Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions

Abstract

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for glnK, which encodes a P II -like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria-apparently a paralogue of GlnK-cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the glnD gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnK Y51N , which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK

“…Both EcGlnK and KpGlnK proteins have high sequence identity to EcPII. However, EcPII expressed from the chromosome is unable to substitute for the GlnKs with respect to NifLA [13,16] . This protein was called GlnK because it has higher identity to EcGlnK than EcPII and it is encoded by a glnK gene which is located on the glnKamtB operon.…”

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

“…In the presence of O 2 , however, NifL appears to be oxidized directly by O 2 and dissociates from the membrane [21]. Concerning the nitrogen signal transduction, it is known that uridylylated GlnK transduces the signal of nitrogenlimitation to the nif regulon [8][9][10]21]. Experimental data indicate that under nitrogen-limitation, GlnK interacts with the inhibitory NifL-NifA complex, resulting in the dissociation of the complex (J. Stips and R. A. Schmitz, unpublished observation).…”

Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae

“…PII-like nitrogen sensory proteins have been demonstrated to play a major role in regulating nitrogen metabolism and are considered to be the most widespread signalling proteins in nature [25][26][27]. Recent studies have revealed that the nitrogen status in the diazotrophs K. pneumoniae and Azotobacter vinelandii is transduced towards the regulatory proteins NifL and NifA by the PII-like protein GlnK, a GlnB paralogue [14,17,[28][29][30][31][32]. By contrast to the glnK gene of A. vinelandii, enteric glnK genes are under the control of the general nitrogen regulatory system, and are therefore only expressed under conditions of nitrogen starvation [25,33,34].…”

“…However, in response to the internal nitrogen status, GlnK is covalently modified by uridylylation, as is GlnB at the conserved tyrosine residue located in the T-loop (Y51), by the uridylyltransferase ⁄ uridylyl-removing enzyme GlnD [25,35,36]. It has been demonstrated that GlnK mediates the cellular nitrogen status towards the NifL ⁄ NifA regulatory system by direct protein interaction; however, the mechanism differs between K. pneumoniae and A. vinelandii [14,17,[28][29][30][31][32]. In A. vinelandii, the nonuridylylated form of GlnK activates the inhibitory function of NifL under nitrogen excess by direct protein-protein interaction [37,38], whereas, under nitrogen limitation, NifA is relieved from NifL inhibition by elevated levels of 2-oxoglutarate binding to the GAFdomain of NifA [4,14,18,32,[37][38][39][40][41].…”

“…In A. vinelandii, the nonuridylylated form of GlnK activates the inhibitory function of NifL under nitrogen excess by direct protein-protein interaction [37,38], whereas, under nitrogen limitation, NifA is relieved from NifL inhibition by elevated levels of 2-oxoglutarate binding to the GAFdomain of NifA [4,14,18,32,[37][38][39][40][41]. In K. pneumoniae, however, relief of NifA from NifL inhibition under nitrogen limiting conditions depends on the presence of GlnK independent of its uridylylation state [28][29][30][31]. We recently demonstrated that K. pneumoniae GlnK interacts with both NifL and NifA, and obtained evidence that the GlnK interaction with the inhibitory NifL ⁄ NifA complex under nitrogen limitation results in dissociation of the complex by destabilizing the NifA-NifL interactions [17].…”