Suppression of the effects of glutamate dehydrogenase (GDH) overproduction in Klebsiella aerogenes.

Glutamate dehydrogenase (GDH) of Klebsiella aerogenes synthesizes glutamate by the reductive amination of a -ketoglutarate, made by the tricarboxylic acid (TCA) cycle. When present in high copy number, the gene encoding GDH, gdhA, caused a growth defect in K. aerogenes grown in glucose ammonia minimal medium. We hypothesized that high levels of GDH activity caused an a -ketoglutarate starvation (and succinyl-CoA starvation, the next metabolite in the TCA cycle). Several lines of evidence support this hypothesis. A cloned gdhA allele (gdh-1), which makes as much GDH polypeptide but 600-fold less activity, has no growth defect. When gdhA + was cloned at lower copy number, the extent of the defect correlated with the amount of GDH activity. Exogenously added glutamate partially suppressed the growth defect. Exogenously added lysine, methionine and d -aminolevulinic acid (derived from succinyl-CoA) to growth medium containing glutamate suppressed the growth defect. Growth under nitrogen limitation to restrict the ammonia available to GDH, thus slowing the depletion of a -ketoglutarate, suppressed the growth defect. Growth on citrate and succinate which activates the TCA cycle and thus, should increase flux through the a -ketoglutarate pool, suppressed the growth defect. The arguments described above support that the growth defect of the GDH overproducer is caused by starvation for a -ketoglutarate. One complication to our hypothesis is that GDH activity was reduced under all the conditions described above, however, reduction of GDH activity did not account fully for the suppression of the defect. From the studies described above I found that lysine repressed GDH activity. Deletions and point mutants in plasmids containing gdhA:: lacZ fusions showed that: lysine mediated repression (LMR) of GDH is at the transcriptional level; the nucleotides necessary for LMR overlap the binding site for the transcriptional regulatory protein Nitrogen Assimilation Control (NAC) protein; and the effector of LMR competes with NAC for binding at this site. The E. coli gdhA gene contains the target but not the factor that causes LMR. In conclusion, my data shows that the growth defect of a K. aerogenes strain with high levels of GDH activity is caused by depletion of a -ketoglutarate. Feeding the auxotrophy and repression of GDH contributed to the suppression of the growth defect, showing that regulation of GDH is complex (e.g. LMR competes with NAC for repression of GDH), as expected for a central enzyme in metabolism.Ph.D.Biological SciencesGeneticsMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131761/2/9929928.pd