FACTORS CONTRIBUTING TO THE ACCUMULATION OF GLUTAMATE IN BRADYRHIZOBIUM-JAPONICUM BACTEROIDS UNDER MICROAEROBIC CONDITIONS

Previous studies with labelled N and C have indicated synthesis and accumulation of glutamate in Bradyrhizobium 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 vitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [14C]Glutamate supplied to bacteroids was metabolized to CO2 slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4-aminobutyrate (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 glutamate.