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

Abstract

ABSTRACT Pea plants incubated in 15 N 2 rapidly accumulated labeled γ-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 ω-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.