter by sequence analysis. This observation indicates that GlnR must bind with sufficient affinity to the single operator in the tnrA promoter region to regulate tnrA transcription. Because multiple factors, including operator location, promoter activity, and repressor affinity, can influence the level of repression observed at negatively regulated promoters (23), it is difficult to provide a cohesive explanation for the difference in the location and number of binding sites required for GlnR-mediated regulation at the glnRA, ure P3, and tnrA promoters. However, the observation that a single site is sufficient for GlnR-dependent regulation of tnrA expression implies that the cooperative binding of GlnR at two appropriately spaced operators is not an inherent requirement for GlnR-mediated regulation of gene expression.

Interaction of TnrA with the glnRA promoter region. When TnrA binding to the wild-type glnRA promoter region was examined in gel mobility shift experiments, two shifted bands were observed at high TnrA concentrations (Fig. 5A). This result indicates that the glnRA promoter fragment contains two TnrA binding sites. DNA fragments with mutations in either glnRAo1 (GLN37) or glnRAo2 (GLN34) had reduced affinity for TnrA (Fig. 5B and C), indicating that these two operators are the sites where TnrA binds. Saturated levels of binding were not observed at the highest TnrA protein concentrations used in these experiments. Thus, both glnRA operators function as poor binding sites for TnrA with binding constants greater than 3 µM.


Figure 5
View larger version (111K):
[in this window]
[in a new window]
  		FIG. 5. Gel mobility shift analysis of the binding of TnrA to wild-type and mutant glnRA promoter fragments. The binding reactions contained DNA fragments GLN17 (A), GLN34 (B), and GLN37 (C). For each diagram, the lowest row of bands corresponds to free DNA, the middle row to DNA having TnrA bound to one site, and the top row to DNA having TnrA bound at two sites. Concentrations of TnrA used in each reaction are as follows: no TnrA (lane 1), 50 nM (lane 2), 100 nM (lane 3), 200 nM (lane 4), 400 nM (lane 5), 800 nM (lane 6), 1,600 nM (lane 7), and 3,200 nM (lane 8).

GlnR and TnrA have different DNA binding specificities. Several observations indicate that GlnR and TnrA have the ability to discriminate between different GlnR/TnrA binding sites. First, although the tnrA site 1 and glnRAo1 binding sites have perfect matches with the GlnR/TnrA consensus sequence and contain the A+T-rich region between the two half-sites previously shown to be required for optimal TnrA binding in vitro (43), TnrA binds significantly more tightly to tnrA site 1 (KD of 55 nM) than to the glnRAo1 operator (KD > 3 µM). In addition, previous studies have reported that GlnR binds very tightly to the glnRA promoter region (KD of 10 pM), while TnrA binds weakly to same promoter region (KD > 3 µM) (6). Although different in vitro conditions were used to determine the DNA binding affinity of GlnR and TnrA for the glnRA promoter region, these results are consistent with in vivo results which indicate that GlnR strongly represses glnRA expression, while TnrA is only a weak inhibitor. These observations suggest that, in addition to the TGTNAN7TNACA consensus sequence, GlnR/TnrA sites contain other sequence determinants that allow preferential binding by either GlnR or TnrA.

The interactions between protein residues and DNA bases play a major role in determining DNA binding specificity. The ability of DNA-binding proteins within the same family to recognize different target sequences results from differences in base-contacting amino acid residues (24). Thus, the ability of GlnR and TnrA to discriminate between different binding sites should be reflected in the sequences of their DNA target sites and their DNA-binding domains. GlnR and TnrA are members of the MerR family of transcription factors (5, 34, 40). The crystallographic structures of several MerR family proteins have revealed that their DNA-binding domains contain a winged-helix motif (8, 16, 20, 30). The DNA-bound structures of BmrR and MtaN have demonstrated that protein-DNA contacts are mediated by a helix-turn-helix motif, a ß-loop wing, and a second wing consisting of helices 3 and 4 (Fig. 6). Although amino acid residues that contact the DNA backbone are located throughout the DNA binding domains of BmrR and MtaN, the residues that have sequence-specific interactions with DNA bases are only found in the recognition helix of the helix-turn-helix motif and the ß-loop wing (30).


Figure 6
View larger version (19K):
[in this window]
[in a new window]
  		FIG. 6. Alignment of the DNA-binding domains of MerR family proteins. Amino acid residues of MtaN and BmrR that make contacts with specific DNA bases are highlighted with gray shading (30). A graphic representation of the secondary structures of MtaN and BmrR is shown at the bottom where {alpha}-helices and ß-strands are depicted as cylinders and arrows, respectively.

