Regulation Of Ammonium Assimilation Research Articles

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK1 and glnK2. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK1 and GlnK2. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK1 and GlnK2 produced equivalent results.

In vitroproof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeonHaloferax mediterranei

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.

Nitrogenase Switch-Off and Regulation of Ammonium Assimilation in Response to Light Deprivation in Rhodospirillum rubrum Are Influenced by the Nitrogen Source Used during Growth

Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P(II) modification is dependent on the nitrogen source used for growth, N(2) or glutamate, although both lead to nitrogenase derepression.

ラン藻 Synechosystis sp. PCC 6803 における強光によるアンモニア同化の制御に対するPIIタンパク質の機能

ラン藻 Synechosystis sp. PCC 6803 における強光によるアンモニア同化の制御に対するPIIタンパク質の機能

Function of the PII Protein in Regulation of Ammonium Assimilation in Synechocystis sp. PCC 6803

Function of the PII Protein in Regulation of Ammonium Assimilation in Synechocystis sp. PCC 6803

Modular Response Analysis of Cellular Regulatory Networks

The sheer complexity of intracellular regulatory networks, which involve signal transducing, metabolic, and genetic circuits, hampers our ability to carry out a quantitative analysis of their functions. Here, we describe an approach that greatly simplifies this type of analysis by capitalizing on the modular organization of such networks. Steady-state responses of the network as a whole are accounted for in terms of intermodular interactions between the modules alone; processes operating solely within modules need not be considered when analysing signal transfer through the entire network. The intermodular interactions are quantified through (local) response coefficients which populate an interaction map (matrix). This matrix can be derived from a biochemical or molecular biological analysis of (macro) molecular interactions that constitute the regulatory network. The approach is illustrated by two examples: (i) mitogenic signalling through the mitogen-activated protein kinase cascade in the epidermal growth factor receptor network and (ii) regulation of ammonium assimilation in Escherichia coli.

Modular Response Analysis of Cellular Regulatory Networks

The sheer complexity of intracellular regulatory networks, which involve signal transducing, metabolic, and genetic circuits, hampers our ability to carry out a quantitative analysis of their functions. Here, we describe an approach that greatly simplifies this type of analysis by capitalizing on the modular organization of such networks. Steady-state responses of the network as a whole are accounted for in terms of intermodular interactions between the modules alone; processes operating solely within modules need not be considered when analysing signal transfer through the entire network. The intermodular interactions are quantified through (local) response coefficients which populate an interaction map (matrix). This matrix can be derived from a biochemical or molecular biological analysis of (macro) molecular interactions that constitute the regulatory network. The approach is illustrated by two examples: (i) mitogenic signalling through the mitogen-activated protein kinase cascade in the epidermal growth factor receptor network and (ii) regulation of ammonium assimilation in Escherichia coli.

Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72.

PII-like signal transmitter proteins are involved in the regulation of ammonium assimilation and nitrogen fixation. We report the identification of three PII-like proteins in the diazotrophic, endophytic proteobacterium Azoarcus sp. BH72, encoded by glnB (monocistronically transcribed) or in the glnKamtB and glnYamtY operons. Phylogenetic analysis revealed that glnB, glnK and glnY represent distinct lineages within the Proteobacteria. A combined approach of two-dimensional gel electrophoresis, Western blotting with paralogue-specific antibodies, N-terminal sequencing and marker exchange mutagenesis allowed us to analyse PII protein expression of Azoarcus sp. BH72 in vivo. GlnK and GlnB were present on all nitrogen sources. Knock-out mutant analysis revealed that GlnB was the only detectable PII protein in a glnK- background, whereas GlnY was only present in a glnK/glnB- double mutant. Nitrogen limitation enhanced transcript abundance of glnK strongly, glnY moderately and glnB not at all in wild-type, glnB-/glnK- or glnK- backgrounds respectively. Phenotypic characterization of knock-out mutants revealed that, unlike in other Proteobacteria, neither glnK nor glnB were essential for nitrogen fixation. As the growth of a double mutant was drastically impaired only on minimal media, both proteins are probably involved in the control of ammonium and nitrate assimilation. The PII-like proteins differed from each other in details of N-sensing. They were covalently modified by uridylylation upon nitrogen limitation, as shown by mass spectrometry; however, the modification patterns in relation to the supplied nitrogen source differed. The novel paralogue GlnY was unusual, as it only occurred in the uridylylated state in vivo and thus lacked a deuridylylation response to nitrogen excess.

Pathways and Regulation of Ammonium Assimilation in Streptomyces clavuligerus

Glutamine synthetase, glutamate synthase and alanine dehydrogenase activities were detected in crude extracts of Streptomyces clavuligerus. Glutamate dehydrogenase and alanine: 2-oxoglutarate aminotransferase could not be found. Glutamate synthase levels were independent of the nitrogen source in the culture medium. Glutamine synthetase activity was repressed in the presence of ammonium, and a rapid inactivation of the enzyme was seen after ammonium shock. Alanine dehydrogenase was induced by alanine or ammonium concentrations higher than 20 mm. Glutamate and, in lower amounts, glutamine, alanine, ?gM-aminobutyrate and aspartate were the main constituents of the intracellular pool of free amino acids. Only the pool concentrations of glutamine and alanine were markedly influenced by the nitrogen source used for growth. Mutants devoid of glutamine synthetase, glutamate synthase, or alanine dehydrogenase were isolated. The pattern of utilization of nitrogen sources by the three classes of mutants indicated that the glutamine synthetase–glutamate synthase pathway is the only means of assimilation of ammonium in S. clavuligerus. Alanine dehydrogenase is not directly involved in that process, but a possible related function is discussed.