Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions

Abstract

Previous studies with labelled N and C have indicated synthesis and accumulation of glutamate in Brudyrhizobium japonicum bacteroids under microaerobic conditions similar to those found in soybean nodules. Low 2-oxoglutarate dehydrogenase (OGDH) activity might have accounted for this observation, but similar levels of enzyme activity were found in bacteroids isolated anaerobically or aerobically and in cultured bacteria. However, OGDH from B.japonicum bacteroids was strongly inhibited by NADH, and the degree of inhibition depended on the NADH: NAD ratio. Determination of endogenous levels of NAD and NADH gave NADH: NAD ratios of 0.19 and 0.83 in bacteroids isolated under aerobic and anaerobic conditions, respectively. A ratio of 0.83 resulted in more than 50% inhibition of OGDH in uitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [ 14C)Glutamate supplied to bacteroids was metabolized to CO, slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4aminobutyrate (GAB) was found. Attempts to trap 14C in GAB by supplying unlabelled GAB or transaminase inhibitors with [ 14C]glutamate were unsuccessful. The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil. The inhibition of OGDH by a high NADH:NAD ratio and the absence of the GAB shunt are complementary mechanisms which probably account for the accumulation of glut ama te .

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with 14CO2

“…Thus, it appears that a significant portion of carbon entering bacteroids in pea nodules is diverted to glutamate, a result previously noted in B. japonicum bacteroids (24). If glutamate accumulation results from inhibition of 2-oxoglutarate dehydrogenase complex activity (25), then this would not be surprising, because both B. japonicum and R. leguminosarum bacteroids must function under microaerobic conditions. The overall results for metabolism of dicarboxylic acids in detached, intact nodules confirm our previous suggestion that the operation of the TCA cycle in bacteroids under microaerobic conditions results in significant diversion of carbon into glutamate (24,27).…”

“…1). Isolated bacteroids under microaerobic conditions took up [U-'4C]proline and converted it to CO2 (unpublished data), but only at rates far below those of aspartate and glutamate (24,25).…”

Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules

“…The strong inhibition of glutamate utilization with AOA, therefore, indicates that glutamate catabolism in bacteroids involves a transamination process(es) as an essential step, and GDH in the bacteroids does not operate in the direction of oxidative deamination. Recently, Salminen & Streeter (1990) oids, substantial incorporation of 4C into glutamate was found (Table 5), also suggesting the formation of glutamate from 2-oxoglutarate in the bacteroids.…”

“…Amino-transfer to oxaloacetate is the most probable first reaction in glutamate catabolism in bacteroids because of the presence of relatively high activity of aspartate-oxoglutarate aminotransferase (Table 7 ; see also Salminen & Streeter, 1990). Nitrogen derived from glutamate would need to be released from the bacteroids by rapid deamination of aspartate.…”

“…meliloti bacteroids (Miller et al, 1991). In B. japonicum bacteroids, however, it is suggested that the GABA-shunt does not operate because of the lack of GDC activity (Salminen & Streeter, 1990). In the present work GDC activity in the bacteroids was very low but detectable (net radioactivity in GABA formed in a standard GDC assay was about 2.5-fold of control) and activities of two other GABA-shunt enzymes, GABA-AT and SSDH, were more than tenfold higher in bacteroids than in the plant cytosol (Table 7).…”