Characterization of a Mutant Form of the glnG Fene Whose Product, Nitrogen Regulator I (NR1), is the transcriptional activator of the glnALG operon responsible for nitrogen assimilation in Escherichia coli

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Authors
Navai, Yasaman Hannah
Issue Date
1994
Type
Thesis
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en_US
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Abstract
Glutamate is a vital amino acid which serves as a nitrogen donor in the formation of other amino acids and compounds. In conditions of excess nitrogen, such as when ammonia is plentiful, glutamate dehydrogenase provides the primary pathway for glutamate formation. In nitrogen limiting conditions, this enzyme is ineffective in assimilating ammonia. Instead, in enteric bacteria such as Escherichia coli, two different enzymes, glutamine synthetase and glutamate synthase, function in the capacity of nitrogen assimilation and glutamate formation in times of low ammonia. The importance of glutamine synthetase, which is part of the Nitrogen Regulation (Ntr) regulon responsible for nitrogen assimilation, is reflected through its elaborate regulation. GS is encoded by the gInA gene. Transcription of this gene, part of the gInALG operon is activated by the phosphorylated form of Nitrogen Regulator I (NRI) protein (product of the gInG gene). Two compounds, Nitrogen Regulator II (NRII) and acetyl-P, have been demonstrated as donating phosphoryl groups to NRI. This study focused on the characterization of a mutant form of gInG, gInG1128, whose product NRI1128 was believed to activate transcription regardless of its state of phosphorylation. Using a quantitative glutamine synthetase transferase assay in various nitrogen conditions, it was demonstrated that the mutant NRIl128 protein regulated glutamine synthetase transcription less efficiently than did the wild type. Additionally, a second quantitative GS assay revealed that the mutant was not phosphorylation independent, while qualitative assays, demonstrated phosphorylation independence. Examination of both assays and differing growth conditions implicated the possibility of a third compound with the capability of phosphorylating the mutant NRI1128, or even wild type NRI.
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v, 39 p.
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Kalamazoo College
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