REGULATION OF THE TCA CYCLE AND THE GENERAL AMINO-ACID PERMEASE BY OVERFLOW METABOLISM IN RHIZOBIUM-LEGUMINOSARUM

Mutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to sucA end sucD, which are part of a gene clust er mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-C oA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogena se complex (sucAB). Mutation of either sucC or sucD prevents expressio n of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of suc A or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes m dh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type , excrete large quantities of glutamate and 2-oxoglutarate. Concomitan t with mutation of sucA or sucD, the intracellular concentration of gl utamate but not 2-oxoglutarate was highly elevated, suggesting that 2- oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excre tion as part of an overflow pathway for regulation of the TCA cycle. A mino acid uptake via the Aap of R. leguminosarum was strongly inhibite d in the suc mutants, even though the transcription level of the aap o peron was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another cla ss of mutant impaired in uptake by the Aap is mutated in polyhydroxybu tyrate synthase (phaC). Mutants of succinyl-CoA synthetase (sucD) or 2 -oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon sour ce in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy so urce in the bacteroid. Instead glutamate synthesis, like polyhydroxybu tyrate synthesis, appears to be a sink for carbon and reductant, forme d when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high con centrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxyb utyrate biosynthesis which act as overflow pathways for excess carbon and reductant.