To identify amino acid residues responsible for the DNA sequence discrimination by GlnR and TnrA, the sequences of the DNA binding domains of GlnR and TnrA from bacteria which encode both of these transcriptional regulators were aligned (see Materials and Methods). The amino acid sequences of the recognition helices of TnrA (residues 28 to 35) and GlnR (residues 26 to 33) were found to be completely conserved between all of these proteins (data not shown). In contrast, comparison of the amino acid consensus sequence logos of the ß-loop wings revealed that there is significant sequence divergence in this segment of the DNA binding domain (Fig. 7). This observation suggests that TnrA and GlnR discriminate between distinct DNA binding sites by using one or more amino acid residues in their ß-loop wings to preferentially interact with different DNA bases. Interestingly, the structures of the BmrR-DNA and MtaN-DNA complexes reveal that the ß-loop wings make contact with bases located at the outside ends of their DNA binding sites (30). Thus, it is tempting to speculate that the ability of GlnR and TnrA to discriminate between different binding sites is dependent upon DNA residues located outside of the conserved TGTNAN7TNACA consensus sequence. Comparison of known DNA binding sites for GlnR and TnrA did not identify any differentially conserved bases either within or adjacent to the consensus sequence that could account for the observed binding site discrimination. This analysis of GlnR and TnrA binding sites was limited in that only a small number of GlnR regulatory sites have been identified and quantitative binding data are available for only a few of the known GlnR and TnrA sites. Thus, a more extensive analysis will be required to precisely define the differences in DNA-binding by GlnR and TnrA.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7. Amino acid consensus sequence logos for the ß-loop wings of GlnR and TnrA homologs. The numbers shown below each logo correspond to the residues of the B. subtilis proteins.

It has been previously reported that GlnR-dependent regulation of glnRA expression is relieved by mutational replacement of the thymine at position –41 by a cytosine (GLN38 in Fig. 4) (35). Interestingly, this thymine lies immediately outside of the TnrA/GlnR consensus sequence of the glnRAo1 operator. Since the analogous mutation within the TnrA binding site of the amtB promoter was shown to have no effect upon the regulation of this promoter (42), a lacZ fusion containing this mutation, (glnRA-lacZ)38, was constructed to examine its role in nitrogen regulation of glnRA expression. When either glutamate or glutamine was used as the nitrogen source, ß-galactosidase levels in cells containing the (glnRA-lacZ)38 fusion were essentially identical to the levels obtained with the wild-type (glnRA-lacZ)17 fusion (Table 3). These results indicate that the thymine-to-cytosine mutation at position –41 does not affect the GlnR-dependent regulation of glnRA expression and argue that this nucleotide change does not alter binding site discrimination by GlnR. We have no explanation for the difference between our results and the previously published observations of Schreier et al. (35).

Cross-regulation of tnrA and glnRA expression by GlnR and TnrA. The use of the TnrA and GlnR dual-regulator system provides the cells with considerable flexibility in regulating the expression of genes involved in nitrogen metabolism. Although GlnR and TnrA share sequence similarity, these proteins differ in that each protein is functional under different nutritional conditions. One consequence of this dual regulatory system is the need for each protein to have a distinctive DNA binding site specificity. For example, if GlnR and TnrA had identical DNA binding specificities, then glnRA expression would be severely repressed during nitrogen-limited growth by TnrA. Since increased levels of glutamine synthetase is the first adaptive response to nitrogen-limited growth (1, 25), there would have been severe selective pressure against high-level repression of glnRA transcription by TnrA during the evolution of bacteria encoding both GlnR and TnrA. Thus, the weak repression of glnRA expression by TnrA is most likely a vestige of the common evolutionary ancestry of GlnR and TnrA.

