Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

Abstract

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

Figure 1

(A) Theoretical trypsin digest fragment of M. smegmatis GlnK shows the GAEYSVDFVPK Tyrosine-51 containing peptide with a mass of 1210.6 Da. (B) MS/MS analysis of the native GAEYSVDFVPK peptide obtained from M. tuberculosis grown in nitrogen excess.

Figure 2

MS/MS analysis of the adenylylated GlnK peptide obtained from M. tuberculosis grown in nitrogen limitation.

Figure 3

Dynamics of GlnK adenylylation in (A)M. smegmatis and (B)M. tuberculosis during nitrogen limitation. GlnK-AMP is represented by light grey bars and GlnK is represented by dark grey bars. Adenylylation of GlnK is induced upon external nitrogen exhaustion (**).

Figure 4

MRM of the GlnK peptide obtained from M. tuberculosis grown in nitrogen limitation. (A) Before SVPDE treatment (B) After SVPDE treatment. Different colour lines represent different peptide-specific transition pairs used in the analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 5

Western blot of GS adenylylation and GS protein levels in M. tuberculosis wild type (lanes 1–4) and glnD deletion mutant (lanes 5–8) during nitrogen limitation (representative gel; n = 3). (A) GS adenylylation levels significantly decreased upon external ammonium depletion (lanes 3 and 7) and increased upon replenishment of ammonium (+++) for both strains (lanes 4 and 8). (B) GS levels were not significantly changed during ammonium depletion or replenishment for either strain (lanes 1–8). Samples were collected on day 6 (lanes 1, 5), day 8 (lane 2, 6), day 9 before (lanes 3, 7), and after (lanes 4, 8) addition of ammonium.