Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh -1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiel la aerogenes. The properties of these two mutations were consistent with th ose of a locus containing either a regulatory gene or a structural gene. Th e gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mu tation was generated and shown to be linked to trp. A region of gdhA from a strain bearing gdh-1 was sequenced and shown to have a single-base-pair ch ange, confirming that the locus defined by gdh-1 is the structural gene for GDH. Mutants with the same phenotype as rev-2 were isolated, and their seq uences showed that the mutations were located in the promoter region of the gdhA gene. The linkage of gdhA to trp in K. aerogenes was explained by pos tulating an inversion of the genetic map relative to other enteric bacteria . Strains that bore high-copy-number clones of gdhA displayed an auxotrophy that was interpreted as a limitation for alpha -ketoglutarate and conseque ntly for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy number clones of the enzymatically inactive gdhA1 allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the alpha -ketoglutarate supply or reduce th e succinyl-CoA requirement relieved the auxotrophy.