TnrA is regulated at two different levels. First, the transcription of the tnrA gene is repressed by GlnR when nitrogen is in excess and activated by TnrA during nitrogen-limited growth. Second, during growth with excess nitrogen, the activity of TnrA is posttranslationally inhibited by a protein-protein interaction with the feedback-inhibited form of glutamine synthetase (44). GlnR and TnrA control tnrA transcription by binding to the same regulatory site. Interestingly, the TnrA-activated amtB, ansZ, gabP, nasA, nasB, ykzB, and tnrA promoters all contain GlnR/TnrA sites upstream of the –35 promoter element in a similar position with respect to the transcriptional start site. Despite these similarities, GlnR-dependent repression has only been observed at the tnrA promoter (1, 15, 28; L. V. Wray and S. H. Fisher, unpublished observations). It is not known if this difference in regulation reflects a difference in the ability of GlnR to bind to these regulatory sites or whether GlnR-dependent regulation is only observed at the tnrA promoter because this promoter has a higher basal level of expression than the other promoters. Indeed, it would make good sense for the tnrA promoter to have a high basal level of transcription because TnrA autoregulates its own expression. Since TnrA would be unable to activate its own expression unless GlnR-dependent repression was relieved, it is possible that the repression of tnrA transcription by GlnR prevents inappropriate tnrA expression under conditions of nitrogen excess. However, the observation that regulation of amtB expression is not altered when the tnrA gene is transcribed from a constitutive promoter argues that the modulation of TnrA autoregulation by GlnR does not play a significant role in controlling the expression of TnrA-activated promoters (43). Taken together, these results argue that the regulation of TnrA activity by the feedback-inhibited form of GS is the primary mechanism for controlling the levels of active TnrA and the expression of TnrA-regulated promoters.


arrow
ACKNOWLEDGMENTS
 
We thank Vesa Kontinen for providing plasmid pJVK75.

The research was supported by Public Health Service research grant GMA51127 from the National Institutes of Health.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Phone: (617) 638-5498. Fax: (617) 638-4286. E-mail: shfisher{at}bu.edu. Back

