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Mutation in Acetate+ aceK∆ Strain Decreases IDH Activity in Escherichia coli Allowing for Growth on Acetate

Menghini, Mark
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The glyoxylate bypass via the enzymes isocitrate lyase and malate synthase allows bacteria to grow on acetate by bypassing the CO2 producing steps and, therefore, accumulating carbon skeletons required for biosynthesis. In order to utilize isocitrate lyase in this bypass and subsequently allow growth on acetate, isocitrate dehydrogenase must be regulated via reversible phosphorylation completed by isocitrate dehydrogenase kinase. IDH activity was observed where the four acetate+ aceK∆ mutants selected for by growth on acetate media had significantly less IDH activity than the aceK∆ parent strain which had full IDH activity, but more than the icd∆ mutant strain which had approximately no IDH activity. The selected acetate+ aceK∆ mutant MTM2 was chosen for further analysis because it contained <25% IDH activity relative to the parent strain aceK∆. Sequencing of the icd gene in MTM2 contained a SNP in the 5’UTR of the icd. Upon Western blot analysis, the relative florescent signal of the MTM2 lane against the parent strain control lane was reduced indicating decreased protein abundance. Since the MTM2 mutation is upstream of the coding region, decreased IDH protein levels were suspected. Future experimentation utilizing qRT-PCR, EMSA, and DNA footprinting may reveal an unknown transcription regulatory binding region upstream of the icd coding sequence.
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University of Wyoming. Libraries
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Keywords
Escherichia coli , Isocitrate dehydrogenase , Isocitrate lyase , Glyoxylate bypass , Acetate , Mutant , aceK\u2206 , IDH activity , Sequencing , Western blot
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