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Journal of Bacteriology, July 2008, p. 4894-4902, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00166-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Analysis of GlnE, an Essential Adenylyl Transferase in Mycobacterium tuberculosis ^,

Paul Carroll, Carey A. Pashley, and Tanya Parish^*

Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, Blizard Building, 4 Newark Street, Whitechapel, London E1 2AT, United Kingdom

Received 2 February 2008/ Accepted 1 May 2008

Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.

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* Corresponding author. Mailing address: Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, Blizard Building, 4 Newark Street, Whitechapel, London E1 2AT, United Kingdom. Phone: 44 (0) 20 7882 2306. Fax: 44 (0) 20 7882 2189. E-mail: t.parish{at}qmul.ac.uk

Published ahead of print on 9 May 2008.

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

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Journal of Bacteriology, July 2008, p. 4894-4902, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00166-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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