A Feedback-resistant Mutant of Bacillus subtilis Glutamine Synthetase with Pleiotropic Defects in Nitrogen-regulated Gene Expression

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.

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

“…In contrast, GlnR and FBI-GS have a weak or transient interaction that could only be demonstrated by chemical cross-linking. Surprisingly, all of the mutations in glnA that are defective in the regulation of TnrA also are defective in regulating GlnR (18)(19)(20). Thus, although there is a fundamental difference in the mode of the interaction of FBI-GS with GlnR and TnrA, these genetic studies indicate that this interaction involves a common region of GS located near the glutamate substrate site (18)(19)(20).…”

“…Surprisingly, all of the mutations in glnA that are defective in the regulation of TnrA also are defective in regulating GlnR (18)(19)(20). Thus, although there is a fundamental difference in the mode of the interaction of FBI-GS with GlnR and TnrA, these genetic studies indicate that this interaction involves a common region of GS located near the glutamate substrate site (18)(19)(20). Interestingly, comparison of the amino acid sequence of B. subtilis GS with the sequences of GS enzymes present in bacteria that lack GlnR and TnrA orthologs indicates that the region of the B. subtilis GS region involved in the interaction with GlnR and TnrA is present in all GS enzymes (L.V.W., unpublished observations).…”

“…Previous studies have identified single amino acid substitution mutants of GS that are defective in regulating GlnR activity in vivo (18)(19)(20). To validate the specificity of the in vitro assay, the ability of two of these mutant proteins to activate GlnR DNA binding was examined.…”

“…The wild-type GS protein was purified as described (13). The concentration of the GS protein was determined by measuring its UV absorbance at 280 nM (18). The S186F and E424K GS proteins were purified by using the same procedure as the wild-type protein.…”

Feedback-Resistant Mutations in Bacillus subtilis Glutamine Synthetase Are Clustered in the Active Site

“…Glutamine synthetase (GS) is a metalloenzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonium. Although Mg 2ϩ or Mn 2ϩ can be used to assay GS in vitro, the Mg 2ϩ -dependent reaction is the physiologically relevant enzymatic activity (34,48).…”