Susan H. Fisher

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two‐component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen‐limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR‐dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild‐type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR‐ and TnrA‐regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen‐limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY‐dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient‐limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.

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.

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.

The Streptomyces coelicolor glnR gene encodes a protein similar to other bacterial response regulators.

The Streptomyces coelicolor glnR gene positively regulates the transcription of the glutamine synthetase-encoding glnA gene. The nucleotide sequence of a 1682-bp DNA segment containing glnR was determined. The deduced amino acid sequence of the GlnR protein was found to be similar to the sequence of several bacterial response regulators that are known to function as transcriptional activators. Primer extension analysis of glnR mRNA identified three transcriptional start points (tsp) upstream from the glnR coding sequence.

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.

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.

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.

Glutamine synthesis in Streptomyces--a review.

The synthesis of glutamine synthetase (GS), a key enzyme in ammonium (NH4+) assimilation, is regulated by nitrogen availability in several Streptomyces strains. In addition, the enzymatic activity of the GS enzyme is post-translationally regulated by adenylylation. Nitrogen regulation of GS synthesis is mediated at the transcriptional level in S. coelicolor, and transcription of the GS structural gene (glnA) requires a positive regulatory protein, GlnR. The amino acid sequence of the GlnR protein is similar to that of the Escherichia coli positive regulatory proteins, OmpR and PhoB, which belong to the family of bacterial two-component regulatory systems. DNA encoding a GSII-like enzyme has been cloned from S. viridochromogenes and S. hygroscopicus, but the role of this GS isoenzyme in NH4+ assimilation in Streptomyces is unclear.

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism.

Background: B. subtilis GS catalyzes the production of glutamine, a key metabolite in nitrogen assimilation. Results: B. subtilis GS forms a dodecamer that undergoes large intersubunit conformational changes during catalysis. Conclusion: B. subtilis GS structures reveal a heretofore unseen active site restructuring that is linked to a novel feedback regulatory mechanism. Significance: The GS specific regulatory/catalytic mechanism may be used to target Gram-positive pathogens. 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-β. 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, Arg62, from an adjacent subunit. Notably, Arg62 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.