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Journal of Bacteriology, August 2006, p. 5966-5974, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00544-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Susan H. Fisher* and Lewis V. Wray Jr

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118

Received 16 April 2006/ Accepted 31 May 2006

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 Km 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.

* Corresponding author. Mailing address: Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Phone: (617) 638-5498. Fax: (617) 638-4286. E-mail: shfisher{at}bu.edu.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, August 2006, p. 5966-5974, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00544-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fisher, S. H., Wray, L. V. Jr. (2009). Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression. J. Bacteriol. 191: 2485-2492 [Abstract] [Full Text]  
  • Fisher, S. H., Wray, L. V. Jr. (2008). Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes. Proc. Natl. Acad. Sci. USA 105: 1014-1019 [Abstract] [Full Text]  


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