Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis

Abstract

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.

FIG. 1.

Functional analysis of GlnE. (A) The proposed deadenylylation and adenylylation domains and the location of the conserved nucleotidyl transferase motif in GlnE are indicated. The three domain deletions are shown below. The original NotI deletion in pGLN16 is also marked. This is the region deleted in the chromosome of strain Glue14. The drawing is not to scale. (B) Primary amino acid sequence of GlnE from M. tuberculosis. The potential nucleotidyl transferase motif found in the adenylylation domain of GlnE is boxed. The two conserved aspartates in this motif which were individually mutated to alanines are shown in bold. Residues which were mutated in the temperature-sensitive strains, TS1 and TS2, are underlined.

FIG. 2.

Growth of the M. tuberculosis glnE del-int strain (Glue14) in liquid medium with different nitrogen sources. Media were 7H9 (black diamonds), 30 mM (high) ammonia (black squares), 0.1 mM (low) ammonia (open triangles), 3 mM glutamate (open diamonds), or 3 mM glutamine (open squares). Data are the averages and standard deviations from three independent cultures.

FIG. 3.

Growth of M. tuberculosis strains in liquid medium. (A and B) The control strain, Glue14 (black squares), and the recombinant strain carrying the mutated glnE allele (pLEDGE3M2) (open diamonds) were grown with 0.1 mM (low) ammonia (A) or 3 mM glutamine (B). (C) The control strain (Glue14 [glnE^+int]) (black squares) and the temperature-sensitive strain TS2 (open squares) were grown with 0.1 mM (low) ammonia. Data are the averages and standard deviations from three independent cultures.

FIG. 4.

GS activity in M. tuberculosis strains grown in different nitrogen sources. Active GS and total GS were assayed in cell extracts from strains grown in liquid media with different nitrogen sources. (A) Control strain Glue14 carrying wild-type GlnE. (B) Strains 3D1, carrying plasmid pLEDGE3D1 (GlnE deadenylylation domain deletion), and 3M2, carrying plasmid pLEDGE3M2 (D732A mutation) (C). Temperature-sensitive strains TS1 and TS2. Data are the averages and standard deviations from three independent cultures, assayed in duplicate (n = 6). GS activity is given in μmol min⁻¹ mg⁻¹.

FIG. 5.

Growth of M. tuberculosis strains in the presence or absence of GS inhibition. (A) Wild-type (squares) and Glue21 (ΔglnE) (circles) growth in liquid culture. Strains were grown in 7H9 medium with (open symbols) or without (filled symbols) 200 μM MetSox and 3 mM l-Gln (MSG). Data are the averages and standard deviations from three independent cultures. (B) Long-term survival of wild-type and Glue21 strains. Stationary-phase cultures were kept for 4 months and CFU/ml calculated at each time point. Data are the averages and standard deviations from three independent cultures.

FIG. 6.

Active GS in M. tuberculosis strains after removal of MetSox. Strains were grown in 7H9 medium with 200 μM MetSox and 3 mM l-Gln for 7 days, washed, and subcultured in fresh 7H9 medium without supplementation for up to 7 days. Active GS was assayed in cell extracts prepared using the biosynthetic reaction. Data are the averages and standard deviations from three independent cultures, assayed in duplicate (n = 6). GS activity is given in μmol min⁻¹ mg⁻¹.