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 Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. Th...

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Bibliographic Details
Published in:Journal of bacteriology 2001-04, Vol.183 (8), p.2709-2714
Main Authors: Janes, B K, Pomposiello, P J, Perez-Matos, A, Najarian, D J, Goss, T J, Bender, R A
Format: Article
Language:English
Subjects:
a-ketoglutarate
a-Ketoglutaric acid
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriology
Base Sequence
Cloning, Molecular
Enterobacter aerogenes - genetics
Enterobacter aerogenes - growth & development
Enterobacter aerogenes - metabolism
gdhA gene
Gene Expression Regulation, Bacterial
Genes
Genes, Bacterial
Glutamate Dehydrogenase - genetics
Glutamate Dehydrogenase - metabolism
Klebsiella aerogenes
Molecular Sequence Data
Mutation
nitrogen assimilation control protein
Physiology and Metabolism
rev gene
Sequence Analysis, DNA
succinyl-CoA
trp gene
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Summary:Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh-1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mutation 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 change, 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 sequences 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 postulating 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 consequently 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 the succinyl-CoA requirement relieved the auxotrophy.
ISSN:0021-9193
1098-5530
DOI:10.1128/JB.183.8.2709-2714.2001