Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum

Abstract

SummaryP II -type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P II -type protein in Corynebacterium glutamicum , is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnKglnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F 1 b b b b , are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.

The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel

“…We therefore have suggested that AmtB activity might be regulated by GlnK (16). Indeed, in vivo, GlnK binds rapidly and reversibly to AmtB in response to an extracellular ammonium shock (Ն50 M extracellular ammonium) (17), and this interaction has also been shown in a number of other eubacterial species (16,(18)(19)(20). Methylammonium uptake by AmtB is also inhibited by a GlnK variant that forms an irreversible complex, supporting the concept that GlnK regulates ammonium flux through AmtB (17).…”

“…The occurrence of the AmtB-GlnK pair is highly conserved in both eubacteria and archaea, and the interaction of these proteins has already been demonstrated in a number of cases (16,(18)(19)(20). Hence, we might expect that the mode of binding is also conserved.…”

Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor

“…Such an interaction was indeed identified in E. coli (14), C. glutamicum (15) as well as a number of other prokaryotes and plays a role in regulating the transport of ammonium into the cell. Under conditions of nitrogen excess, GlnK is not posttranslationally modified (see ''PII Proteins and GlnD'' below) and in this native form is sequestered to the membrane by AmtB (15,16). GlnK binds reversibly to AmtB to prevent the transport of ammonium into the cell (14).…”

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

“…This suggests that ATP binding stabilizes the base of the T-loop (residues 36-39), but that the rest of the T-loop must be stabilized by a target protein. Given that PII proteins can bind a multitude of target proteins (particularly, AmtB, and GlnD of the same operon 13 ), it is logical to think that the T-loop exists in different conformations depending on the target protein it complexes with. For example, in the Arabidopsis thaliana PII:NAGK complex structure, the T-loop adopts a very different conformation compared to the E. coli PII:AmtB complex structure.…”

“…12 In the E. coli GlnK, the Tyr51 residue is uridylylated by the GlnD enzyme, but in the Gram-positive soil bacteria Corynebacterium glutamicum and Streptomyces coelicolor GlnD has been shown to act as an adenylyl transferase, adenylylating instead of uridylylating the GlnK protein at this conserved Tyr51 residue. [13][14][15] Under nitrogen limiting conditions, the GlnK protein is uridylylated/adenylylated at the Tyr51 residue, which prevents its interaction with AmtB allowing an unhindered flow of extracellular ammonium into the cell through AmtB. 16 The AmtB:GlnK complex is also implicated in the membrane sequestration of enzymes involved in nitrogen fixation, such as the glycohydrolase DraG in Azospirillum brasilense.…”