Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis.
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.
Carroll
Paul
P
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, United Kingdom.
Pashley
Carey A
CA
Parish
Tanya
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D005798:Genes, Bacterial; D020043:Genes, Essential; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006358:Hot Temperature; D008717:Methionine Sulfoximine / Q000494:pharmacology; D050296:Microbial Viability; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D005973:Glutamine; D008717:Methionine Sulfoximine; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D005974:Glutamate-Ammonia Ligase