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J Bacteriol, April 1998, p. 1841-1847, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in Escherichia coli

Dana Ault-Riché, Cresson D. Fraley, Chi-Meng Tzeng, and Arthur Kornberg*

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307

Received 24 November 1997/Accepted 27 January 1998

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.


* Corresponding author. Mailing address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307. Phone: (650) 723-6161. Fax: (650) 723-6783. E-mail: akornber{at}cmgm.stanford.edu.




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