{dagger} Present address: Lahey Clinic, 41 Mall Road, Burlington, MA 01805. Back


arrow
REFERENCES
 
    1
  1. Atkinson, M. R., and S. H. Fisher. 1991. Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis. J. Bacteriol. 173:23-27.[Abstract/Free Full Text]
    2. 2
  2. Atkinson, M. R., L. V. Wray, Jr., and S. H. Fisher. 1990. Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis. J. Bacteriol. 172:4758-4765.[Abstract/Free Full Text]
    3. 3
  3. Biaudet, V., F. Samson, C. Anagnostopoulos, S. D. Erlich, and P. Bessières. 1996. Computerized map of Bacillus subtilis. Microbiology 142:2669-2729.[Free Full Text]
    4. 4
  4. Brandenburg, J. L., L. V. Wray, Jr., L. Beier, H. Jarmer, H. H. Saxlid, and S. H. Fisher. 2002. Roles of PucR, GlnR, and TnrA in regulation expression of the Bacillus subtilis ure P3 promoter. J. Bacteriol. 184:6060-6064.[Abstract/Free Full Text]
    5. 5
  5. Brown, N. L., J. V. Stoyanov, S. P. Kidd, and J. L. Hobman. 2003. The MerR family of transcriptional regulators. FEMS Microbiol. Rev. 27:145-163.[CrossRef][Medline]
    6. 6
  6. Brown, S. W., and A. L. Sonenshein. 1996. Autogenous regulation of the Bacillus subtilis glnRA operon. J. Bacteriol. 178:2450-2454.[Abstract/Free Full Text]
    7. 7
  7. Chambers, S. P., S. E. Prior, D. A. Barstow, and N. P. Minton. 1988. The pMTL nic– cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139-149.[CrossRef][Medline]
    8. 8
  8. Changela, A., K. Chen, Y. Xue, J. Holschen, C. E. Outten, T. V. O'Halloran, and A. Mondragón. 2003. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301:1383-1387.[Abstract/Free Full Text]
    9. 9
  9. Copeland, R. A. 2000. Enzymes: a practical introduction to structure, mechanism, and data analysis, p. 84-85. Wiley-VCH, Inc., New York, N.Y.
    10. 10
  10. Crooks, G. E., G. Hon, J.-M. Chandonia, and S. E. Brenner. 2004. WebLogo: a sequence logo generator. Genome Res. 14:1188-1190.[Abstract/Free Full Text]
    11. 11
  11. Ferson, A. E., L. V. Wray, Jr., and S. H. Fisher. 1996. Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability. Mol. Microbiol. 22:693-701.[CrossRef][Medline]
    12. 12
  12. Fisher, S. H. 1999. Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence! Mol. Microbiol. 32:223-232.[CrossRef][Medline]
    13. 13
  13. Fisher, S. H., M. S. Rosenkrantz, and A. L. 1984. Sonenshein. Glutamine synthetase gene of Bacillus subtilis. Gene 32:427-438.[CrossRef][Medline]
    14. 14
  14. Fisher, S. H., and L. V. Wray, Jr. 1989. Regulation of glutamine synthetase in Streptomyces coelicolor. J. Bacteriol. 171:2378-2383.[Abstract/Free Full Text]
    15. 15
  15. Fisher, S. H., and L. V. Wray, Jr. 2002. Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase. J. Bacteriol. 184:2148-2154.[Abstract/Free Full Text]
    16. 16
  16. Godsey, M. H., N. N. Baranova, A. A. Neyfakh, and R. G. Brennan. 2001. Crystal structure of MtaN, a global multidrug transporter gene activator. J. Biol. Chem. 276:47178-47184.[Abstract/Free Full Text]
    17. 17
  17. Guo, H.-C., and J. W. Roberts. 1990. Heterogeneous initiation due to slippage at the bacteriophage 82 late gene promoter in vitro. Biochemistry 29:10702-10709.[CrossRef][Medline]
    18. 18
  18. Gutowski, J. C., and H. J. Schreier. 1992. Interaction of the Bacillus subtilis glnRA repressor with operator and promoter regions in vivo. J. Bacteriol. 174:671-681.[Abstract/Free Full Text]
    19. 19
  19. Haldenwang, W. G. 1995. The sigma factors of Bacillus subtilis. Microbiol. Rev. 59:1-30.[Abstract/Free Full Text]
    20. 20
  20. Heldwein, E. E. Z., and R. G. Brennan. Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409:378-382.
    21. 21
  21. Helmann, J. D. 1995. Compilation and analysis of Bacillus subtilis FA-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23:2351-2360.[Abstract/Free Full Text]
    22. 22
  22. Kontinen, V. P., I. M. Helander, and H. Tokuda. 1996. The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis. FEBS Lett. 389:281-284.[CrossRef][Medline]
    23. 23
  23. Lanzer, M., and H. Bujard. 1988. Promoters largely determine the efficiency of repressor action. Proc. Natl. Acad. Sci. USA 85:8973-8977.[Abstract/Free Full Text]
    24. 24
  24. Luscombe, N. M., and J. M. Thornton. 2002. Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity. J. Mol. Biol. 320:991-1009.[CrossRef][Medline]
    25. 25
  25. Magasanik, B., and F. C. Neidhardt. 1987. Regulation of caron and nitrogen utilization, p. 1318-1325. In F. C. Neidhardt, J. L. Ingraham, K. Brooks, B. Magasanik, M. Schaechter, and H. E. Umbarger, (ed.), Escherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology Press, Washington, D.C.
    26. 26
  26. Meng, Q., C. L. Turnbough, Jr., and R. L. Switzer. 2004. Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription. Proc. Natl. Acad. Sci. USA 101:10943-10948.[Abstract/Free Full Text]
    27. 27
  27. Nakano, M. M., T. Hoffmann, Y. Zhu, and D. Jahn. 1998. Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE. J. Bacteriol. 180:5344-5350.[Abstract/Free Full Text]
    28. 28
  28. Nakano, M. M., F. Yang, P. Hardin, and P. Zuber. 1995. Nitrogen regulation of nasA and nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis. J. Bacteriol. 177:573-579.[Abstract/Free Full Text]
    29. 29
  29. Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119:736-747.[Abstract/Free Full Text]
    30. 30
  30. Newberry, K. J., and R. G. Brennan. 2004. The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. J. Biol. Chem. 279:20356-20362.[Abstract/Free Full Text]
    31. 31
  31. Reitzer, L. 2003. Nitrogen assimilation and global regulation in Escherichia coli. Annu. Rev. Microbiol. 57:155-176.[CrossRef][Medline]
    32. 32
  32. Robichon, D., M. Arnaud, R. Gardan, Z. Pragal, M. O'Reilly, G. Rapoport, and M. Débarbouillé. 2000. Expression of a new operon from Bacillus subtilis, ykzB-ykoL, under the control of the TnrA and PhoP-PhoR global regulators. J. Bacteriol. 182:1226-1231.[Abstract/Free Full Text]
    33. 33
  33. Schneider, T. D., and R. M. Stephens. 1990. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18:6097-6100.[Abstract/Free Full Text]
    34. 34
  34. Schreier, H. J., S. W. Brown, K. D. Hirschi, J. F. Nomellini, and A. L. Sonenshein. 1989. Regulation of Bacillus subtilis glutamine synthetase gene expression by the product of the glnR gene. J. Mol. Biol. 210:51-63.[CrossRef][Medline]
    35. 35
  35. Schreier, H. J., C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi. 1991. Identification of DNA sequences involved in regulating Bacillus subtilis glnRA expression by nitrogen source. J. Mol. Biol. 220:241-253.[CrossRef][Medline]
    36. 36
  36. Strauch, M. A., A. I. Aronson, S. W. Brown, H. J. Schreier, and A. L. Sonenshein. 1988. Sequence of the Bacillus subtilis glutamine synthetase gene region. Gene 71:257-265.[CrossRef][Medline]
    37. 37
  37. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    38. 38
  38. Tojo, S., T. Satomura, K. Morisaki, K.-I. Yoshida, K. Hirooka, and Y. Fujita. 2004. Negative transcriptional regulation of the ilv-leu operon for biosynthesis of branched-chain amino acids through the Bacillus subtilis global regulator TnrA. J. Bacteriol. 186:7971-7979.[Abstract/Free Full Text]
    39. 39
  39. Wray, L. V., Jr., A. E. Ferson, and S. H. Fisher. 1997. Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H. J. Bacteriol. 179:5494-5501.[Abstract/Free Full Text]
    40. 40
  40. Wray, L. V., Jr., A. E. Ferson, K. Rohrer, and S. H. Fisher. 1996. TnrA, a transcriptional factor required for global nitrogen regulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 93:8841-8845.[Abstract/Free Full Text]
    41. 41
  41. Wray, L. V., Jr., F. K. Pettengill, and S. H. Fisher. 1994. Catabolite repression of the Bacillus subtilis hut operon requires a cis-acting site located downstream of the transcription initiation site. J. Bacteriol. 176:1894-1902.[Abstract/Free Full Text]
    42. 42
  42. Wray, L. V., Jr., J. M. Zalieckas, A. E. Ferson, and S. H. Fisher. 1998. Mutational analysis of the TnrA-binding sites in the Bacillus subtilis nrgAB and gabP promoter regions. J. Bacteriol. 180:2943-2949.[Abstract/Free Full Text]
    43. 43
  43. Wray, L. V., Jr., J. M. Zalieckas, and S. H. Fisher. 2000. Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor. J. Mol. Biol. 300:29-40.[CrossRef][Medline]
    44. 44
  44. Wray, L. V., Jr., J. M. Zalieckas, and S. H. Fisher. 2001. Bacillus subtilis glutamine synthetase controls gene expression through a protein-protein interaction with transcription factor TnrA. Cell 107:427-435.[CrossRef][Medline]
    45. 45
  45. Xiong, X. F., and W. S. Reznikoff. 1993. Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo. J. Mol. Biol. 231:569-580.[CrossRef][Medline]

