Pathway of γ-Aminobutyrate Metabolism in Rhizobium leguminosarum 3841 and Its Role in Symbiosis

Abstract

Pea plants incubated in 15N 2 rapidly accumulated labeled ␥-aminobutyrate (GABA) in the plant cytosol and in bacteroids of Rhizobium leguminosarum bv. viciae 3841. Two pathways of GABA metabolism were identified in R. leguminosarum 3841. In the first, glutamate is formed by GABA aminotransferase (GabT), transferring the amino group from GABA to 2-oxoglutarate. In the second, alanine is formed by two -aminotransferases (OpaA and OpaB), transferring the amino group from GABA to pyruvate. While the gabT mutant and the gabT opaA double mutant grew on GABA as a nitrogen source, the final triple mutant did not. The semialdehyde released from GABA by transamination is oxidized by succinate semialdehyde dehydrogenase (GabD). Five of six potential GabD proteins in R. leguminosarum bv. viciae 3841 (GabD1, -D2, -D3, -D4, and -D5) were shown by expression analysis to have this activity. However, only mutations of GabD1, GabD2, and GabD4 were required to prevent utilization of GABA as the sole nitrogen source in culture. The specific enzyme activities of GabT, Opa, and GabD were highly elevated in bacteroids relative to cultured bacteria. This was due to elevated expression of gabT, opaA, gabD1, and gabD2 in nodules. Strains mutated in aminotransferase and succinate semialdehyde dehydrogenases (gabT, opaA, or opaB and gabD1, gabD2, or gabD4, respectively) that cannot use GABA in culture still fixed nitrogen on plants. While GABA catabolism alone is not essential for N 2 fixation in bacteroids, it may have a role in energy generation and in bypassing the decarboxylating arm of the tricarboxylic acid cycle.Rhizobia are alphaproteobacteria that form beneficial symbioses with higher plants, mainly of the legume family. The symbiosis occurs in root structures, called nodules, which provide appropriate conditions, such as a low oxygen concentration, for nitrogen fixation and proliferation of bacteria. To establish a root nodule, a highly specific exchange of signaling compounds is required; rhizobia attach to root hairs and grow down plant-derived infection threads into the root cortex (29). Bacteria are engulfed by plant cells and surrounded by a plantderived membrane (symbiosome membrane) with the whole structure called a symbiosome (13). Within symbiosomes, bacteria differentiate into bacteroids that reduce N 2 to ammonia, which is supplied to the plant in exchange for a dicarboxylate (e.g., L-malate, succinate, and fumarate) for use as a carbon source (1, 47). Consistent with this, dicarboxylate transport via DctA is essential for bacteroid function (9, 34), and labeling studies support a high turnover of malate (35). Recently, it was shown that nutrient exchange is more complex, with amino acid movement between Rhizobium leguminosarum bv. viciae 3841 and the plant cytosol essential in peas (Pisum sativum) for an effective symbiosis (26). This was apparent because mutation of the two main broad-range amino acid ABC transporters (AapJQMP and BraDEFGC) led to a severely impaired symbiotic phenotype. The nitrogen fixation rate of...

Does GABA increase the efficiency of symbiotic N2fixation in legumes?

“…They incubated the nodulated roots of the plant in 15 N 2 for 30 min and detected a rapid labeling level of GABA in the cytosol and bacteroids fraction of R. leguminosarum. 33 The high expression levels of the main enzymes that involve in the GABA metabolism in the bacteroids are consistent with these findings. Despite its relative abundance in nodules, no apparent function for GABA has indicated for a long time.…”

“…32,38 For the amino acid candidate, initially glutamate (or one of its metabolites) was proposed and the recent advances identified GABA as one of the strongest possible amino acids that could take part in cycling model. 32,33 The above mention rapid detection of GABA in the bacteroid fraction (after 15 N 2 application) provides strong support for this thesis. Further evidence was provided by Hosie et al 39 who mentioned that branched-chain amino acid transporter (Bra) was be able to transport GABA across the PBM.…”

“…enzymes involved include: GabD, succinate semialdehyde dehydrogenase; GabT, GABA oxoglutarate aminotransferase; GDC, glutamate decarboxylase; GOGAT, glutamate oxoglutarate amidotransferase; GS, glutamine synthetase; N 2 ase, nitrogenase; OpaA, omega amino acid pyruvate aminotransferase. Adapted from Prell et al 33 …”

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

“…The role of GABA in animal-microbe interactions remains to be explored. In R. leguminosarum, the Atu2422 ortholog Rl3745 is involved in the transport of GABA, which accumulates in the nitrogen-fixing nodules induced on the host plant (42), but the role of GABA in the symbiosis is still unclear (8). In A. tumefaciens, the ratio between exogenous GABA and GABA antagonists controls GABA signaling and modulation of the emergence of plant tumors (10,14).…”

“…In the ameba Dictyostelium, GABA binds to the protein GrlE, which controls terminal steps of sporulation (4). In plants, GABA accumulates in response to abiotic and biotic stresses (6), including wind, injury, and infection by fungi (7) and symbiotic and pathogenic bacteria, such as Rhizobium leguminosarum (8) and Agrobacterium tumefaciens (9). In A. tumefaciens, GABA acts as a signal that triggers gene expression (10,11).…”