Lewis V. Wray

Bacillus subtilis Glutamine Synthetase Controls Gene Expression through a Protein-Protein Interaction with Transcription Factor TnrA

Bacillus subtilis TnrA, a global regulator of transcription, responds to nitrogen availability, but the specific signal to which it responds has been elusive. Genetic studies indicate that glutamine synthetase is required for the regulation of TnrA activity in vivo. We report here that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA and blocks the DNA binding activity of TnrA. Mutations in the tnrA gene (tnrA(C)) that allow constitutive high level expression of tnrA-activated genes were isolated and characterized. Feedback-inhibited glutamine synthetase had a significantly reduced ability to block the in vitro DNA binding by three of the TnrA(C) proteins. Thus, glutamine synthetase, an enzyme of central metabolism, directly interacts with and regulates the DNA binding activity of TnrA.

Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability

Expression from the Bacillus subtilis nrg‐21 locus increases 26‐fold during nitrogen‐limited growth. The DNA corresponding to this locus was cloned and sequenced. The nucleotide sequence revealed a gene that could encode a protein with sequence similarity to the Escherichia coliγ‐aminobutyric acid (GABA) permease. A transposon insertion in this locus eliminated the uptake of GABA and severely inhibited the utilization of GABA as a nitrogen source. Primer extension analysis revealed that the B. subtilis gabP gene was transcribed from two overlapping promoters. Transcription from the P1 promoter was repressed during growth in the presence of amino acids. The product of the codY gene proved to be required for this repression. Transcription from the P2 promoter increased during nitrogen‐limited growth and was dependent upon the product of the tnrA gene. Deletion analysis revealed that activation of the P2 promoter during nitrogen‐limited growth requires a nucleotide sequence located upstream of its −35 region. Regulation of gabP expression by the CodY and TnrA regulatory systems, which respond to different physiological signals, allows for a wide range of gabP expression during growth on various nitrogen sources.

Role of TnrA in Nitrogen Source-Dependent Repression of Bacillus subtilis Glutamate Synthase Gene Expression

Synthesis of glutamate, the cells major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

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.

Transcription-repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons.

A Bacillus subtilis mutant that partially relieves carbon catabolite repression (CCR) of the hut operon was isolated by transposon mutagenesis. Characterization of this mutant revealed that the transposon had inserted into the gene, mfd, that encodes transcription–repair coupling factor. The Mfd protein is known to promote strand‐specific DNA repair by displacing RNA polymerase stalled at a nucleotide lesion and directing the (A)BC excinuclease to the DNA damage site. A set of transcriptional lacZ fusions was used to demonstrate that the mfd mutation relieves CCR of hut and gnt expression at the cis‐acting cre sequences located downstream of the transcriptional start site but does not affect CCR at sites located at the promoters. CCR of the amyE and bglPH genes, which contain cre sequences that overlap their promoters, is not altered by the mfd mutation. These results support a model in which the Mfd protein displaces RNA polymerase stalled at downstream cre sites that function as transcriptional roadblocks and reveal a new role for Mfd in cellular physiology.

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.

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 others expression and that there are differences in their DNA-binding specificities.

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.

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.

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.