Journal of Bacteriology, April 2006, p. 2578-2585, Vol. 188, No. 7
0021-9193/06/$08.00+0     doi:10.1128/JB.188.7.2578-2585.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Somerville, G. A., Proctor, R. A. (2009). At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci. Microbiol. Mol. Biol. Rev. 73: 233-248 [Abstract] [Full Text]  
  • Fisher, S. H., Wray, L. V. Jr. (2009). Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression. J. Bacteriol. 191: 2485-2492 [Abstract] [Full Text]  
  • El Qaidi, S., Plumbridge, J. (2008). Switching Control of Expression of ptsG from the Mlc Regulon to the NagC Regulon. J. Bacteriol. 190: 4677-4686 [Abstract] [Full Text]  
  • Hendriksen, W. T., Kloosterman, T. G., Bootsma, H. J., Estevao, S., de Groot, R., Kuipers, O. P., Hermans, P. W. M. (2008). Site-Specific Contributions of Glutamine-Dependent Regulator GlnR and GlnR-Regulated Genes to Virulence of Streptococcus pneumoniae. Infect. Immun. 76: 1230-1238 [Abstract] [Full Text]  
  • Fisher, S. H., Wray, L. V. Jr. (2008). Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes. Proc. Natl. Acad. Sci. USA 105: 1014-1019 [Abstract] [Full Text]  
  • Wray, L. V. Jr., Fisher, S. H. (2007). Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein. J. Bacteriol. 189: 20-27 [Abstract] [Full Text]  
  • Fisher, S. H., Wray, L. V. Jr (2006). Feedback-Resistant Mutations in Bacillus subtilis Glutamine Synthetase Are Clustered in the Active Site.. J. Bacteriol. 188: 5966-5974 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zalieckas, J. M.
Right arrow Articles by Fisher, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zalieckas, J. M.
Right arrow Articles by Fisher, S. H.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»