Nitrogen metabolism in the low G+C Gram-positive bacterium Bacillus subtilis is controlled by a novel regulatory system where the enzyme glutamine synthetase is required for both glutamine synthesis and the direct control of the activity of two transcription factors GlnR and TnrA. The feedback-inhibited form of glutamine synthetase inhibits the activity of TnrA by forming a stable TnrA-glutamine synthetase complex. In contrast, GlnR DNA binding is activated by a transient association with feedback-inhibited glutamine synthetase which stabilizes the GlnR-DNA complexes. Interestingly, this same GlnR-glutamine synthetase nitrogen regulatory system is present in a number of important low G+C Gram-positive pathogens. The major focus of the next project period will be directed toward a detailed analysis of the molecular mechanisms by which feedback-inhibited glutamine synthetase regulates the activity of GlnR and TnrA. The mechanism by which the C-terminal region of GlnR autoinhibits GlnR dimerization will be investigated by identifying the intramolecular interactions that occur between the GlnR C-terminal and N-terminal domains and the amino acid residues required for these interaction(s). The protein-protein interfaces present in the complexes of both TnrA-glutamine synthetase and GlnR-glutamine synthetase will be characterized using mutational, biochemical and structural approaches. Site-directed mutagenesis will be used to identify amino acid residues in the active site of glutamine synthetase required for feedback inhibition. Characterization of biofilm development in B. subtilis indicates that this process is influenced by the nitrogen status of the cell. The interrelationship between nitrogen metabolism and biofilm formation will be explored by identifying the mechanisms responsible for nitrogen regulation of the biofilm matrix protein TasA and the reduced expression of glutamine synthetase in sinR mutants. Genetic experiments suggest that constitutive biofilm formation seen in glutamine synthetase mutants results from increased levels of Spo0A phosphorylation. This will be confirmed by examining expression of Spo0A~P-dependent lacZ fusions in wild-type and mutant cells.

Public Health Relevance

A fundamental question in physiology is how bacteria adapt to growth on different sources of nitrogen. The proposed research will investigate a novel mechanism of nitrogen signal transduction in the low G+C Gram-positive bacterium Bacillus subtilis where the enzyme glutamine synthetase directly controls the activity of the transcription factors TnrA and GlnR. Since the GlnR-glutamine synthetase regulatory system is present in a number of important low G+C Gram-positive pathogens, these studies will provide insight into how nitrogen metabolism is regulated in these bacteria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Reddy, Michael K
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Boston University
Schools of Medicine
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Wray Jr, Lewis V; Fisher, Susan H (2011) Bacillus subtilis CodY operators contain overlapping CodY binding sites. J Bacteriol 193:4841-8
Wray Jr, Lewis V; Fisher, Susan H (2010) Functional roles of the conserved Glu304 loop of Bacillus subtilis glutamine synthetase. J Bacteriol 192:5018-25
Fisher, Susan H; Wray Jr, Lewis V (2009) Novel trans-Acting Bacillus subtilis glnA mutations that derepress glnRA expression. J Bacteriol 191:2485-92
Wray Jr, Lewis V; Fisher, Susan H (2008) Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase. Mol Microbiol 68:277-85
Fisher, Susan H; Wray Jr, Lewis V (2008) Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes. Proc Natl Acad Sci U S A 105:1014-9
Wray Jr, Lewis V; Fisher, Susan H (2007) Functional analysis of the carboxy-terminal region of Bacillus subtilis TnrA, a MerR family protein. J Bacteriol 189:20-7
Fisher, Susan H; Wray Jr, Lewis V (2006) Feedback-resistant mutations in Bacillus subtilis glutamine synthetase are clustered in the active site. J Bacteriol 188:5966-74
Wray Jr, Lewis V; Fisher, Susan H (2005) A feedback-resistant mutant of Bacillus subtilis glutamine synthetase with pleiotropic defects in nitrogen-regulated gene expression. J Biol Chem 280:33298-304
Fisher, Susan H; Wray Jr, Lewis V (2002) Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase. J Bacteriol 184:2148-54
Brandenburg, Jaclyn L; Wray Jr, Lewis V; Beier, Lars et al. (2002) Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter. J Bacteriol 184:6060-4

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