Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism.

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-ß. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

Bacillus subtilis CodY operators contain overlapping CodY binding sites.

CodY is a global transcriptional regulator that is activated by branched-chain amino acids. A palindromic 15-bp sequence motif, AATTTTCNGAAAATT, is associated with CodY DNA binding. A gel mobility shift assay was used to examine the effect of pH on the binding of Bacillus subtilis CodY to the hutPp and ureAp(3) promoters. CodY at pH 6.0 has higher affinity for DNA, more enhanced activation by isoleucine, and a lower propensity for nonspecific DNA binding than CodY at pH 8.0. DNase I footprinting was used to identify the CodY-protected regions in the hutPp and ureAp(3) promoters. The CodY-protected sequences for both promoters were found to contain multiple copies of the 15-bp motif with 6-bp overlaps. Mutational analysis of the hutPp regulatory region revealed that two overlapping sequence motifs were required for CodY-mediated regulation. The presence of overlapping sequence motifs in the regulatory regions of many B. subtilis CodY-regulated genes suggests that CodY binds to native operators that contain overlapping binding sites.

Functional roles of the conserved Glu304 loop of Bacillus subtilis glutamine synthetase.

The enzymatic activity of Bacillus subtilis glutamine synthetase (GS), which catalyzes the synthesis of glutamine from ammonium and glutamate, is regulated by glutamine feedback inhibition. The feedback-inhibited form of B. subtilis GS regulates the DNA-binding activities of the TnrA and GlnR nitrogen transcriptional factors. Bacterial GS proteins contain a flexible seven-residue loop, the Glu304 flap, that closes over the glutamate entrance to the active site. Amino acid substitutions in Glu304 flap residues were examined for their effects on gene regulation, enzymatic activity, and feedback inhibition. Substitutions in five of the Glu304 loop residues resulted in constitutive expression of both TnrA- and GlnR-regulated genes, indicating that this flap is important for regulating the activity of these transcription factors. The residues in the highly conserved Glu304 flap appear to be optimized for glutamate binding because mutant enzymes with substitutions in five of the flap residues had increased glutamate Km values compared to that for wild-type GS. The E304A and E304D substitutions increased the ammonium Km values compared to that for wild-type GS and conferred high-level resistance to inhibition by glutamine, glycine, and methionine sulfoximine. A model for the role of the Glu304 residue in glutamine feedback inhibition is proposed.

Novel trans-Acting Bacillus subtilis glnA mutations that derepress glnRA expression.

Bacillus subtilis contains two nitrogen transcription factors, GlnR and TnrA. The activities of GlnR and TnrA are regulated by direct protein-protein interactions with the feedback-inhibited form of glutamine synthetase (GS). To look for other factors involved in regulating GlnR activity, we isolated mutants with constitutive glnRA expression (Gln(C)). The twenty-seven Gln(C) mutants isolated in this mutant screen all contained mutations tightly linked to the glnRA operon which encodes GlnR (glnR) and GS (glnA). Four Gln(C) mutants contained mutations in the glnR gene that most likely impair the ability of GlnR to bind DNA. Three other Gln(C) mutants contained novel glnA mutations (S55F, V173I, and L174F). GlnR regulation was completely relieved in the three glnA mutants, while only modest defects in TnrA regulation were observed. In vitro enzymatic assays showed that the purified S55F mutant enzyme was catalytically defective while the V173I and L174F enzymes were highly resistant to feedback inhibition. The V173I and L174F GS proteins were found to require higher glutamine concentrations than the wild-type GS to regulate the DNA-binding activities of GlnR and TnrA in vitro. These results are consistent with a model where feedback-inhibited GS is the only cellular factor involved in regulating the activity of GlnR in B. subtilis.

Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

Functional analysis of the carboxy-terminal region of Bacillus subtilis TnrA, a MerR family protein.

The Bacillus subtilis TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When B. subtilis cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated amtB gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III tnrA mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.

Feedback-resistant mutations in Bacillus subtilis glutamine synthetase are clustered in the active site.

The feedback-inhibited form of Bacillus subtilis glutamine synthetase regulates the activity of the TnrA transcription factor through a protein-protein interaction that prevents TnrA from binding to DNA. Five mutants containing feedback-resistant glutamine synthetases (E65G, S66P, M68I, H195Y, and P318S) were isolated by screening for colonies capable of cross-feeding Gln(-) cells. In vitro enzymatic assays revealed that the mutant enzymes had increased resistance to inhibition by glutamine, AMP, and methionine sulfoximine. The mutant proteins had a variety of enzymatic alterations that included changes in the levels of enzymatic activity and in substrate K(m) values. Constitutive expression of TnrA- and GlnR-regulated genes was seen in all five mutants. In gel mobility shift assays, the E65G and S66P enzymes were unable to inhibit TnrA DNA binding, while the other three mutant proteins (M68I, H195Y, and P318S) showed partial inhibition of TnrA DNA binding. A homology model of B. subtilis glutamine synthetase revealed that the five mutated amino acid residues are located in the enzyme active site. These observations are consistent with the hypothesis that glutamine and AMP bind at the active site to bring about feedback inhibition of glutamine synthetase.

Cross-regulation of the Bacillus subtilis glnRA and tnrA genes provides evidence for DNA binding site discrimination by GlnR and TnrA.

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 muM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

A feedback-resistant mutant of Bacillus subtilis glutamine synthetase with pleiotropic defects in nitrogen-regulated gene expression.

The Bacillus subtilis TnrA transcription factor regulates gene expression during nitrogen-limited growth. When cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA and prevents TnrA from binding to DNA. A mutation in glutamine synthetase with a phenylalanine replacement at the Ser-186 residue (S186F) was isolated by screening for B. subtilis mutants with constitutive TnrA activity. Although S186F glutamine synthetase has kinetic properties that are similar to the wild-type protein, the S186F enzyme is resistant to feedback inhibition by glutamine and AMP. Ligand binding experiments revealed that the S186F protein had a lower affinity for glutamine and AMP than the wild-type enzyme. S186F glutamine synthetase was defective in its ability to block DNA binding by TnrA in vitro. The properties of the feedback-resistant S186F mutant support the model in which the feedback-inhibited form of glutamine synthetase regulates TnrA activity in vivo.

Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter.

Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes. Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold. Since urea is produced during purine degradation in B. subtilis, regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia. The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression. The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR. Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites. B. subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation.

Mutations in Bacillus subtilis glutamine synthetase that block its interaction with transcription factor TnrA.

In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.

Mutations in the Bacillus subtilis glnRA operon that cause nitrogen source-dependent defects in regulation of TnrA activity.

The Bacillus subtilis nitrogen transcriptional factor TnrA is inactive in cells grown with excess nitrogen, e.g., glutamine or glutamate plus ammonium, because feedback-inhibited glutamine synthetase (product of glnA) binds to TnrA and blocks its DNA-binding activity. Two conditional mutations that allow TnrA-dependent gene expression in cells grown with glutamate plus ammonium, but not in glutamine-grown cells, were characterized. One mutant contained a mutation in the glnA ribosome binding site, while the other mutant synthesized a truncated GlnR protein that constitutively repressed glnRA expression. The levels of glutamine synthetase were reduced in both mutants. As a result, when these mutants are grown with excess nitrogen in the absence of glutamine, there is insufficient production of the feedback inhibitors necessary to convert glutamine synthetase into its feedback-inhibited form and TnrA-activated genes are expressed at high levels.

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.