STRINGENT CONTROL AND GUANOSINE NUCLEOTIDE REGULATION OF THE NITROGEN ASSIMILATORY ENZYMES OF SALMONELLA TYPHIMURIUM

MYRNA SALES, Purdue University

Abstract

The influence of stringent control of the specific activities of the nitrogen assimilatory enzymes has been examined by adjusting nutritional conditions to cause limitations in carbon source, nitrogen source, and general amino acid supply. Limitation of the amino acid supply caused stringent (rel('+)) strains of Salmonella typhimurium to accumulate guanosine 5'-diphosphate -3'-diphosphate (ppGpp), a well characterized indicator of stringent regulation. The specific activites of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase were measured during carbon and amino acid shifts. Although the activities of all three enzymes increase in the rel('+) strain after nutritional downshifts, glutamate dehydrogenase is seen to have the most significant increase. Furthermore, the increase in glutamate dehydrogenase activity is shown to begin shortly after the ppGpp levels increase. Glutamine synthetase activity is seen to increase 3-fold, glutamate synthase activity transiently increases, and glutamate dehydrogenase activity is unchanged upon nitrogen source shifts. Evidence that the shift from glucose-ammonia to glucose-arginine media stimulates stringent regulation is shown by changes in the guanosine nucleotide pools and by a sharp decrease in the rates of RNA and protein synthesis. A novel guanine nucleotide was observed after nitrogen source limitations. The compound was observed only in extracts of rel('+) cells. Chemical and physical studies tentatively identifies the compound as guanosine 5'-triphosphate -3'monophosphate (pppGp). Synthesis of pppGp is shown to be dependent on the post-shift nitrogen source. Evidence is presented which correlates synthesis of this compound with the changes in the activity of glutamine synthetase during nitrogen source shifts. Upon examination of six regulatory mutants of glutamine synthetase during the nitrogen source shifts, mutants classified as glnF('-) were unable to synthesize pppGp. The possible function of pppGp in regulating glutamine synthetase activity is discussed.

Degree

Ph.D.

Subject Area

Microbiology

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