Role of GOGAT in carbon and nitrogen partitioning in Rhizobium etli The GenBank accession number for the sequence reported in this paper is AF107264.

Abstract

The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 72 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40 % of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N 2 is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.

Metabolic Reconstruction and Modeling of Nitrogen Fixation in Rhizobium etli

“…It has been shown experimentally that the glutamine synthetase and glutamate synthase pathway (GS-GOGAT) constitutes the central mechanism of ammonium assimilation in free-living Rhizobia [30][31][32]. FBA predicts that there is no activity in the ammonium assimilation pathway during symbiosis (a non-free-living state), which agrees with recent reported measurements where ammonium assimilation was not observed during nitrogen fixation [20,32].…”

Key Role of Bacterial NH 4 + Metabolism in Rhizobium-Plant Symbiosis

“…3). S. meliloti and R. etli mutants lacking GOGAT are not altered in their symbiotic behavior (24,95). Thus, during symbiosis, glutamate is provided in the nodule by the plant and is synthesized through a different pathway, such as amino acid degradation.…”

“…Thus, during symbiosis, glutamate is provided in the nodule by the plant and is synthesized through a different pathway, such as amino acid degradation. In the case of R. etli, lack of GOGAT activity causes an enhancement of nodulation efficiency, probably correlated to an increased ability of BTs to release fixed N (24). In contrast, strains of B. japonicum and A. caulinodans lacking GOGAT are defective in symbiotic N 2 fixation (71,133).…”

“…GOGAT activity is present in both free-living rhizobia and isolated SBs (20), and the structural genes, gltB and gltD, coding for GOGAT in R. etli have been isolated (24). Due to the absence of assimilatory GDH activity, mutants lacking GOGAT activity are glutamate auxotrophs (24,45,88,95,133,136) (Fig.…”

“…Due to the absence of assimilatory GDH activity, mutants lacking GOGAT activity are glutamate auxotrophs (24,45,88,95,133,136) (Fig. 3).…”