Abstract
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.
Highlights
The low G ϩ C Gram-positive bacterium Bacillus subtilis utilizes two transcriptional factors, GlnR and TnrA, to control the expression of nitrogen-regulated genes [5]
Feedback inhibition of B. subtilis glutamine synthetase (GS) is analogous to the adenylylation of GS in enteric bacteria in that both processes posttranslationally regulate the activity of these enzymes
Similar alterations in nitrogen metabolism are observed in the B. subtilis glnA186 mutant and in strains of S. typhimurium that contain mutations in glnE, the gene encoding GS adenylyltransferase [36]
Summary
Bacterial Strains and Growth—The glnA186 mutation was isolated using SF402S cells (amyE::[(amtB-lacZ)402 cat] tnrA(spc) trpC2) mutagenized with N-methyl-NЈ-nitro-N-nitrosoguanidine [13]. Gel mobility shift experiments to examine the ability of wild-type and S186F GS to inhibit the DNA binding by TnrA were performed as described previously [13]. The inhibition constant for L-methionine-S-sulfoximine was determined from glutamate saturation curves using inhibitor concentrations of 15, 50, and 150 M The data from these experiments were globally fit to the velocity equation for competitive inhibitors, v ϭ (VmaxS)/ (SϩKm(1ϩI/Ki)), where I is the inhibitor concentration and Ki is the inhibition constant. The IC50 values for glutamine and AMP were determined with the Mg2ϩ-dependent biosynthetic reaction with glutamate and ATP concentrations of 150 and 18 mM, respectively. To reliably measure ligand binding, this method requires that the amount of bound ligand must be a significant fraction of the total ligand and is limited to the detection of binding constants that are similar to the concentration of the protein binding sites used in the experiment. The measurement of glutamine binding to wild-type GS in the presence of TnrA was not attempted because it was not practical to isolate the large amounts of TnrA that would be required for this experiment
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